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

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.

Characterization of Epitope-Specific Anti-Respiratory Syncytial Virus (Anti-RSV) Antibody Responses after Natural Infection and after Vaccination with Formalin-Inactivated RSV

“…Flys-GCN proteins (16,21) and the heavy and light chains of palivizumab (22) and D25 and AM22 (23) antibody have been described previously. The expression constructs encoding the 101F antibody heavy and light chains were constructed on the basis of the published sequence (46) as described before (16,21).…”

“…Flys-GCN proteins (16,21) and the heavy and light chains of palivizumab (22) and D25 and AM22 (23) antibody have been described previously. The expression constructs encoding the 101F antibody heavy and light chains were constructed on the basis of the published sequence (46) as described before (16,21). The cDNA clones encoding a subtype A RSV G ectodomain (residues 64 to 298; GenBank accession number P03423.1) or the prefusion F protein DSCav1-T4fd (17) were synthesized by GenScript USA, Inc., using human-preferred codons and cloned in the appropriate expression vectors.…”

“…1A and B). Monoclonal anti-bodies (MAbs) against the different antigenic sites differ in their neutralizing capacities, with pre-and postfusion-specific antibodies displaying the highest and lowest neutralizing capacities, respectively (16). In agreement, vaccination with F proteins stabilized in a prefusion-like conformation, which presumably results in the induction of highly neutralizing prefusion-specific antibodies, appeared to be more effective than vaccination with postfusion F proteins (17,18).…”

“…The structures of these two F protein conformations have been solved (13)(14)(15). While some epitopes are found on both structures (antigenic sites II and IV), others appear to be specific for the prefusion form (antigenic site Ø) or the postfusion form (antigenic site I) of F (14,16) (Fig. 1A and B).…”

Characterization of Pre-F-GCN4t, a Modified Human Respiratory Syncytial Virus Fusion Protein Stabilized in a Noncleaved Prefusion Conformation

“…The generation of a soluble prefusion F protein has long been a challenging task due to the transient nature of the prefusion state and its spontaneous rearrangement into the postfusion conformation when produced as a soluble recombinant protein. Nevertheless, several stabilized pre-F proteins were described recently (40)(41)(42)(43)(44)(45). We have also designed and produced a molecule with pre-F characteristics, called Pre-F-GCN4t, that has now reached clinical development.…”