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Active vaccination with vaccinia virus A33 protects mice against lethal vaccinia and ectromelia viruses but not against cowpoxvirus; elucidation of the specific adaptive immune response.

Paran N, Lustig S, Zvi A, Erez N, Israely T, Melamed S, Politi B, Ben-Nathan D, Schneider P, Lachmi B, Israeli O, Stein D, Levin R, Olshevsky U - Virol. J. (2013)

Bottom Line: We identified a single protective region located between residues 104-120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions.Both epitopes in A33CPXV are mutated and predicted to be non-functional.This epitope's critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious Diseases, Israel Institute for Biological Research, P.O. box 19, Ness-Ziona 74100, Israel. nirp@iibr.gov.il

ABSTRACT
Vaccinia virus protein A33 (A33VACV) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. In this study we show that single-dose vaccination with recombinant Sindbis virus expressing A33VACV, is sufficient to protect mice against lethal challenge with vaccinia virus WR (VACV-WR) and ectromelia virus (ECTV) but not against cowpox virus (CPXV), a closely related orthopoxvirus. Moreover, a subunit vaccine based on the cowpox virus A33 ortholog (A33CPXV) failed to protect against cowpox and only partially protected mice against VACV-WR challenge. We mapped regions of sequence variation between A33VACV and A33CPXVand analyzed the role of such variations in protection. We identified a single protective region located between residues 104-120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions. Both epitopes in A33CPXV are mutated and predicted to be non-functional. Whereas vaccination with A33VACV did not induce in-vivo CTL activity to the predicted epitope, inhibition of virus spread in-vitro, and protection from lethal VACV challenge pointed to the B cell epitope highlighting the critical role of residue L118 and of adjacent compensatory residues in protection. This epitope's critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here. These findings should be considered when developing new subunit vaccines and monoclonal antibody based therapeutics against orthopoxviruses, especially variola virus, the etiologic agent of smallpox.

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Expression and induction of antibodies with comet inhibition activity by Sindbis A33 harboring point mutations within the CP II region. Multiple sequence alignment of A33 CP II region showing amino acid changes between several Orthopoxviruses-VACV-WR (Vaccinia virus – Western Reserve), CPXV-BR (CPXV Brighton red), MPXV-Zar (monkeypox Zaire), ECTV-Moscow and VARV-BGD74-sol (Bangladesh 74) (A). Expression analysis of the various A33 genes in lysates of Sindbis A33 infected BHK21 cells separated by SDS-PAGE and immunoblotted as indicated. MW – Molecular weight (Kda). Arrowheads indicate bands of the different proteins (B). Geometric mean titer (GMT) (ELISA) in sera of vaccinated mice 21 days post vaccination. Bars represent GeoStDev (C). Comet inhibition activity in sera of Sindbis A33 vaccinated mice (D, same sera as in C). Serum dilutions are indicated to the left and the type of Sindbis A33 used for vaccination is indicated at the top.
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Figure 6: Expression and induction of antibodies with comet inhibition activity by Sindbis A33 harboring point mutations within the CP II region. Multiple sequence alignment of A33 CP II region showing amino acid changes between several Orthopoxviruses-VACV-WR (Vaccinia virus – Western Reserve), CPXV-BR (CPXV Brighton red), MPXV-Zar (monkeypox Zaire), ECTV-Moscow and VARV-BGD74-sol (Bangladesh 74) (A). Expression analysis of the various A33 genes in lysates of Sindbis A33 infected BHK21 cells separated by SDS-PAGE and immunoblotted as indicated. MW – Molecular weight (Kda). Arrowheads indicate bands of the different proteins (B). Geometric mean titer (GMT) (ELISA) in sera of vaccinated mice 21 days post vaccination. Bars represent GeoStDev (C). Comet inhibition activity in sera of Sindbis A33 vaccinated mice (D, same sera as in C). Serum dilutions are indicated to the left and the type of Sindbis A33 used for vaccination is indicated at the top.

Mentions: Next, we wanted to identify which of the residues in CP-II contribute to protection. The CP-II region in A33CPXV harbors the following amino acid substitutions compared to A33VACV: L112F, Q117K and L118S (Figure 6A). The L112F substitution is shared also by variola virus (VARV-BGD74_sol) while the Q117K and L118S substitutions are found in monkeypox (MPXV-ZAR) and ectromelia (ECTV-Moscow) but not in VARV. Both MPXV and ECTV share also the S120E substitution. To check which of these substitutions abrogates protection, we generated modified A33VACV genes harboring the L112F, Q117K and L118S substitutions on the backbone of A33VACV . In addition, we also generated a double mutant A33 gene (Q117K-L118S). These modified A33VACV genes were cloned into the Sindbis expression system. Recombinant Sindbis viruses were produced and expression of the modified A33 genes was confirmed by infection of BHK 21 cells followed by Western blot analysis of the cell lysates (Figure 6B). A33 is post-translationally modified in cells (N’ and O’ glycosylations and acylation) resulting in a well-known multiple band pattern in SDS-PAGE [14,16,19]. All Sindbis viruses expressed the modified A33VACV genes, yet certain differences in the multiple band pattern could be observed, mainly in the double mutant A33 (Q117K-L118S). Expression of the E1 and E2 sindbis virus proteins and β-Tubulin confirm the specificity of A33 detection and serve as infection and loading controls.


