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Recombinant Soluble Respiratory Syncytial Virus F Protein That Lacks Heptad Repeat B, Contains a GCN4 Trimerization Motif and Is Not Cleaved Displays Prefusion-Like Characteristics.

Widjaja I, Rigter A, Jacobino S, van Kuppeveld FJ, Leenhouts K, Palomo C, Melero JA, Leusen JH, Haijema BJ, Rottier PJ, de Haan CA - PLoS ONE (2015)

Bottom Line: In contrast, a similar F protein completely lacking HRB displayed high reactivity with prefusion-specific antibodies recognizing antigenic site Ø, but did not expose postfusion-specific antigenic site I, in agreement with this protein maintaining a prefusion-like conformation.In conclusion, proteins that are not able to form the 6HB, due to mutation of HRB, may still display postfusion-specific antigenic site I.Replacement of HRB by the GCN4 trimerization domain in a non-cleaved soluble F protein resulted, however, in a protein with prefusion-like characteristics, suggesting that this HRB-lacking protein may represent a potential prefusion F protein subunit vaccine candidate.

View Article: PubMed Central - PubMed

Affiliation: Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, 3894 CL, Utrecht, The Netherlands; Mucosis B.V., Meditech Center, L.J. Zielstraweg 1, 9713 GX Groningen, The Netherlands.

ABSTRACT
The respiratory syncytial virus (RSV) fusion protein F is considered an attractive vaccine candidate especially in its prefusion conformation. We studied whether recombinant soluble RSV F proteins could be stabilized in a prefusion-like conformation by mutation of heptad repeat B (HRB). The results show that soluble, trimeric, non-cleaved RSV F protein, produced by expression of the furin cleavage site-mutated F ectodomain extended with a GCN4 trimerization sequence, is efficiently recognized by pre- as well as postfusion-specific antibodies. In contrast, a similar F protein completely lacking HRB displayed high reactivity with prefusion-specific antibodies recognizing antigenic site Ø, but did not expose postfusion-specific antigenic site I, in agreement with this protein maintaining a prefusion-like conformation. These features were dependent on the presence of the GCN4 trimerization domain. Absence of cleavage also contributed to binding of prefusion-specific antibodies. Similar antibody reactivity profiles were observed when the prefusion form of F was stabilized by the introduction of cysteine pairs in HRB. To study whether the inability to form the 6HB was responsible for the prefusion-like antibody reactivity profile, alanine mutations were introduced in HRB. Although introduction of alanine residues in HRB inhibited the formation of the 6HB, the exposure of postfusion-specific antigenic site I was not prevented. In conclusion, proteins that are not able to form the 6HB, due to mutation of HRB, may still display postfusion-specific antigenic site I. Replacement of HRB by the GCN4 trimerization domain in a non-cleaved soluble F protein resulted, however, in a protein with prefusion-like characteristics, suggesting that this HRB-lacking protein may represent a potential prefusion F protein subunit vaccine candidate.

No MeSH data available.


Related in: MedlinePlus

Antigenic analysis of F proteins with mutated HRB.ELISA analysis of purified F proteins Flys.ΔHRB-GCN, Flys.HRBcys-GCN and Flys.HRBala-GCN was performed similarly as described in the legend to Fig 5. Error bars indicate standard deviations (n = 3-5).
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pone.0130829.g007: Antigenic analysis of F proteins with mutated HRB.ELISA analysis of purified F proteins Flys.ΔHRB-GCN, Flys.HRBcys-GCN and Flys.HRBala-GCN was performed similarly as described in the legend to Fig 5. Error bars indicate standard deviations (n = 3-5).

Mentions: Next, the reactivity of the HRB mutant proteins was analyzed with the panel of conformation-specific antibodies (Fig 7 and S6 Fig). Flys.HRBala-GCN and Flys.HRBcys-GCN displayed similar reactivities with Palivizumab, AM22 and D25, which were comparable to those of Flys.ΔHRB-GCN. None of the HRB mutant proteins were able to form the 6HB as demonstrated by their negligible reactivity with α6HB. However, the different F proteins dramatically differed in their reactivity with the postfusion-specific antibody 131-2a (antigenic site I). In contrast to Flys.HRBcys-GCN and Flys.ΔHRB-GCN, Flys.HRBala-GCN was efficiently recognized by 131-2a. From these results, we conclude that mutation of HRB diminishes the formation of the 6HB, but does not necessarily affect the presentation of antigenic site I, as is exemplified by Flys.HRBala-GCN. Of note, Flys.HRBcys-GCN and Flys.ΔHRB-GCN exhibited similar reactivities for all antibodies tested, indicating that these two proteins adopt similar prefusion-like conformations. While the one protein is stabilized by deletion of HRB (Flys.ΔHRB-GCN), the other protein is stabilized by the formation of disulfide bonds in HRB (Flys.HRBcys-GCN).


