Chimeric Pneumoviridae fusion proteins as immunogens to induce cross‐neutralizing antibody responses
2017; Springer Nature; Volume: 10; Issue: 2 Linguagem: Inglês
10.15252/emmm.201708078
ISSN1757-4684
AutoresEduardo Olmedillas, Olga Cano, Isidoro Martínez, Daniel Luque, María C. Terrón, Jason S. McLellan, José A. Melero, Vicente Más,
Tópico(s)Pneumonia and Respiratory Infections
ResumoResearch Article7 December 2017Open Access Transparent process Chimeric Pneumoviridae fusion proteins as immunogens to induce cross-neutralizing antibody responses Eduardo Olmedillas Eduardo Olmedillas Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Olga Cano Olga Cano Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Isidoro Martínez Isidoro Martínez Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Daniel Luque Daniel Luque Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author María C Terrón María C Terrón Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Jason S McLellan Jason S McLellan Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA Search for more papers by this author José A Melero Corresponding Author José A Melero [email protected] orcid.org/0000-0001-8148-6521 Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Vicente Más Vicente Más Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Eduardo Olmedillas Eduardo Olmedillas Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Olga Cano Olga Cano Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Isidoro Martínez Isidoro Martínez Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Daniel Luque Daniel Luque Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author María C Terrón María C Terrón Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Jason S McLellan Jason S McLellan Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA Search for more papers by this author José A Melero Corresponding Author José A Melero [email protected] orcid.org/0000-0001-8148-6521 Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Vicente Más Vicente Más Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Author Information Eduardo Olmedillas1,2, Olga Cano1,2, Isidoro Martínez1, Daniel Luque1, María C Terrón1, Jason S McLellan3, José A Melero *,1,2 and Vicente Más1,2 1Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain 2CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain 3Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA *Corresponding author. Tel: +34 9182 23908; E-mail: [email protected] EMBO Mol Med (2018)10:175-187https://doi.org/10.15252/emmm.201708078 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV), two members of the Pneumoviridae family, account for the majority of severe lower respiratory tract infections worldwide in very young children. They are also a frequent cause of morbidity and mortality in the elderly and immunocompromised adults. High levels of neutralizing antibodies, mostly directed against the viral fusion (F) glycoprotein, correlate with protection against either hRSV or hMPV. However, no cross-neutralization is observed in polyclonal antibody responses raised after virus infection or immunization with purified F proteins. Based on crystal structures of hRSV F and hMPV F, we designed chimeric F proteins in which certain residues of well-characterized antigenic sites were swapped between the two antigens. The antigenic changes were monitored by ELISA with virus-specific monoclonal antibodies. Inoculation of mice with these chimeras induced polyclonal cross-neutralizing antibody responses, and mice were protected against challenge with the virus used for grafting of the heterologous antigenic site. These results provide a proof of principle for chimeric fusion proteins as single immunogens that can induce cross-neutralizing antibody and protective responses against more than one human pneumovirus. Synopsis Chimeric fusion (F) proteins bearing neutralizing epitopes of hRSV and hMPV offer the possibility of using a single immunogen to induce cross-protective antibody responses against both viruses. Postfusion-stabilized hMPV F bearing antigenic site II of hRSV induced antibodies that neutralized hRSV and hMPV and protected against an hRSV challenge. Prefusion-stabilized hRSV F bearing antigenic site IV of hMPV also induced antibodies that cross-neutralized hRSV and hMPV. Introduction Human respiratory syncytial virus (hRSV) is the leading cause worldwide of severe acute lower respiratory tract infections (ALRI, mainly bronchiolitis and pneumonia) in infants and very young children. The estimated new cases of ALRI each year owing to hRSV infections are more than 33 million in children