Active vaccination with vaccinia virus A33 protects mice against lethal vaccinia and ectromelia viruses but not against cowpoxvirus; elucidation of the specific adaptive immune response.

Paran N, Lustig S, Zvi A, Erez N, Israely T, Melamed S, Politi B, Ben-Nathan D, Schneider P, Lachmi B, Israeli O, Stein D, Levin R, Olshevsky U - Virol. J. (2013)

Expression and induction of antibodies with comet inhibition activity by Sindbis A33 harboring point mutations within the CP II region. Multiple sequence alignment of A33 CP II region showing amino acid changes between several Orthopoxviruses-VACV-WR (Vaccinia virus – Western Reserve), CPXV-BR (CPXV Brighton red), MPXV-Zar (monkeypox Zaire), ECTV-Moscow and VARV-BGD74-sol (Bangladesh 74) (A). Expression analysis of the various A33 genes in lysates of Sindbis A33 infected BHK21 cells separated by SDS-PAGE and immunoblotted as indicated. MW – Molecular weight (Kda). Arrowheads indicate bands of the different proteins (B). Geometric mean titer (GMT) (ELISA) in sera of vaccinated mice 21 days post vaccination. Bars represent GeoStDev (C). Comet inhibition activity in sera of Sindbis A33 vaccinated mice (D, same sera as in C). Serum dilutions are indicated to the left and the type of Sindbis A33 used for vaccination is indicated at the top.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3722049&req=5

Figure 6: Expression and induction of antibodies with comet inhibition activity by Sindbis A33 harboring point mutations within the CP II region. Multiple sequence alignment of A33 CP II region showing amino acid changes between several Orthopoxviruses-VACV-WR (Vaccinia virus – Western Reserve), CPXV-BR (CPXV Brighton red), MPXV-Zar (monkeypox Zaire), ECTV-Moscow and VARV-BGD74-sol (Bangladesh 74) (A). Expression analysis of the various A33 genes in lysates of Sindbis A33 infected BHK21 cells separated by SDS-PAGE and immunoblotted as indicated. MW – Molecular weight (Kda). Arrowheads indicate bands of the different proteins (B). Geometric mean titer (GMT) (ELISA) in sera of vaccinated mice 21 days post vaccination. Bars represent GeoStDev (C). Comet inhibition activity in sera of Sindbis A33 vaccinated mice (D, same sera as in C). Serum dilutions are indicated to the left and the type of Sindbis A33 used for vaccination is indicated at the top.
Mentions: Next, we wanted to identify which of the residues in CP-II contribute to protection. The CP-II region in A33CPXV harbors the following amino acid substitutions compared to A33VACV: L112F, Q117K and L118S (Figure 6A). The L112F substitution is shared also by variola virus (VARV-BGD74_sol) while the Q117K and L118S substitutions are found in monkeypox (MPXV-ZAR) and ectromelia (ECTV-Moscow) but not in VARV. Both MPXV and ECTV share also the S120E substitution. To check which of these substitutions abrogates protection, we generated modified A33VACV genes harboring the L112F, Q117K and L118S substitutions on the backbone of A33VACV . In addition, we also generated a double mutant A33 gene (Q117K-L118S). These modified A33VACV genes were cloned into the Sindbis expression system. Recombinant Sindbis viruses were produced and expression of the modified A33 genes was confirmed by infection of BHK 21 cells followed by Western blot analysis of the cell lysates (Figure 6B). A33 is post-translationally modified in cells (N’ and O’ glycosylations and acylation) resulting in a well-known multiple band pattern in SDS-PAGE [14,16,19]. All Sindbis viruses expressed the modified A33VACV genes, yet certain differences in the multiple band pattern could be observed, mainly in the double mutant A33 (Q117K-L118S). Expression of the E1 and E2 sindbis virus proteins and β-Tubulin confirm the specificity of A33 detection and serve as infection and loading controls.

Bottom Line: We identified a single protective region located between residues 104-120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions.Both epitopes in A33CPXV are mutated and predicted to be non-functional.This epitope's critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious Diseases, Israel Institute for Biological Research, P.O. box 19, Ness-Ziona 74100, Israel. nirp@iibr.gov.il

ABSTRACT
Vaccinia virus protein A33 (A33VACV) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. In this study we show that single-dose vaccination with recombinant Sindbis virus expressing A33VACV, is sufficient to protect mice against lethal challenge with vaccinia virus WR (VACV-WR) and ectromelia virus (ECTV) but not against cowpox virus (CPXV), a closely related orthopoxvirus. Moreover, a subunit vaccine based on the cowpox virus A33 ortholog (A33CPXV) failed to protect against cowpox and only partially protected mice against VACV-WR challenge. We mapped regions of sequence variation between A33VACV and A33CPXVand analyzed the role of such variations in protection. We identified a single protective region located between residues 104-120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions. Both epitopes in A33CPXV are mutated and predicted to be non-functional. Whereas vaccination with A33VACV did not induce in-vivo CTL activity to the predicted epitope, inhibition of virus spread in-vitro, and protection from lethal VACV challenge pointed to the B cell epitope highlighting the critical role of residue L118 and of adjacent compensatory residues in protection. This epitope's critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here. These findings should be considered when developing new subunit vaccines and monoclonal antibody based therapeutics against orthopoxviruses, especially variola virus, the etiologic agent of smallpox.

Show MeSH
Related in: MedlinePlus