Pre-fusion RSV F strongly boosts pre-fusion specific neutralizing responses in cattle pre-exposed to bovine RSV.
ABSTRACT: Human respiratory syncytial virus (hRSV) is responsible for serious lower respiratory tract disease in infants and in older adults, and remains an important vaccine need. RSV fusion (F) glycoprotein is a key target for neutralizing antibodies. RSV F stabilized in its pre-fusion conformation (DS-Cav1 F) induces high neutralizing antibody titers in naïve animals, but it remains unknown to what extent pre-fusion F can boost pre-existing neutralizing responses in RSV seropositive adults. We here assess DS-Cav1 F immunogenicity in seropositive cattle pre-exposed to bovine RSV, a virus closely related to hRSV. A single immunization with non-adjuvanted DS-Cav1 F strongly boosts RSV neutralizing responses, directed towards pre-fusion F-specific epitopes, whereas a post-fusion F is unable to do so. Vaccination with pre-fusion F thus represents a promising strategy for maternal immunization and for other RSV vaccine target populations such as older adults.
Project description:Efforts to develop a vaccine for respiratory syncytial virus (RSV) have primarily focused on the RSV fusion protein. The pre-fusion conformation of this protein induces the most potent neutralizing antibodies and is the focus of recent efforts in vaccine development. Following the first identification of mutations in the RSV F protein (DS-Cav1 mutant protein) that stabilized the pre-fusion conformation, other mutant stabilized pre-fusion F proteins have been described. To determine if there are differences in alternate versions of stabilized pre-fusion F proteins, we explored the use, as vaccine candidates, of virus-like particles (VLPs) containing five different pre-fusion F proteins, including the DS-Cav1 protein. The expression of these five pre-F proteins, their assembly into VLPs, their pre-fusion conformation stability in VLPs, their reactivity with anti-F monoclonal antibodies, and their induction of immune responses after the immunization of mice, were characterized, comparing VLPs containing the DS-Cav1 pre-F protein with VLPs containing four alternative pre-fusion F proteins. The concentrations of anti-F IgG induced by each VLP that blocked the binding of prototype monoclonal antibodies using two different soluble pre-fusion F proteins as targets were measured. Our results indicate that both the conformation and immunogenicity of alternative VLP associated stabilized pre-fusion RSV F proteins are different from those of DS-Cav1 VLPs.
Project description:Pre-fusion stabilizing mutations (DS-Cav1) in soluble fusion (F) proteins of human respiratory syncytial virus (RSV) were previously reported. Here we investigated the antigenic and immunogenic properties of pre-fusion like RSV F proteins on enveloped virus-like particles (VLP). Additional mutations were introduced to DS-Cav1 (F-dcmTM VLP); fusion peptide deletion and cleavage mutation site 1 (F1d-dcmTM VLP) or both sites (F12d-dcmTM VLP). F1d-dcmTM VLP and F12d-dcmTM VLP displayed higher reactivity against pre-fusion specific site Ø and antigenic site I and II specific monoclonal antibodies, compared to F-dcmTM VLP with DS-Cav1 only. Mice immunized with F1d-dcmTM VLP and F12d-dcmTM VLP induced higher levels of DS-Cav1 pre-fusion specific IgG antibodies, RSV neutralizing activity titers, and effective lung viral clearance after challenge. These results suggest that cleavage site mutations and fusion peptide deletion in addition to DS-Cav1 mutations have contributed to structural stabilization of pre-fusion like F conformation on enveloped VLP, capable of inducing high levels of pre-fusion F specific and RSV neutralizing antibodies.
Project description:Recombinant subunit vaccines should contain minimal non-pathogen motifs to reduce potential off-target reactivity. We recently developed a vaccine antigen against respiratory syncytial virus (RSV), which comprised the fusion (F) glycoprotein stabilized in its pre-fusion trimeric conformation by "DS-Cav1" mutations and by an appended C-terminal trimerization motif or "foldon" from T4-bacteriophage fibritin. Here we investigate the creation of a cysteine zipper to allow for the removal of the phage foldon, while maintaining the immunogenicity of the parent DS-Cav1+foldon antigen. Constructs without foldon yielded RSV F monomers, and enzymatic removal of the phage foldon from pre-fusion F trimers resulted in their dissociation into monomers. Because the native C terminus of the pre-fusion RSV F ectodomain encompasses a viral trimeric coiled-coil, we explored whether introduction of cysteine residues capable of forming inter-protomer disulfides might allow for stable trimers. Structural modeling indicated the introduced cysteines to form disulfide "rings", with each ring comprising a different set of inward facing residues of the coiled-coil. Three sets of rings could be placed within the native RSV F coiled-coil, and additional rings could be added by duplicating portions of the coiled-coil. High levels of neutralizing activity in mice, equivalent to that of the parent DS-Cav1+foldon antigen, were elicited by a 4-ring stabilized RSV F trimer with no foldon. Structure-based alteration of a viral coiled-coil to create a cysteine zipper thus allows a phage trimerization motif to be removed from a candidate vaccine antigen.