younger than 5 years, with 10% of these resulting in hospital admissions. hRSV infections also cause 60,000–120,000 deaths each year in young children, with 90% occurring in developing countries (Nair et al, 2010; Shi et al, 2017). Human metapneumovirus (hMPV) is also a virus of clinical significance (van den Hoogen et al, 2001), second only to hRSV as a cause of ALRI in young children (Edwards et al, 2013; Schuster & Williams, 2013). Both hRSV and hMPV also have a clinical impact in the elderly and in adults with cardiopulmonary disease or impaired immune systems (Hall et al, 1986; Dowell et al, 1996; Falsey et al, 2005; Panda et al, 2014). hRSV and hMPV have been recently reclassified in the orthopneumovirus and metapneumovirus genera, respectively, of the newly created Pneumoviridae family, which was detached from the original Paramyxoviridae family (Afonso et al, 2016). Hence, hRSV and hMPV share clinical and biological features, although they also have important differences, for example, in the number of genes or in the proteolytic processing of their respective fusion (F) glycoproteins (van den Hoogen et al, 2002). Both hRSV and hMPV genomes encode three glycoproteins (SH, G, and F) that are inserted in the viral membrane. SH is a small hydrophobic protein whose function is still unclear and is incorporated in low amounts into the virus particle (Bao et al, 2008; Gan et al, 2012). G is a heavily glycosylated and highly variable type II glycoprotein that resembles mucins and serves as the viral attachment protein (Levine et al, 1987; Thammawat et al, 2008). Finally, F is a type I glycoprotein that fuses the viral and cell membranes at the initial stages of an infectious cycle and is the main target of neutralizing antibodies (for a recent review Melero & Mas, 2015). The F protein is synthesized as a F0 precursor that requires proteolytic processing to become functional. While hMPV F is cleaved only once by trypsinlike proteases outside the cell (Shirogane et al, 2008), hRSV F is cleaved twice inside the cell at two polybasic sites recognized by furinlike proteases (Gonzalez-Reyes et al, 2001; Zimmer et al, 2001; Begona Ruiz-Arguello et al, 2002). Both hRSV F and hMPV F are trimers of disulfide-linked heterodimers, and these F proteins initially fold into a metastable prefusion conformation. During membrane fusion, the F glycoprotein refolds through a series of unstable intermediates into a highly stable postfusion conformation that shares some neutralizing epitopes with the prefusion conformation (Lamb et al, 2006; McLellan et al, 2013b). There are no available vaccines for either hRSV or hMPV despite being greatly needed. A wealth of data support the conclusion that neutralizing antibodies are the major players in protection against hRSV and hMPV infections (for a recent review Melero & Mas, 2015). The majority of these antibodies are directed against the F glycoprotein (Walsh & Hruska, 1983), and it was recently shown that most of the hRSV-neutralizing activity present in human sera was due to antibodies specific for the metastable prefusion F conformation (Magro et al, 2012; Ngwuta et al, 2015). The search for new hRSV vaccines has been reinvigorated by recent advances in structure-based design of soluble hRSV F proteins folded in either the prefusion (McLellan et al, 2013a; Krarup et al, 2015) or postfusion conformations (McLellan et al, 2011b; Swanson et al, 2011). Stabilized soluble forms of prefusion hRSV F were shown to induce higher levels of neutralizing antibodies than soluble postfusion hRSV F in mice, cotton rats, and non-human primates (McLellan et al, 2013a; Krarup et al, 2015; Palomo et al, 2016). However, postfusion F, which has the advantage of being highly stable, can induce sizeable levels of neutralizing antibodies and afford protection against hRSV because it shares certain epitopes with prefusion F (Swanson et al, 2011). Recently, the structure of a soluble form of postfusion hMPV F was determined, revealing extensive similarity with postfusion hRSV F despite having only ~38% sequence identity (Mas et al, 2016). In addition, the purified postfusion hMPV F protein was able to elicit high titers of neutralizing antibodies in mice (Mas et al, 2016). A few monoclonal antibodies capable of neutralizing both hRSV and hMPV have been reported (Corti et al, 2013; Schuster et al, 2014; Mas et al, 2016); however, no significant cross-neutralization was detected in polyclonal antibody responses elicited by soluble postfusion forms of either hRSV F or hMPV F (Mas et al, 2016). Based on knowledge gained about the antigenic structure of these two proteins, we engineered