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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

Reactivity of full length RSV F proteins with pre- and postfusion-specific antibodies.(A) Cells transfected with full length F protein expression plasmids or (B) infected with RSV (strain Long) were fixed and processed for immunofluorescence analysis as described in the Materials and Methods using MAbs AM22 and 131-2a. Nuclei were stained with DAPI. Wild type F protein (Fwt) or F proteins containing cysteine pairs in HRB (Fcys; [17]) were expressed. (C) Sandwich ELISA of RSV virus particles. RSV particles (+RSV) were captured using 131-2a or AM22. Capture of virus particles was detected using a PAb against RSV. As controls the experiment was performed without adding RSV particles (-RSV) or after heating of the particles (+heated RSV).
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pone.0130829.g003: Reactivity of full length RSV F proteins with pre- and postfusion-specific antibodies.(A) Cells transfected with full length F protein expression plasmids or (B) infected with RSV (strain Long) were fixed and processed for immunofluorescence analysis as described in the Materials and Methods using MAbs AM22 and 131-2a. Nuclei were stained with DAPI. Wild type F protein (Fwt) or F proteins containing cysteine pairs in HRB (Fcys; [17]) were expressed. (C) Sandwich ELISA of RSV virus particles. RSV particles (+RSV) were captured using 131-2a or AM22. Capture of virus particles was detected using a PAb against RSV. As controls the experiment was performed without adding RSV particles (-RSV) or after heating of the particles (+heated RSV).

Mentions: The poor neutralizing capacity of MAb 131-2a is in agreement with the assumption that this antibody predominantly recognizes the postfusion form of F [9]. Although recombinant F proteins that were efficiently recognized by prefusion-specific antibodies AM22 and D25 were in general not efficiently bound by 131-2a, some of these have been reported to display high reactivity with prefusion-specific antibodies as well as with 131-2a [18]. To establish that MAb 131-2a is indeed a postfusion-specific antibody that is not able to bind membrane-associated prefusion F we analyzed the ability of this antibody to recognize full-length RSV F stabilized in the prefusion conformation. To this end, cells were transfected with plasmids expressing the full length, wild-type F protein (Fwt) or a mutant version thereof, which contains cysteine residues in HRB (Fcys). These cysteine residues were previously reported to stabilize the prefusion-structure of RSV F by the formation of intersubunit disulfide bridges [17]. Cells expressing wild-type F were recognized by AM22 and 131-2a (Fig 3A and S1 Fig) as well as by D25 and Palivizumab (not shown). In contrast, cells expressing prefusion Fcys were bound by AM22 (and D25), but not by 131-2a. These results indicate that epitope I recognized by 131-2a is not exposed on prefusion F, while cell-associated wild-type F proteins may be recognized both by prefusion- and postfusion-specific antibodies.


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)

Reactivity of full length RSV F proteins with pre- and postfusion-specific antibodies.(A) Cells transfected with full length F protein expression plasmids or (B) infected with RSV (strain Long) were fixed and processed for immunofluorescence analysis as described in the Materials and Methods using MAbs AM22 and 131-2a. Nuclei were stained with DAPI. Wild type F protein (Fwt) or F proteins containing cysteine pairs in HRB (Fcys; [17]) were expressed. (C) Sandwich ELISA of RSV virus particles. RSV particles (+RSV) were captured using 131-2a or AM22. Capture of virus particles was detected using a PAb against RSV. As controls the experiment was performed without adding RSV particles (-RSV) or after heating of the particles (+heated RSV).
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Related In: Results  -  Collection

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pone.0130829.g003: Reactivity of full length RSV F proteins with pre- and postfusion-specific antibodies.(A) Cells transfected with full length F protein expression plasmids or (B) infected with RSV (strain Long) were fixed and processed for immunofluorescence analysis as described in the Materials and Methods using MAbs AM22 and 131-2a. Nuclei were stained with DAPI. Wild type F protein (Fwt) or F proteins containing cysteine pairs in HRB (Fcys; [17]) were expressed. (C) Sandwich ELISA of RSV virus particles. RSV particles (+RSV) were captured using 131-2a or AM22. Capture of virus particles was detected using a PAb against RSV. As controls the experiment was performed without adding RSV particles (-RSV) or after heating of the particles (+heated RSV).
Mentions: The poor neutralizing capacity of MAb 131-2a is in agreement with the assumption that this antibody predominantly recognizes the postfusion form of F [9]. Although recombinant F proteins that were efficiently recognized by prefusion-specific antibodies AM22 and D25 were in general not efficiently bound by 131-2a, some of these have been reported to display high reactivity with prefusion-specific antibodies as well as with 131-2a [18]. To establish that MAb 131-2a is indeed a postfusion-specific antibody that is not able to bind membrane-associated prefusion F we analyzed the ability of this antibody to recognize full-length RSV F stabilized in the prefusion conformation. To this end, cells were transfected with plasmids expressing the full length, wild-type F protein (Fwt) or a mutant version thereof, which contains cysteine residues in HRB (Fcys). These cysteine residues were previously reported to stabilize the prefusion-structure of RSV F by the formation of intersubunit disulfide bridges [17]. Cells expressing wild-type F were recognized by AM22 and 131-2a (Fig 3A and S1 Fig) as well as by D25 and Palivizumab (not shown). In contrast, cells expressing prefusion Fcys were bound by AM22 (and D25), but not by 131-2a. These results indicate that epitope I recognized by 131-2a is not exposed on prefusion F, while cell-associated wild-type F proteins may be recognized both by prefusion- and postfusion-specific antibodies.

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