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Recombinant rabies virus particles presenting botulinum neurotoxin antigens elicit a protective humoral response in vivo.

Hudacek AW, Al-Saleem FH, Willet M, Eisemann T, Mattis JA, Simpson LL, Schnell MJ - Mol Ther Methods Clin Dev (2014)

Bottom Line: Our previous research has shown that recombinant rabies virus-based particles can effectively present heterologous antigens.Administration of a combination of these vectors also demonstrated antibody responses against all three serotypes based on enzyme-linked immunosorbent assay (ELISA) measurements, with minimal decay within the study timeline.Complete protection was achieved against toxin challenge from the serotypes /A and /B and partial protection for /E, indicating that a multivalent approach is feasible.

View Article: PubMed Central - PubMed

Affiliation: Molecular Targeting Technologies, Inc. , West Chester, Pennsylvania, USA ; Department of Microbiology and Immunology, Sidney Kimmel Medical College, Thomas Jefferson University , Philadelphia, Pennsylvania, USA.

ABSTRACT
Botulinum neurotoxins are one of the most potent toxins found in nature, with broad medical applications from cosmetics to the treatment of various neuropathies. Additionally, these toxins are classified as Category A-Tier 1 agents, with human lethal doses calculated at as little as 90 ng depending upon the route of administration. Of the eight distinct botulinum neurotoxin serotypes, the most common causes of human illness are from serotypes /A, /B, and /E. Protection can be achieved by eliciting antibody responses against the receptor-binding domain of the neurotoxin. Our previous research has shown that recombinant rabies virus-based particles can effectively present heterologous antigens. Here, we describe a novel strategy using recombinant rabies virus particles that elicits a durable humoral immune response against the botulinum neurotoxin receptor binding domains from serotypes /A, /B, and /E. Following intramuscular administration of β-propiolactone-inactivated rabies virus particles, mice elicited specific immune responses against the cognate antigen. Administration of a combination of these vectors also demonstrated antibody responses against all three serotypes based on enzyme-linked immunosorbent assay (ELISA) measurements, with minimal decay within the study timeline. Complete protection was achieved against toxin challenge from the serotypes /A and /B and partial protection for /E, indicating that a multivalent approach is feasible.

No MeSH data available.


Related in: MedlinePlus

Recombinant HC50/RABV-G expression analyses on BSR cells. BSR cells were infected at an multiplicity of infection of 10 for 48 hours with the viruses indicated and probed with (a) anti-HC50/A1 (NR-9353), (b) anti-HC50/B1 (NR-9354), or (c) anti-BoNT/E1 (NR-17613). Duplicate blots were also probed with anti-HA-tag monoclonal antibody to detect recombinant HC50/RABV-G proteins; as infection and loading controls, blots were also probed with anti-RABV-M and anti-β-actin.
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fig2: Recombinant HC50/RABV-G expression analyses on BSR cells. BSR cells were infected at an multiplicity of infection of 10 for 48 hours with the viruses indicated and probed with (a) anti-HC50/A1 (NR-9353), (b) anti-HC50/B1 (NR-9354), or (c) anti-BoNT/E1 (NR-17613). Duplicate blots were also probed with anti-HA-tag monoclonal antibody to detect recombinant HC50/RABV-G proteins; as infection and loading controls, blots were also probed with anti-RABV-M and anti-β-actin.

Mentions: As increased levels of surface expression are associated with enhanced incorporation of fusion RABV G/HC50 proteins into RABV particles,27 we next sought to further maximize protein expression by codon optimizing the different HC50 proteins to compensate for mammalian codon bias. Codon-optimized and wild-type versions of the three HC50 domains were analyzed by western blot of infected BSR cell lysates. We found that in the cases of serotypes /A and /B, codon optimization resulted in increases in recombinant protein expression (Figure 2a,b, serotype specific and HA specific immunoblots). Although rescued from a cDNA clone with an intact codon optimized HC50/E transgene, we observed a loss of expression from the codon optimized HC50/E transgene (Figure 2c, serotype-specific and HA-tag-specific immunoblots) by the second passage of the virus. While unexpected, similar observations have been reported previously in which nonsense mutations were acquired in the measles virus F transgene during recombinant VSV replication.27,32 In order to determine the mechanism behind the silencing of the codon optimized HC50/E transgene, we analyzed the viral RNA and found that there was a frame-shift mutation caused by the incorporation of an additional adenosine residue into an adenosine-rich section of the coding sequence resulting in premature termination (data not shown). While the recombinant RABV G-HC50/E protein could be initially detected in the first passage of the virus, this mutation effectively silenced expression of the codon-optimized region by the second passage of the virus (data not shown). We further confirmed that the wild-type BNSP-333-HC50/E virus did indeed contain the wild-type gene by sequencing the genomic material from the HC50/E high expressing particles (data not shown). Because of these data, we continued to focus on the wild-type HC50/E virus.