a series of chimeric proteins in which certain epitopes of hRSV F were grafted onto hMPV F and vice versa. When mice were inoculated with the purified chimeras, they elicited antibodies that neutralized both hRSV and hMPV. Furthermore, in cases that were tested, immunization of mice with the chimeric proteins afforded protection against a challenge with the virus used for grafting of the heterologous antigenic site. Passive transfer of the mouse sera also reduced significantly lung virus titer after challenge, demonstrating the prominent role of antibodies in the protection provided by the chimeric F proteins. Results The plasmids encoding each of the chimeric proteins shown in Fig EV1 were generated and tested for transient expression of the matching F proteins in CV-1 cells, as described in Materials and Methods. Some chimeric proteins were expressed at very low or undetectable levels and were not further analyzed. Chimeric proteins that reached expression levels above 10% of the wild-type protein were incorporated into vaccinia virus recombinants for expression and further antigenic and immunogenic characterization as described in subsequent sections. Click here to expand this figure. Figure EV1. List of wild-type and chimeric proteins encoded in pRB21 plasmidsThe code of each chimeric protein is indicated in the left-hand column and distributed in groups. In each group, the name in the upper line denotes the wild-type protein used as backbone and the bottom line the protein from which antigenic sites were grafted in the chimeras. Numbers above and below sequences correspond to residues of the indicated F proteins. The first part of the chimera name refers to the source of the backbone and the second part to the grafted antigenic site. hRSV F and hMPV F sequences are colored red and blue, respectively. Residues shared by both sequences are colored purple. Transient expression levels were measured in culture supernatants by ELISA as indicated in Materials and Methods, using an anti-foldon mAb (common to all proteins) to capture the proteins that were developed with an anti-His mAb. Expression levels of chimeric proteins were normalized to that of the corresponding wild-type backbone protein, taken as 100%. Numbers shown in boldface indicate the chimeras that were incorporated to vaccinia virus recombinants. *Denotes a chimera that was not included in vaccinia virus for reasons explained in the text. Download figure Download PowerPoint Expression and characterization of postfusion hMPV F with antigenic site II from hRSV F Based on the similarity of hMPV F and hRSV F antigenic site II structures (Mas et al, 2016), it was expected that certain amino acids could be exchanged between the two proteins without disrupting the overall local folding (Mas et al, 2016). Indeed, the chimeric proteins F-414, F-415, and F-416 (Fig EV1), with increasing numbers of hRSV F residues replacing the equivalent postfusion hMPV F residues, were expressed at high level and therefore initially analyzed. Since the expression level of F-414 turned out to be the same as that of F-415, the former protein was not further considered. Much of the C-terminal helix of hMPV F site II in F-415 was made hRSV-like by swapping seven amino acids unique to each protein (Fig 1A and B). Additionally, the chimeric F-416 protein incorporated six further amino acid changes in the hMPV F backbone to reproduce almost entirely the amino acid sequence of hRSV F site II (Fig 1A and B). Both F-415 and F-416 were readily purified to homogeneity by Ni2+ chromatography followed by gel filtration and showed the characteristic cone shape of postfusion hMPV F (Mas et al, 2016) when examined by negative stain EM (Fig EV2). Figure 1. Antigenic characterization of postfusion hMPV F chimeras with amino acids from hRSV F antigenic site II Partial amino acid sequences of the hMPV F (blue) and hRSV F (red) antigenic site II (Toiron et al, 1996; Lopez et al, 1998; McLellan et al, 2011b). Partial sequences of the F-415 and F-416 chimeras are color coded to indicate the origin of their antigenic site II residues. Amino acids shared by hMPV F and hRSV F are colored purple. The remaining sequences of F-415 and F-416 are derived from hMPV F. Surface representation of the hMPV F structure in the postfusion conformation (Mas et al, 2016). One of the protomers is shown as a blue ribbon. Antigenic site II is magnified and colored as in (A) for the F-415 and F-416 chimeras. ELISA binding results of mAbs specific for hRSV F (left panels) or hMPV F (right panels) with the proteins indicated on the left. mAbs specific for hRSV F bind epitopes of antigenic site II (Mota and 47F) or antigenic site IV (101F). mAs specific for hMPV F bind epitopes of antigenic site II (MF9, MF12, MF14, and MF15) or antigenic site IV (MF16). