Limits...
Protein Crystallography in Vaccine Research and Development.

Malito E, Carfi A, Bottomley MJ - Int J Mol Sci (2015)

Bottom Line: The use of protein X-ray crystallography for structure-based design of small-molecule drugs is well-documented and includes several notable success stories.However, it is less well-known that structural biology has emerged as a major tool for the design of novel vaccine antigens.We discuss several examples of the crystallographic characterization of vaccine antigen structures, alone or in complexes with ligands or receptors.

View Article: PubMed Central - PubMed

Affiliation: Protein Biochemistry Department, Novartis Vaccines & Diagnostics s.r.l. (a GSK Company), Via Fiorentina 1, 53100 Siena, Italy. enrico.x.malito@gsk.com.

ABSTRACT
The use of protein X-ray crystallography for structure-based design of small-molecule drugs is well-documented and includes several notable success stories. However, it is less well-known that structural biology has emerged as a major tool for the design of novel vaccine antigens. Here, we review the important contributions that protein crystallography has made so far to vaccine research and development. We discuss several examples of the crystallographic characterization of vaccine antigen structures, alone or in complexes with ligands or receptors. We cover the critical role of high-resolution epitope mapping by reviewing structures of complexes between antigens and their cognate neutralizing, or protective, antibody fragments. Most importantly, we provide recent examples where structural insights obtained via protein crystallography have been used to design novel optimized vaccine antigens. This review aims to illustrate the value of protein crystallography in the emerging discipline of structural vaccinology and its impact on the rational design of vaccines.

No MeSH data available.


Related in: MedlinePlus

(A) Stabilized respiratory syncytial virus (RSV) F pre-fusion (pdb 4MMV) is shown as light and dark surfaces for two chains, and as yellow cartoon for the third chain of the trimer. Sites that were mutated to stabilize the pre-fusion configuration are colored in blue, green, and pink, for the S190F-V207L pair (Cav1), the S155C-S290C double mutant (DS), and the D486H-E487Q-F488W-D489H mutant (TriC), respectively [86]. Known epitope surfaces for Fabs D25, and for palivizumab and motavizumab, are colored in cyan and green, respectively. A zoomed view of the region of DS and Cav1 mutants (central box) provides details of the cavity-filling mutation S190F and of the introduction of the disulfide bridge C155-C290; (B) Post-fusion RSV F (pdb 3RKI) [79] is shown as surface, color-coded as in panel A.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4490488&req=5

ijms-16-13106-f003: (A) Stabilized respiratory syncytial virus (RSV) F pre-fusion (pdb 4MMV) is shown as light and dark surfaces for two chains, and as yellow cartoon for the third chain of the trimer. Sites that were mutated to stabilize the pre-fusion configuration are colored in blue, green, and pink, for the S190F-V207L pair (Cav1), the S155C-S290C double mutant (DS), and the D486H-E487Q-F488W-D489H mutant (TriC), respectively [86]. Known epitope surfaces for Fabs D25, and for palivizumab and motavizumab, are colored in cyan and green, respectively. A zoomed view of the region of DS and Cav1 mutants (central box) provides details of the cavity-filling mutation S190F and of the introduction of the disulfide bridge C155-C290; (B) Post-fusion RSV F (pdb 3RKI) [79] is shown as surface, color-coded as in panel A.

Mentions: Significant efforts have also been made to harness the vaccine potential of the more elusive “metastable” pre-fusion F antigen. Conceptually, the pre-fusion F conformation would be a better vaccine target as it exposes all the functional sites and neutralizing epitopes present on virion F. Although engineered post-fusion F can elicit high titers of neutralizing antibodies in animal models [79], a subsequent report demonstrated that antibodies specific for the pre-fusion F form account for most of the neutralizing activity of human sera from seropositive subjects [84]. It thus appeared that some critical neutralizing mAb binding sites were absent in the post-fusion F form and consequently attempts to design a stable pre-fusion F antigen intensified. Important “turning points” that ultimately enabled informed antigen design were the discoveries of a few new anti-F neutralizing mAbs (mouse 5C4, human D25 and AM22) with the unique property of not recognizing a stabilized post-fusion F form. These mAbs were used for structural studies to trap the F molecule in its pre-fusion state. Crucially, after co-expression and co-purification, the crystal structure of Fab D25 bound to RSV F in the pre-fusion conformation was determined [85]. Although the structure revealed that the palivizumab and motavizumab epitopes were well exposed in pre-fusion F, there was a dramatic overall change in conformation (Figure 3). Analysis of the epitope–paratope interface in this complex explained why D25 does not bind to post-fusion F and thus the crystal structure provided mechanistic insights, suggesting that D25 neutralizes RSV by restraining F in the pre-fusion state. The epitope recognized by D25, site Ø, which is also the target of 5C4 and AM22, is on the most exposed apex of F, which may underlie the higher effectiveness of neutralizing antibodies against this region, despite having a binding affinity similar to that of motavizumab. These structural studies led to the proposal that F antigens stabilized in the pre-fusion conformation may further improve the immunogenicity of this molecule. Indeed, stabilization of the trimer by addition of a trimerization tag (a foldon) replacing the transmembrane region, structure-based insertion of hydrophobic packing mutations and judicious insertion of a novel disulfide bond, forms the basis of a leading pre-fusion F candidate antigen (Figure 3). Of note, similarly to the iterative approach of structure-based design used for the development of high-affinity drugs, the authors developed a method to screen hundreds of structure-guided mutations to identify those resulting in protein stabilization and favorable expression levels. Most importantly, in mouse and nonhuman primate animal models, a stabilized pre-fusion F molecule elicited RSV-specific neutralizing titers significantly greater than those elicited by a post-fusion F protein and well above the protective threshold [86].