Project description:Appropriate adjuvant selection may be essential to optimize the potency and to tailor the immune response of subunit vaccines. To induce protective responses against respiratory syncytial virus (RSV)-a highly prevalent childhood pathogen without a licensed vaccine-we previously engineered a pre-fusion-stabilized trimeric RSV F (pre-F) "DS-Cav1" immunogen, which induced high titer RSV-neutralizing antibodies, in mice and non-human primates, when formulated with adjuvants Poly (I:C) and Poly (IC:LC), respectively. To assess the impact of different adjuvants, here we formulated RSV F DS-Cav1 with multiple adjuvants and assessed immune responses. Very high RSV-neutralizing antibody responses (19,006 EC50) were observed in naïve mice immunized with 2 doses of DS-Cav1 adjuvanted with Sigma adjuvant system (SAS), an oil-in-water adjuvant, plus Carbopol; high responses (3658-7108) were observed with DS-Cav1 adjuvanted with Alum, SAS alone, Adjuplex, Poly (I:C) and Poly (IC:LC); and moderate responses (1251-2129) were observed with DS-Cav1 adjuvanted with the TLR4 agonist MPLA, Alum plus MPLA or AddaVax. In contrast, DS-Cav1 without adjuvant induced low-level responses (6). A balanced IgG1 and IgG2a (Th2/Th1) immune response was elicited in most of the high to very high response groups (all but Alum and Adjuplex). We also tested the immune response induced by DS-Cav1 in elderly mice with pre-existing DS-Cav1 immunity; we observed that DS-Cav1 adjuvanted with SAS plus Carbopol boosted the response 2-3-fold, whereas DS-Cav1 adjuvanted with alum boosted the response 5-fold. Finally, we tested whether a mixture of ISA 71 VG and Carbopol would enhanced the antibody response in DS-Cav1 immunized calves. While pre-F-stabilized bovine RSV F induced very high titers in mice when adjuvanted with SAS plus Carbopol, the addition of Carbopol to ISA 71 VG did not enhance immune responses in calves. The vaccine response to pre-F-stabilized RSV F is augmented by adjuvant, but the degree of adjuvant-induced enhancement appears to be both context-dependent and species-specific.
Project description:Respiratory syncytial virus (RSV) is a significant cause of severe respiratory illness worldwide, particularly in infants, young children, and the elderly. Although no licensed vaccine is currently available, an engineered version of the metastable RSV fusion (F) surface glycoprotein-stabilized in the pre-fusion (pre-F) conformation by "DS-Cav1" mutations-elicits high titer RSV-neutralizing responses. Moreover, pre-F-specific antibodies, often against the neutralization-sensitive antigenic site Ø in the membrane-distal head region of trimeric F glycoprotein, comprise a substantial portion of the human response to natural RSV infection. To focus the vaccine-elicited response to antigenic site Ø, we designed a series of RSV F immunogens that comprised the membrane-distal head of the F glycoprotein in its pre-F conformation. These "head-only" immunogens formed monomers, dimers, and trimers. Antigenic analysis revealed that a majority of the 70 engineered head-only immunogens displayed reactivity to site Ø-targeting antibodies, which was similar to that of the parent RSV F DS-Cav1 trimers, often with increased thermostability. We evaluated four of these head-only immunogens in detail, probing their recognition by antibodies, their physical stability, structure, and immunogenicity. When tested in naïve mice, a head-only trimer, half the size of the parent RSV F trimer, induced RSV titers, which were statistically comparable to those induced by DS-Cav1. When used to boost DS-Cav1-primed mice, two head-only RSV F immunogens, a dimer and a trimer, boosted RSV-neutralizing titers to levels that were comparable to those boosted by DS-Cav1, although with higher site Ø-directed responses. Our results provide proof-of-concept for the ability of the smaller head-only RSV F immunogens to focus the vaccine-elicited response to antigenic site Ø. Decent primary immunogenicity, enhanced physical stability, potential ease of manufacture, and potent immunogenicity upon boosting suggest these head-only RSV F immunogens, engineered to retain the pre-fusion conformation, may have advantages as candidate RSV vaccines.