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Production and characterization of F-415 and F-416 chimeras A. Coomassie-stained SDS–PAGE of the proteins indicated in each lane. Numbers on the left correspond to molecular mass markers (kilodalton, kDa). The position of F1 and F2 subunits is indicated on the right. B. Gel filtration chromatography profiles of the indicated proteins. C, D. Negative-stained EM images of purified F-415 (C) and F-416 (D). Scale bar, 50 nm. Download figure Download PowerPoint When tested for binding to site-specific mAbs, F-415 had lost reactivity with the hMPV F site II mAbs MF9, MF12, MF14, and MF15 but retained reactivity with the hMPV F site IV-specific mAb MF16; in addition, it gained full and partial reactivity with the hRSV F site II mAbs motavizumab (Mz) and 47F, respectively (Fig 1C). F-416 also lost reactivity with hMPV F site II-specific mAbs but gained full reactivity not only with Mz but also with 47F (Fig 1C). To have a better estimate of the affinities of Mz and 47F for the chimeric proteins, the binding of their respective Fab fragments to wild-type postfusion hRSV F and the chimeric F proteins was assessed by surface plasmon resonance (SPR) (Fig 2). Mz Fab bound to the F-415 and F-416 chimeras with a kon similar to that observed for the wild-type postfusion hRSV F but showed a much faster koff for F-415 than for the wild-type and F-416 proteins. Consequently, Mz affinity for the F-416 chimera was similar to that for postfusion hRSV F but was markedly decreased for F-415 (see KD values). Figure 2. Binding of Fabs to wild-type and chimeric proteins measured by surface plasmon resonanceBinding of Mz and 47F Fabs to the immobilized proteins indicated on the left. Sensorgrams of eight different concentrations are shown in each panel. The calculated binding parameters are shown in the table. Red lines indicate the best fit of the data to a 1:1 binding model. RUs, response units. Download figure Download PowerPoint No binding of 47F Fab to F-415 was detectable (Fig 2), and although binding of 47F Fab to F-416 was quantifiable—with a slightly faster kon compared with wild-type postfusion hRSV F—a much faster koff reduced its affinity for F-416 by 10-fold compared to postfusion hRSV F. Therefore, although site II of F-415 and particularly F-416 protein resembled the antigenic properties of this site in hRSV F, there were some remaining distinguishable differences between wild-type and chimeric proteins. We noted that although ELISA binding of Mz and 47F to the chimeric proteins followed the same trend as that of their Fab fragments in SPR, significant differences could be perceived with the two methods. Thus, whereas binding of 47F Fab to F-415 was negligible by SPR (Fig 2), substantial binding of mAb 47F to F-415 was observed in the ELISA (Fig 1C). Similarly, although binding of mAb Mz to F-415 was comparable to postfusion hRSV F in the ELISA, a substantial reduction in Mz Fab affinity for this chimeric protein was observed by SPR. These differences are likely due to the higher density of proteins bound to the ELISA plates than to the SPR chips and the bivalent nature of antibodies compared with the monovalent Fabs. To assess the immunogenicity of the F-415 and F-416 chimeras, groups of five mice were inoculated twice (4 weeks apart) i.m. with 10 μg/dose of each purified protein adjuvanted with CpG. The same inoculation regimen was used for postfusion hRSV F and hMPV F, as controls. Three weeks after the last immunization, mice were bled and their sera tested in ELISA for antibody binding to the wild-type postfusion forms of either hRSV F or hMPV F (Fig 3A). As reported, the wild-type postfusion F proteins induced high levels of antibodies that bound to the homologous protein but failed to bind (or bound very poorly in the case of serum from mice inoculated with hMPV F and tested against hRSV F) to the heterologous protein (Mas et al, 2016). In contrast, the antibodies induced by the F-415 chimera showed substantial binding not only to hMPV F but also to the hRSV postfusion F protein (Fig 3A). The level of antibody binding to postfusion hRSV F was even higher with sera from mice inoculated with F-416 and significantly higher (P < 0.001) than sera from mice inoculated with wild-type hMPV F. Figure 3. Antibody responses of mice inoculated twice with either wild-type proteins or chimeric proteins with a postfusion hMPV F backboneGroups of BALB/c female mice (n = 