Recombinant rabies virus particles presenting botulinum neurotoxin antigens elicit a protective humoral response in vivo.

Hudacek AW, Al-Saleem FH, Willet M, Eisemann T, Mattis JA, Simpson LL, Schnell MJ - Mol Ther Methods Clin Dev (2014)

Recombinant HC50/RABV-G expression analyses on BSR cells. BSR cells were infected at an multiplicity of infection of 10 for 48 hours with the viruses indicated and probed with (a) anti-HC50/A1 (NR-9353), (b) anti-HC50/B1 (NR-9354), or (c) anti-BoNT/E1 (NR-17613). Duplicate blots were also probed with anti-HA-tag monoclonal antibody to detect recombinant HC50/RABV-G proteins; as infection and loading controls, blots were also probed with anti-RABV-M and anti-β-actin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362357&req=5

fig2: Recombinant HC50/RABV-G expression analyses on BSR cells. BSR cells were infected at an multiplicity of infection of 10 for 48 hours with the viruses indicated and probed with (a) anti-HC50/A1 (NR-9353), (b) anti-HC50/B1 (NR-9354), or (c) anti-BoNT/E1 (NR-17613). Duplicate blots were also probed with anti-HA-tag monoclonal antibody to detect recombinant HC50/RABV-G proteins; as infection and loading controls, blots were also probed with anti-RABV-M and anti-β-actin.
Mentions: As increased levels of surface expression are associated with enhanced incorporation of fusion RABV G/HC50 proteins into RABV particles,27 we next sought to further maximize protein expression by codon optimizing the different HC50 proteins to compensate for mammalian codon bias. Codon-optimized and wild-type versions of the three HC50 domains were analyzed by western blot of infected BSR cell lysates. We found that in the cases of serotypes /A and /B, codon optimization resulted in increases in recombinant protein expression (Figure 2a,b, serotype specific and HA specific immunoblots). Although rescued from a cDNA clone with an intact codon optimized HC50/E transgene, we observed a loss of expression from the codon optimized HC50/E transgene (Figure 2c, serotype-specific and HA-tag-specific immunoblots) by the second passage of the virus. While unexpected, similar observations have been reported previously in which nonsense mutations were acquired in the measles virus F transgene during recombinant VSV replication.27,32 In order to determine the mechanism behind the silencing of the codon optimized HC50/E transgene, we analyzed the viral RNA and found that there was a frame-shift mutation caused by the incorporation of an additional adenosine residue into an adenosine-rich section of the coding sequence resulting in premature termination (data not shown). While the recombinant RABV G-HC50/E protein could be initially detected in the first passage of the virus, this mutation effectively silenced expression of the codon-optimized region by the second passage of the virus (data not shown). We further confirmed that the wild-type BNSP-333-HC50/E virus did indeed contain the wild-type gene by sequencing the genomic material from the HC50/E high expressing particles (data not shown). Because of these data, we continued to focus on the wild-type HC50/E virus.

Bottom Line: Our previous research has shown that recombinant rabies virus-based particles can effectively present heterologous antigens.Administration of a combination of these vectors also demonstrated antibody responses against all three serotypes based on enzyme-linked immunosorbent assay (ELISA) measurements, with minimal decay within the study timeline.Complete protection was achieved against toxin challenge from the serotypes /A and /B and partial protection for /E, indicating that a multivalent approach is feasible.

View Article: PubMed Central - PubMed

Affiliation: Molecular Targeting Technologies, Inc. , West Chester, Pennsylvania, USA ; Department of Microbiology and Immunology, Sidney Kimmel Medical College, Thomas Jefferson University , Philadelphia, Pennsylvania, USA.

ABSTRACT
Botulinum neurotoxins are one of the most potent toxins found in nature, with broad medical applications from cosmetics to the treatment of various neuropathies. Additionally, these toxins are classified as Category A-Tier 1 agents, with human lethal doses calculated at as little as 90 ng depending upon the route of administration. Of the eight distinct botulinum neurotoxin serotypes, the most common causes of human illness are from serotypes /A, /B, and /E. Protection can be achieved by eliciting antibody responses against the receptor-binding domain of the neurotoxin. Our previous research has shown that recombinant rabies virus-based particles can effectively present heterologous antigens. Here, we describe a novel strategy using recombinant rabies virus particles that elicits a durable humoral immune response against the botulinum neurotoxin receptor binding domains from serotypes /A, /B, and /E. Following intramuscular administration of β-propiolactone-inactivated rabies virus particles, mice elicited specific immune responses against the cognate antigen. Administration of a combination of these vectors also demonstrated antibody responses against all three serotypes based on enzyme-linked immunosorbent assay (ELISA) measurements, with minimal decay within the study timeline. Complete protection was achieved against toxin challenge from the serotypes /A and /B and partial protection for /E, indicating that a multivalent approach is feasible.

No MeSH data available.


Related in: MedlinePlus