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Mucosal delivery of a vectored RSV vaccine is safe and elicits protective immunity in rodents and nonhuman primates.

Pierantoni A, Esposito ML, Ammendola V, Napolitano F, Grazioli F, Abbate A, Del Sorbo M, Siani L, D'Alise AM, Taglioni A, Perretta G, Siccardi A, Soprana E, Panigada M, Thom M, Scarselli E, Folgori A, Colloca S, Taylor G, Cortese R, Nicosia A, Capone S, Vitelli A - Mol Ther Methods Clin Dev (2015)

Bottom Line: Because RSV infection is restricted to the respiratory tract, we compared intranasal (IN) and intramuscular (M) administration for safety, immunogenicity, and efficacy in different species.In addition, animals primed in the nose developed mucosal IgA against the F protein.In conclusion, we have shown that our vectored RSV vaccine induces potent cellular and humoral responses in a primate model, providing strong support for clinical testing.

View Article: PubMed Central - PubMed

Affiliation: ReiThera Srl , Rome, Italy (former Okairos Srl).

ABSTRACT
Respiratory Syncytial Virus (RSV) is a leading cause of severe respiratory disease in infants and the elderly. No vaccine is presently available to address this major unmet medical need. We generated a new genetic vaccine based on chimpanzee Adenovirus (PanAd3-RSV) and Modified Vaccinia Ankara RSV (MVA-RSV) encoding the F, N, and M2-1 proteins of RSV, for the induction of neutralizing antibodies and broad cellular immunity. Because RSV infection is restricted to the respiratory tract, we compared intranasal (IN) and intramuscular (M) administration for safety, immunogenicity, and efficacy in different species. A single IN or IM vaccination completely protected BALB/c mice and cotton rats against RSV replication in the lungs. However, only IN administration could prevent infection in the upper respiratory tract. IM vaccination with MVA-RSV also protected cotton rats from lower respiratory tract infection in the absence of detectable neutralizing antibodies. Heterologous prime boost with PanAd3-RSV and MVA-RSV elicited high neutralizing antibody titers and broad T-cell responses in nonhuman primates. In addition, animals primed in the nose developed mucosal IgA against the F protein. In conclusion, we have shown that our vectored RSV vaccine induces potent cellular and humoral responses in a primate model, providing strong support for clinical testing.

No MeSH data available.


Related in: MedlinePlus

The RSV vaccine antigen. (a) Schematic diagram of the synthetic DNA fragment used to express the RSV antigens by PanAd3 and MVA vectors. 2A self-cleavage site derives from Foot and Mouth Disease virus sequence; (b) WB analysis of total cell lysates from HeLa cells not transfected (nt) or transfected with an expression plasmid bearing the RSV antigen (RSV) and revealed using a monoclonal antibody against M2-1 (mAb 8). The arrows indicate the bands corresponding to the MW of the unprocessed precursor or to the fused internal proteins N and M2-1; (c) WB analysis of supernatants of Hela cells transfected with an expression plasmid encoding the RSV antigen (RSV) or the F0ΔTM protein (F0), after migration on nonreducing SDS–PAGE. A band of ~170 kDa indicated by the arrow was revealed by the monoclonal antibody mAb 13 raised against the F protein which is consistent with a soluble F trimer.
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fig1: The RSV vaccine antigen. (a) Schematic diagram of the synthetic DNA fragment used to express the RSV antigens by PanAd3 and MVA vectors. 2A self-cleavage site derives from Foot and Mouth Disease virus sequence; (b) WB analysis of total cell lysates from HeLa cells not transfected (nt) or transfected with an expression plasmid bearing the RSV antigen (RSV) and revealed using a monoclonal antibody against M2-1 (mAb 8). The arrows indicate the bands corresponding to the MW of the unprocessed precursor or to the fused internal proteins N and M2-1; (c) WB analysis of supernatants of Hela cells transfected with an expression plasmid encoding the RSV antigen (RSV) or the F0ΔTM protein (F0), after migration on nonreducing SDS–PAGE. A band of ~170 kDa indicated by the arrow was revealed by the monoclonal antibody mAb 13 raised against the F protein which is consistent with a soluble F trimer.

Mentions: We designed a synthetic HRSV antigen composed of three viral proteins: F, N, and M2-1 (F0ΔTM–N–M2-1), which were encoded by a single open reading frame with a self-cleaving Foot and Mouth Disease virus 2A sequence21 between F and N sequences. A short sequence encoding a flexible linker was inserted between the N and the M2-1 sequences to facilitate folding (Figure 1a). The transmembrane and cytoplasmic regions of the F protein were deleted for efficient secretion of a soluble form of the F protein. The synthetic gene was codon-optimized for expression in human cells. Mature N–M2-1 fusion protein was detected by Western blot (WB) in cell lysates of DNA transfected HeLa cells (Figure 1b). The high molecular weight precursor F–N–M2-1 was barely detectable indicating efficient 2A-driven self-processing. Consistent with this observation, the soluble F protein was found in the medium of transfected cells with an apparent molecular weight of ~170 kDa on a nonreducing SDS–PAGE gel, compatible with F protein trimers, and was recognized by the conformation sensitive neutralizing antibody mAb 13 (ref. 22) (Figure 1c). When the soluble F protein was run on a reducing SDS–PAGE, two fragments were revealed consistent with the size of F1 and F2, indicating that the protein was correctly processed at the furin-cleavage sites (see Supplementary Figure S1).