Recombinant Soluble Respiratory Syncytial Virus F Protein That Lacks Heptad Repeat B, Contains a GCN4 Trimerization Motif and Is Not Cleaved Displays Prefusion-Like Characteristics.

Widjaja I, Rigter A, Jacobino S, van Kuppeveld FJ, Leenhouts K, Palomo C, Melero JA, Leusen JH, Haijema BJ, Rottier PJ, de Haan CA - PLoS ONE (2015)

Antigenic analysis of F proteins with mutated HRB.ELISA analysis of purified F proteins Flys.ΔHRB-GCN, Flys.HRBcys-GCN and Flys.HRBala-GCN was performed similarly as described in the legend to Fig 5. Error bars indicate standard deviations (n = 3-5).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4481108&req=5

pone.0130829.g007: Antigenic analysis of F proteins with mutated HRB.ELISA analysis of purified F proteins Flys.ΔHRB-GCN, Flys.HRBcys-GCN and Flys.HRBala-GCN was performed similarly as described in the legend to Fig 5. Error bars indicate standard deviations (n = 3-5).
Mentions: Next, the reactivity of the HRB mutant proteins was analyzed with the panel of conformation-specific antibodies (Fig 7 and S6 Fig). Flys.HRBala-GCN and Flys.HRBcys-GCN displayed similar reactivities with Palivizumab, AM22 and D25, which were comparable to those of Flys.ΔHRB-GCN. None of the HRB mutant proteins were able to form the 6HB as demonstrated by their negligible reactivity with α6HB. However, the different F proteins dramatically differed in their reactivity with the postfusion-specific antibody 131-2a (antigenic site I). In contrast to Flys.HRBcys-GCN and Flys.ΔHRB-GCN, Flys.HRBala-GCN was efficiently recognized by 131-2a. From these results, we conclude that mutation of HRB diminishes the formation of the 6HB, but does not necessarily affect the presentation of antigenic site I, as is exemplified by Flys.HRBala-GCN. Of note, Flys.HRBcys-GCN and Flys.ΔHRB-GCN exhibited similar reactivities for all antibodies tested, indicating that these two proteins adopt similar prefusion-like conformations. While the one protein is stabilized by deletion of HRB (Flys.ΔHRB-GCN), the other protein is stabilized by the formation of disulfide bonds in HRB (Flys.HRBcys-GCN).

Bottom Line: In contrast, a similar F protein completely lacking HRB displayed high reactivity with prefusion-specific antibodies recognizing antigenic site Ø, but did not expose postfusion-specific antigenic site I, in agreement with this protein maintaining a prefusion-like conformation.In conclusion, proteins that are not able to form the 6HB, due to mutation of HRB, may still display postfusion-specific antigenic site I.Replacement of HRB by the GCN4 trimerization domain in a non-cleaved soluble F protein resulted, however, in a protein with prefusion-like characteristics, suggesting that this HRB-lacking protein may represent a potential prefusion F protein subunit vaccine candidate.

View Article: PubMed Central - PubMed

Affiliation: Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, 3894 CL, Utrecht, The Netherlands; Mucosis B.V., Meditech Center, L.J. Zielstraweg 1, 9713 GX Groningen, The Netherlands.

ABSTRACT
The respiratory syncytial virus (RSV) fusion protein F is considered an attractive vaccine candidate especially in its prefusion conformation. We studied whether recombinant soluble RSV F proteins could be stabilized in a prefusion-like conformation by mutation of heptad repeat B (HRB). The results show that soluble, trimeric, non-cleaved RSV F protein, produced by expression of the furin cleavage site-mutated F ectodomain extended with a GCN4 trimerization sequence, is efficiently recognized by pre- as well as postfusion-specific antibodies. In contrast, a similar F protein completely lacking HRB displayed high reactivity with prefusion-specific antibodies recognizing antigenic site Ø, but did not expose postfusion-specific antigenic site I, in agreement with this protein maintaining a prefusion-like conformation. These features were dependent on the presence of the GCN4 trimerization domain. Absence of cleavage also contributed to binding of prefusion-specific antibodies. Similar antibody reactivity profiles were observed when the prefusion form of F was stabilized by the introduction of cysteine pairs in HRB. To study whether the inability to form the 6HB was responsible for the prefusion-like antibody reactivity profile, alanine mutations were introduced in HRB. Although introduction of alanine residues in HRB inhibited the formation of the 6HB, the exposure of postfusion-specific antigenic site I was not prevented. In conclusion, proteins that are not able to form the 6HB, due to mutation of HRB, may still display postfusion-specific antigenic site I. Replacement of HRB by the GCN4 trimerization domain in a non-cleaved soluble F protein resulted, however, in a protein with prefusion-like characteristics, suggesting that this HRB-lacking protein may represent a potential prefusion F protein subunit vaccine candidate.

No MeSH data available.


Related in: MedlinePlus