Project description:Respiratory syncytial virus (RSV) is recognized as an important cause of lower and upper respiratory tract infections in older adults, and a successful vaccine would substantially lower morbidity and mortality in this age group. Recently, two vaccine candidates based on soluble purified glycoprotein F (RSV F), either alone or adjuvanted with glucopyranosyl lipid A formulated in a stable emulsion (GLA-SE), failed to reach their primary endpoints in clinical efficacy studies, despite demonstrating the desired immunogenicity profile and efficacy in young rodent models. Here, one of the RSV F vaccine candidates (post-fusion conformation, RSV post-F), and a stabilized pre-fusion form of RSV F (RSV pre-F, DS-Cav1) were evaluated in aged BALB/c mice. Humoral and cellular immunogenicity elicited after immunization of naïve, aged mice was generally lower compared to young animals. In aged mice, RSV post-F vaccination without adjuvant poorly protected the respiratory tract from virus replication, and addition of GLA-SE only improved protection in the lungs, but not in nasal turbinates. RSV pre-F induced higher neutralizing antibody titers compared to RSV post-F (as previously reported) but interestingly, RSV F-specific CD8 T cell responses were lower compared to RSV post-F responses regardless of age. The vaccines were also tested in RSV seropositive aged mice, in which both antigen forms similarly boosted neutralizing antibody titers, although GLA-SE addition boosted neutralizing activity only in RSV pre-F immunized animals. Cell-mediated immune responses in the aged mice were only slightly boosted and well below levels induced in seronegative young mice. Taken together, the findings suggest that the vaccine candidates were not able to induce a strong anti-RSV immune response in recipient mice with an aged immune system, in agreement with recent human clinical trial results. Therefore, the aged mouse model could be a useful tool to evaluate improved vaccine candidates, targeted to prevent RSV disease in older adults.
Project description:The human respiratory syncytial virus (hRSV) fusion (F) protein is considered a major target of the neutralizing antibody response to hRSV. This glycoprotein undergoes a major structural shift from the prefusion (pre-F) to the postfusion (post-F) state at the time of virus-host cell membrane fusion. Recent evidences suggest that the pre-F state is a superior target for neutralizing antibodies compared to the post-F state. Therefore, for vaccine purposes, we have designed and characterized a recombinant hRSV F protein, called Pre-F-GCN4t, stabilized in a pre-F conformation. To show that Pre-F-GCN4t does not switch to a post-F conformation, it was compared with a recombinant post-F molecule, called Post-F-XC. Pre-F-GCN4t was glycosylated and trimeric and displayed a conformational stability different from that of Post-F-XC, as shown by chemical denaturation. Electron microscopy analysis suggested that Pre-F-GCN4t adopts a lollipop-like structure. In contrast, Post-F-XC had a typical elongated conical shape. Hydrogen/deuterium exchange mass spectrometry demonstrated that the two molecules had common rigid folding core and dynamic regions and provided structural insight for their biophysical and biochemical properties and reactivity. Pre-F-GCN4t was shown to deplete hRSV-neutralizing antibodies from human serum more efficiently than Post-F-XC. Importantly, Pre-F-GCN4t was also shown to bind D25, a highly potent monoclonal antibody specific for the pre-F conformation. In conclusion, this construct presents several pre-F characteristics, does not switch to the post-F conformation, and presents antigenic features required for a protective neutralizing antibody response. Therefore, Pre-F-GCN4t can be considered a promising candidate vaccine antigen.IMPORTANCE Human respiratory syncytial virus (RSV) is a global leading cause of infant mortality and adult morbidity. The development of a safe and efficacious RSV vaccine remains an important goal. The RSV class I fusion (F) glycoprotein is considered one of the most promising vaccine candidates, and recent evidences suggest that the prefusion (pre-F) state is a superior target for neutralizing antibodies. Our study presents the physicochemical characterization of Pre-F-GCN4t, a molecule designed to be stabilized in the pre-F conformation. To confirm its pre-F conformation, Pre-F-GCN4t was analyzed in parallel with Post-F-XC, a molecule in the post-F conformation. Our results show that Pre-F-GCN4t presents characteristics of a stabilized pre-F conformation and support its use as an RSV vaccine antigen. Such an antigen may represent a significant advance in the development of an RSV vaccine.