Mucosal delivery of a vectored RSV vaccine is safe and elicits protective immunity in rodents and nonhuman primates.

Pierantoni A, Esposito ML, Ammendola V, Napolitano F, Grazioli F, Abbate A, Del Sorbo M, Siani L, D'Alise AM, Taglioni A, Perretta G, Siccardi A, Soprana E, Panigada M, Thom M, Scarselli E, Folgori A, Colloca S, Taylor G, Cortese R, Nicosia A, Capone S, Vitelli A - Mol Ther Methods Clin Dev (2015)

The RSV vaccine antigen. (a) Schematic diagram of the synthetic DNA fragment used to express the RSV antigens by PanAd3 and MVA vectors. 2A self-cleavage site derives from Foot and Mouth Disease virus sequence; (b) WB analysis of total cell lysates from HeLa cells not transfected (nt) or transfected with an expression plasmid bearing the RSV antigen (RSV) and revealed using a monoclonal antibody against M2-1 (mAb 8). The arrows indicate the bands corresponding to the MW of the unprocessed precursor or to the fused internal proteins N and M2-1; (c) WB analysis of supernatants of Hela cells transfected with an expression plasmid encoding the RSV antigen (RSV) or the F0ΔTM protein (F0), after migration on nonreducing SDS–PAGE. A band of ~170 kDa indicated by the arrow was revealed by the monoclonal antibody mAb 13 raised against the F protein which is consistent with a soluble F trimer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4441047&req=5

fig1: The RSV vaccine antigen. (a) Schematic diagram of the synthetic DNA fragment used to express the RSV antigens by PanAd3 and MVA vectors. 2A self-cleavage site derives from Foot and Mouth Disease virus sequence; (b) WB analysis of total cell lysates from HeLa cells not transfected (nt) or transfected with an expression plasmid bearing the RSV antigen (RSV) and revealed using a monoclonal antibody against M2-1 (mAb 8). The arrows indicate the bands corresponding to the MW of the unprocessed precursor or to the fused internal proteins N and M2-1; (c) WB analysis of supernatants of Hela cells transfected with an expression plasmid encoding the RSV antigen (RSV) or the F0ΔTM protein (F0), after migration on nonreducing SDS–PAGE. A band of ~170 kDa indicated by the arrow was revealed by the monoclonal antibody mAb 13 raised against the F protein which is consistent with a soluble F trimer.
Mentions: We designed a synthetic HRSV antigen composed of three viral proteins: F, N, and M2-1 (F0ΔTM–N–M2-1), which were encoded by a single open reading frame with a self-cleaving Foot and Mouth Disease virus 2A sequence21 between F and N sequences. A short sequence encoding a flexible linker was inserted between the N and the M2-1 sequences to facilitate folding (Figure 1a). The transmembrane and cytoplasmic regions of the F protein were deleted for efficient secretion of a soluble form of the F protein. The synthetic gene was codon-optimized for expression in human cells. Mature N–M2-1 fusion protein was detected by Western blot (WB) in cell lysates of DNA transfected HeLa cells (Figure 1b). The high molecular weight precursor F–N–M2-1 was barely detectable indicating efficient 2A-driven self-processing. Consistent with this observation, the soluble F protein was found in the medium of transfected cells with an apparent molecular weight of ~170 kDa on a nonreducing SDS–PAGE gel, compatible with F protein trimers, and was recognized by the conformation sensitive neutralizing antibody mAb 13 (ref. 22) (Figure 1c). When the soluble F protein was run on a reducing SDS–PAGE, two fragments were revealed consistent with the size of F1 and F2, indicating that the protein was correctly processed at the furin-cleavage sites (see Supplementary Figure S1).

Bottom Line: Because RSV infection is restricted to the respiratory tract, we compared intranasal (IN) and intramuscular (M) administration for safety, immunogenicity, and efficacy in different species.In addition, animals primed in the nose developed mucosal IgA against the F protein.In conclusion, we have shown that our vectored RSV vaccine induces potent cellular and humoral responses in a primate model, providing strong support for clinical testing.

View Article: PubMed Central - PubMed

Affiliation: ReiThera Srl , Rome, Italy (former Okairos Srl).

ABSTRACT
Respiratory Syncytial Virus (RSV) is a leading cause of severe respiratory disease in infants and the elderly. No vaccine is presently available to address this major unmet medical need. We generated a new genetic vaccine based on chimpanzee Adenovirus (PanAd3-RSV) and Modified Vaccinia Ankara RSV (MVA-RSV) encoding the F, N, and M2-1 proteins of RSV, for the induction of neutralizing antibodies and broad cellular immunity. Because RSV infection is restricted to the respiratory tract, we compared intranasal (IN) and intramuscular (M) administration for safety, immunogenicity, and efficacy in different species. A single IN or IM vaccination completely protected BALB/c mice and cotton rats against RSV replication in the lungs. However, only IN administration could prevent infection in the upper respiratory tract. IM vaccination with MVA-RSV also protected cotton rats from lower respiratory tract infection in the absence of detectable neutralizing antibodies. Heterologous prime boost with PanAd3-RSV and MVA-RSV elicited high neutralizing antibody titers and broad T-cell responses in nonhuman primates. In addition, animals primed in the nose developed mucosal IgA against the F protein. In conclusion, we have shown that our vectored RSV vaccine induces potent cellular and humoral responses in a primate model, providing strong support for clinical testing.

No MeSH data available.


Related in: MedlinePlus