5) were inoculated twice (4 weeks apart) i.m. with 10 μg/dose of the proteins indicated at the top of each panel, folded in their respective postfusion conformations. One week after the last dose, mice were sacrificed and their blood collected. Each mouse serum (identified by a number) was tested in ELISA for antibody binding to the postfusion F proteins indicated at the bottom. The results represented in the y-axis for each individual mouse serum correspond to the inverse dilution that gave 50% of maximal binding, expressed in log10 units (EC50 (log10)). *P = 0.00045. Mouse sera were also tested in microneutralization assays with hRSV and hMPV, as indicated at the bottom. The results represented in the y-axis correspond to the inverse dilution that inhibited 50% of the viral infectivity (IC50 (log10)). P-values: *P = 0.01001 and **P = 0.00271. Data information: Each number represents an individual mouse. Short horizontal bars indicate mean values for each group. P-values were calculated as indicated under "Statistical Analysis". Only relevant P-values are shown. Differences were considered significant when P < 0.05. Long horizontal lines indicate detection limits. Download figure Download PowerPoint To assess whether the antibody binding to hRSV F observed with sera from mice inoculated with F-415 or F-416 was reflected in neutralizing activity against this virus, the mouse sera were tested in a microneutralization assay (Fig 3B). Again, the sera of mice inoculated with the wild-type hMPV F or hRSV F protein had substantial neutralization titers (IC50) only against the homologous virus. In contrast, the sera of mice inoculated with either F-415 or F-416 had sizeable neutralization titers against hRSV, in addition to neutralizing hMPV. In summary, the antigenic characteristics of the F-415 and F-416 chimeras, reflected in their reactivity with hRSV F site II-specific mAbs (Fig 1C), correlated with their capacity to induce murine antibodies that bound hRSV F and neutralized hRSV infectivity, which was particularly prominent in the case of F-416. We note, however, that in some sera no quantitative correlation was found between binding and neutralization titers; for example, serum from mouse #5 inoculated with F-415 had low levels of hRSV F-binding antibodies (Fig 3A) but neutralized hRSV very efficiently (Fig 3B). To evaluate whether induction of hRSV-neutralizing antibodies by the chimeric proteins could be correlated with protection against a challenge with this virus, groups of eight mice were immunized three times, 4 weeks apart, with 20 μg of either F-415 or F-416 adjuvanted with CpG. One week after the last injection, mice were challenged intranasally with hRSV (A2 strain), and 5 days later, the amount of virus in lung extracts was quantified in a plaque assay. In parallel, three groups of five mice were inoculated with either CpG or wild-type postfusion hRSV F or hMPV F, as controls. The results again demonstrated a significant increase in hRSV-neutralizing antibodies in the sera of mice inoculated with F-415 and F-416, just before the challenge, compared with CpG-only and postfusion hMPV F controls (Fig 4A). Although there was some spread of the hRSV-neutralizing titers of individual mice, those inoculated with F-416 had on average higher neutralization titers than those inoculated with F-415. However, in both groups some individual mouse titers reached values close to those of mice inoculated with postfusion hRSV F. Figure 4. Protection against hRSV challenge with hMPV F backbone chimerasGroups of BALB/c mice (n = 5) were inoculated three times, 4 weeks apart, with 20 μg of the postfusion F proteins indicated above each panel. One group (n = 5) was inoculated only with the CpG adjuvant, as indicated. Two other groups of mice (n = 8) were similarly inoculated with either F-415 or F-416, as indicated at top of the panels. Mouse sera, collected just before challenging with hRSV (A2 strain), were used in microneutralization assays against the viruses indicated at bottom. *P = 0.00085 and **P = 0.01986. Five days after virus inoculation, mice were sacrificed and the amount of virus in lung extracts was measured in a plaque assay. Mean virus titer for each group is indicated by short horizontal bar. Data information: Each number represents an individual mouse. Short horizontal bars indicate mean values for each group. P-values were calculated as indicated under "Statistical Analysis". Only relevant P-values are shown. Differences were considered significant when P < 0.05. Long horizontal lines indicate detection limits. Download figure Download PowerPoint Reduction in hRSV