Project description:Background:Respiratory syncytial virus (RSV) subtypes, A and B, co-circulate in annual epidemics and alternate in dominance. We have shown that a subtype A RSV fusion (F) glycoprotein, stabilized in its prefusion conformation by DS-Cav1 mutations, is a promising RSV-vaccine immunogen, capable of boosting RSV-neutralizing titers in healthy adults. In both humans and vaccine-tested animals, neutralizing titers elicited by this subtype A DS-Cav1 immunogen were ~ 2- to 3-fold higher against the homologous subtype A virus than against the heterologous subtype B virus. Methods:To understand the molecular basis for this subtype difference, we introduced DS-Cav1 mutations into RSV strain B18537 F, determined the trimeric crystal structure, and carried out immunogenicity studies. Results:The B18537 DS-Cav1 F structure at 2-Å resolution afforded a precise delineation of prefusion F characteristics, including those of antigenic site Ø, a key trimer-apex site. Structural comparison with the subtype A prefusion F indicated 11% of surface residues to be different, with an alpha-carbon root-mean-square deviation (RMSD) of 1.2 Å; antigenic site Ø, however, differed in 23% of its surface residues and had an alpha-carbon RMSD of 2.2 Å. Immunization of vaccine-tested animals with DS-Cav1-stabilized B18537 F induced neutralizing responses ~100-fold higher than with postfusion B18537 F. Notably, elicited responses neutralized RSV subtypes A and B at similar levels and were directed towards both conserved equatorial and diverse apical regions. Conclusion:We propose that structural differences in apical and equatorial sites-coupled to differently focused immune responses-provide a molecular explanation for observed differences in elicited subtype A and B neutralizing responses.
Project description:Structure-based design of vaccines, particularly the iterative optimization used so successfully in the structure-based design of drugs, has been a long-sought goal. We previously developed a first-generation vaccine antigen called DS-Cav1, comprising a prefusion-stabilized form of the fusion (F) glycoprotein, which elicits high-titer protective responses against respiratory syncytial virus (RSV) in mice and macaques. Here we report the improvement of DS-Cav1 through iterative cycles of structure-based design that significantly increased the titer of RSV-protective responses. The resultant second-generation 'DS2'-stabilized immunogens have their F subunits genetically linked, their fusion peptides deleted and their interprotomer movements stabilized by an additional disulfide bond. These DS2 immunogens are promising vaccine candidates with superior attributes, such as their lack of a requirement for furin cleavage and their increased antigenic stability against heat inactivation. The iterative structure-based improvement described here may have utility in the optimization of other vaccine antigens.
Project description:Human respiratory syncytial virus (RSV) is the most prevalent worldwide cause of severe respiratory tract infection in infants and young children. Human parainfluenza virus type 1 (HPIV1) also causes severe pediatric respiratory illness, especially croup. Both viruses lack vaccines. Here, we describe the preclinical development of a bivalent RSV/HPIV1 vaccine based on a recombinant HPIV1 vector, attenuated by a stabilized mutation, that expresses RSV F protein modified for increased stability in the prefusion (pre-F) conformation by previously described disulfide bond (DS) and hydrophobic cavity-filling (Cav1) mutations. RSV F was expressed from the first or second gene position as the full-length protein or as a chimeric protein with its transmembrane and cytoplasmic tail (TMCT) domains substituted with those of HPIV1 F in an effort to direct packaging in the vector particles. All constructs were recovered by reverse genetics. The TMCT versions of RSV F were packaged in the rHPIV1 particles much more efficiently than their full-length counterparts. In hamsters, the presence of the RSV F gene, and in particular the TMCT versions, was attenuating and resulted in reduced immunogenicity. However, the vector expressing full-length RSV F from the pre-N position was immunogenic for RSV and HPIV1. It conferred complement-independent high-quality RSV-neutralizing antibodies at titers similar to those of wild-type RSV and provided protection against RSV challenge. The vectors exhibited stable RSV F expression in vitro and in vivo In conclusion, an attenuated rHPIV1 vector expressing a pre-F-stabilized form of RSV F demonstrated promising immunogenicity and should be further developed as an intranasal pediatric vaccine.IMPORTANCE RSV and HPIV1 are major viral causes of acute pediatric respiratory illness for which no vaccines or suitable antiviral drugs are available. The RSV F glycoprotein is the major RSV neutralization antigen. We used a rHPIV1 vector, bearing a stabilized attenuating mutation, to express the RSV F glycoprotein bearing amino acid substitutions that increase its stability in the pre-F form, the most immunogenic form that elicits highly functional virus-neutralizing antibodies. RSV F was expressed from the pre-N or N-P gene position of the rHPIV1 vector as a full-length protein or as a chimeric form with its TMCT domain derived from HPIV1 F. TMCT modification greatly increased packaging of RSV F into the vector particles but also increased vector attenuation in vivo, resulting in reduced immunogenicity. In contrast, full-length RSV F expressed from the pre-N position was immunogenic, eliciting complement-independent RSV-neutralizing antibodies and providing protection against RSV challenge.