Protein Crystallography in Vaccine Research and Development.

Malito E, Carfi A, Bottomley MJ - Int J Mol Sci (2015)

(A) Stabilized respiratory syncytial virus (RSV) F pre-fusion (pdb 4MMV) is shown as light and dark surfaces for two chains, and as yellow cartoon for the third chain of the trimer. Sites that were mutated to stabilize the pre-fusion configuration are colored in blue, green, and pink, for the S190F-V207L pair (Cav1), the S155C-S290C double mutant (DS), and the D486H-E487Q-F488W-D489H mutant (TriC), respectively [86]. Known epitope surfaces for Fabs D25, and for palivizumab and motavizumab, are colored in cyan and green, respectively. A zoomed view of the region of DS and Cav1 mutants (central box) provides details of the cavity-filling mutation S190F and of the introduction of the disulfide bridge C155-C290; (B) Post-fusion RSV F (pdb 3RKI) [79] is shown as surface, color-coded as in panel A.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490488&req=5

ijms-16-13106-f003: (A) Stabilized respiratory syncytial virus (RSV) F pre-fusion (pdb 4MMV) is shown as light and dark surfaces for two chains, and as yellow cartoon for the third chain of the trimer. Sites that were mutated to stabilize the pre-fusion configuration are colored in blue, green, and pink, for the S190F-V207L pair (Cav1), the S155C-S290C double mutant (DS), and the D486H-E487Q-F488W-D489H mutant (TriC), respectively [86]. Known epitope surfaces for Fabs D25, and for palivizumab and motavizumab, are colored in cyan and green, respectively. A zoomed view of the region of DS and Cav1 mutants (central box) provides details of the cavity-filling mutation S190F and of the introduction of the disulfide bridge C155-C290; (B) Post-fusion RSV F (pdb 3RKI) [79] is shown as surface, color-coded as in panel A.
Mentions: Significant efforts have also been made to harness the vaccine potential of the more elusive “metastable” pre-fusion F antigen. Conceptually, the pre-fusion F conformation would be a better vaccine target as it exposes all the functional sites and neutralizing epitopes present on virion F. Although engineered post-fusion F can elicit high titers of neutralizing antibodies in animal models [79], a subsequent report demonstrated that antibodies specific for the pre-fusion F form account for most of the neutralizing activity of human sera from seropositive subjects [84]. It thus appeared that some critical neutralizing mAb binding sites were absent in the post-fusion F form and consequently attempts to design a stable pre-fusion F antigen intensified. Important “turning points” that ultimately enabled informed antigen design were the discoveries of a few new anti-F neutralizing mAbs (mouse 5C4, human D25 and AM22) with the unique property of not recognizing a stabilized post-fusion F form. These mAbs were used for structural studies to trap the F molecule in its pre-fusion state. Crucially, after co-expression and co-purification, the crystal structure of Fab D25 bound to RSV F in the pre-fusion conformation was determined [85]. Although the structure revealed that the palivizumab and motavizumab epitopes were well exposed in pre-fusion F, there was a dramatic overall change in conformation (Figure 3). Analysis of the epitope–paratope interface in this complex explained why D25 does not bind to post-fusion F and thus the crystal structure provided mechanistic insights, suggesting that D25 neutralizes RSV by restraining F in the pre-fusion state. The epitope recognized by D25, site Ø, which is also the target of 5C4 and AM22, is on the most exposed apex of F, which may underlie the higher effectiveness of neutralizing antibodies against this region, despite having a binding affinity similar to that of motavizumab. These structural studies led to the proposal that F antigens stabilized in the pre-fusion conformation may further improve the immunogenicity of this molecule. Indeed, stabilization of the trimer by addition of a trimerization tag (a foldon) replacing the transmembrane region, structure-based insertion of hydrophobic packing mutations and judicious insertion of a novel disulfide bond, forms the basis of a leading pre-fusion F candidate antigen (Figure 3). Of note, similarly to the iterative approach of structure-based design used for the development of high-affinity drugs, the authors developed a method to screen hundreds of structure-guided mutations to identify those resulting in protein stabilization and favorable expression levels. Most importantly, in mouse and nonhuman primate animal models, a stabilized pre-fusion F molecule elicited RSV-specific neutralizing titers significantly greater than those elicited by a post-fusion F protein and well above the protective threshold [86].

Bottom Line: The use of protein X-ray crystallography for structure-based design of small-molecule drugs is well-documented and includes several notable success stories.However, it is less well-known that structural biology has emerged as a major tool for the design of novel vaccine antigens.We discuss several examples of the crystallographic characterization of vaccine antigen structures, alone or in complexes with ligands or receptors.

View Article: PubMed Central - PubMed

Affiliation: Protein Biochemistry Department, Novartis Vaccines & Diagnostics s.r.l. (a GSK Company), Via Fiorentina 1, 53100 Siena, Italy. enrico.x.malito@gsk.com.

ABSTRACT
The use of protein X-ray crystallography for structure-based design of small-molecule drugs is well-documented and includes several notable success stories. However, it is less well-known that structural biology has emerged as a major tool for the design of novel vaccine antigens. Here, we review the important contributions that protein crystallography has made so far to vaccine research and development. We discuss several examples of the crystallographic characterization of vaccine antigen structures, alone or in complexes with ligands or receptors. We cover the critical role of high-resolution epitope mapping by reviewing structures of complexes between antigens and their cognate neutralizing, or protective, antibody fragments. Most importantly, we provide recent examples where structural insights obtained via protein crystallography have been used to design novel optimized vaccine antigens. This review aims to illustrate the value of protein crystallography in the emerging discipline of structural vaccinology and its impact on the rational design of vaccines.

No MeSH data available.


Related in: MedlinePlus