replication in the lungs of inoculated mice clearly correlated with the noted induction of antibodies that neutralized hRSV infectivity (Fig 4B). Whereas hRSV reached high titers in the lungs of mice inoculated with either CpG or hMPV F, there was no detectable virus in the lungs of mice inoculated with postfusion hRSV F. Two of the mice inoculated with the F-415 chimera (#6 and #7) had no detectable virus in their lungs. These two mice also had the higher titers of hRSV-neutralizing antibodies in their sera (compare Fig 4A and B). As a group, mice inoculated with F-415 protein had a substantial reduction in lung virus titers compared with CpG and hMPV F controls. Remarkably, the lungs of all mice inoculated with F-416 were free of detectable virus, demonstrating the efficacy of F-416 vaccination for protection against hRSV infection. To further substantiate the relevance of antibodies in protection of mice inoculated with F-415 or F-416 chimeras, sera from each group of mice of Fig 4B were pooled together and passively transferred i.p. to new mice that were challenged the following day with the same amount of virus as in Fig 4. Five days after challenge, the amount of virus in lung extracts was quantified in a plaque assay. The results of Fig EV3 demonstrate that virus replicated to high titers in mice that received sera from those previously inoculated with either CpG or hMPV F. In contrast, no virus was detected in the lungs of mice that received sera from mice previously inoculated with hRSV F. Most importantly, virus titers were significantly reduced in mice that received sera from those previously inoculated with F-415 and particularly with F-416 in which about ten times less virus was found in their lungs than in those from the CpG- and hMPV-negative controls (Fig EV3). It is worth noting though that protection afforded passively by sera from mice previously inoculated with F-416 did not reach the level of protection provided by sera from mice previously inoculated with hRSV F. Click here to expand this figure. Figure EV3. Adoptive transfer protection of mice against a hRSV challengeSera from groups of mice inoculated with the indicated immunogen and bled immediately before the challenge of Fig 4 were pooled together and diluted with an equal volume of PBS. 200 μl of the diluted pool was inoculated i.p. to each mouse 24 h before i.n. challenging with 4 × 106 pfu/mouse of hRSV (A2 strain) as indicated in Materials and Methods. Five days after challenge, mice were sacrificed and the amount of virus in lung extracts quantified by plaque assay. The number of mice per group was five except in the case of those receiving serum from mice inoculated with hRSV F (3 mice) and F-415 (4 mice) due to shortage of sera. Each dot represents an individual mouse. Short horizontal bars indicate mean values for each group. P-values were calculated as indicated under "Statistical Analysis". Only relevant P-values are shown. Differences were considered significant when P < 0.05. Long horizontal lines indicate detection limits. Download figure Download PowerPoint Expression and characterization of prefusion hRSV F with antigenic site IV from hMPV F It has been recently demonstrated that most of the neutralizing antibodies in human sera are directed against the prefusion form of hRSV F (Magro et al, 2012; Ngwuta et al, 2015) and that prefusion-specific site Ø mAbs are the most potent neutralizing antibodies (McLellan et al, 2013b; McLellan, 2015). For this reason, and to test the possibility of extending the chimera approach to other conformations and antigenic sites, the chimeric proteins F-410, F-411, and F-412 of Fig EV1 were produced. These proteins were derived from soluble hRSV F stabilized in the prefusion conformation as previously reported (McLellan et al, 2013a). Although F-412 was expressed at a moderate level, it failed to react with hMPV F site IV mAbs, and thus, it was discontinued from the study. In the F-410 protein, five amino acids of antigenic site IV were swapped with the corresponding residues of hMPV F (Fig 5A and B). Six further amino acid changes were introduced in the F-411 chimera to make it more hMPV-like. Both F-410 and F-411 were purified to homogeneity and had the characteristic globular shape of prefusion hRSV F, as seen by EM (Fig EV4). Figure 5. Antigenic characterization of prefusion hRSV F chimeras with amino acids from hMPV F antigenic site IV Partial amino acid sequences of the hRSV F (red) and hMPV F (blue) antigenic site IV (Lopez et al, 1998; Wu et al, 2007; McLellan et al, 2010). Partial sequences
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