Respiratory syncytial virus fusion nanoparticle vaccine immune responses target multiple neutralizing epitopes that contribute to protection against wild-type and palivizumab-resistant mutant virus challenge
Introduction
Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection (LRTI) amongst infants, young children, adults over 65 years of age, and immune-compromised. Human RSV is a medium sized (120–200 nm) enveloped virus in the Pneumoviridae family [1]. There are two major subgroups: RSV/A with twelve genotypes and RSV/B with twenty genotypes, based upon the heterogeneity of the attachment (G) glycoprotein [2], [3], [4], [5]. The fusion (F) protein is essential for infection and is highly conserved (∼90% homology) with shared antigenic epitopes between RSV/A and B subgroups. The F protein is produced as a single inactive polypeptide (F0) of 574 aa with 5–6 N-linked glycans and a mass of 70 kD. F0 is processed to an active form by cellular furin which cleaves F0 at residues 109 (site I) and 136 (site II) resulting in the removal of a 27 aa fragment (p27) and the generation of the F1 and F2 fragments. The activated F protein mediates fusion of the virus with the host cell membrane [6]. There is controversy as to the timing and cellular location of the furin cleavage events. One cleavage occurs early during post-translational processing, while the second cleavage may occur within the host cell. Cleavage at both sites generates metastable pre-fusion intermediate conformation [7]. Partially cleaved F protein intermediates have been found in purified virus [6], and perhaps within the same oligomers as cleaved F, until complete cleavage and activation at the time of virus entry into the cell [8]. Using site-directed mutagenesis, it was shown that cleavage at site II, adjacent to the fusion domain in F1, is indispensable for F-activation whereas cleavage at site I appears to increase the efficiency of cleavage at site II [8]. Site I furin cleavage results in a fusion-inactive pre-fusogenic F intermediate with the p27 fragment at the N-terminus of F1 (F-p27). Antigenic fingerprinting following primary RSV infection of infants and very young children identified that an immuno-dominant epitope in p27 and immune responses to p27 decline with age [9]. In a recent report, removal of the N-glycosylation sequon in p27 resulted in enhanced antibody responses after DNA immunization [10]. This supports that a partially cleaved pre-fusogenic F with p27 is physiologically relevant and exposed on the surface of infected cells and the virus membrane and is accessible to immune recognition.
Subsequent cleavage of furin cleavage site II results in the metastable pre-fusion (pre-F) structure that undergoes significant conformational rearrangement to the stable post-fusion (post-F) structure without further processing. This final conformational change appears to occur within the infected cell and is essential for host-viral membrane fusion and subsequent release of the viral genome into the host cell [7]. Thus F glycoprotein is essential for infection, structurally conserved, and a major target of protective antibodies. The preclinical and clinical efficacy associated with palivizumab clearly indicates that F-targeted immunity can prevent clinical disease [11], [12].
RSV neutralizing mAbs to the F protein were first described in 1983 [13]. The subsequent identification of broadly-neutralizing antibodies led to the development of palivizumab, which is a humanized mAb derived from murine mAb 1129 [14], [42]. Palivizumab is currently the only approved prophylactic antibody for use in infants at high risk of RSV infection [12]. More recently, the crystal structures of synthetic, truncated pre-fusion and post-fusion F proteins, in association with Fab fragments, has enabled the fine mapping of multiple antigenic sites on the F glycoprotein [15]. These studies have defined the antigenic site II located on the F1 aa 254–277 as the binding site of palivizumab (site IIa) and motavizumab (site IIb). Antigenic site IV is a second neutralizing epitope on F1 (aa 422–438) and is the target of prototype mAbs 101F and murine NVX 1.42 [16], [17]. Antigenic sites II and IV are present on both pre- and post-fusion F conformational structures [18], [19]. Antigenic site II sequence is highly conserved while the site IV sequence is absolutely conserved between subgroups thus implicating these sites as critical targets for a seasonal and potentially changing virus vaccine [20]. Antigen sites zero (Ø) is on the pre-fusion F conformation and the target of potent neutralizing mAbs (D25, AM22 and 5CA). Antigenic site Ø is relatively conserved within the RSV/A subgroup and less conserved within the B subgroup [21], [22]. Antigenic site VIII is also present on the pre-fusion F structure and is the target of neutralizing mAb hRSV90 [23].
Although the F protein is highly conserved, the high mutation rate and genetic drift inherent with RNA viruses may lead to changes within neutralizing epitopes creating a challenge for use of prophylactic mAbs. Palivizumab-resistant RSV (PR-RSV) strains are readily generated by serial passage of wild-type (wt) RSV in cell culture with palivizumab. PR-RSV escape mutants typically have a single-point mutation within antigenic site II (N262S/Y, N268I, K272N/M/T/Q, and S275F/L) and fail to bind, or bind with low affinity, to palivizumab [24], [25]. In vivo, PR-RSV mutants have also been isolated from RSV-infected cotton rats [26] and humans treated with palivizumab [11], [27]. In the US, the emergence of PR-RSV occurs in 1–5% of patients treated with palivizumab or motavizumab [28], [29], [30]. In Japan, the first appearance of the N276S palivizumab resistance mutant was identified in 2007 followed by identification of this mutation in 100% of clinical isolates by 2013 [31]. In Canada, resistant strains have been associated with clinical use of palivizumab [32]. An ideal vaccine will induce polyclonal antibodies to multiple neutralizing sites and minimize the potential for induction of resistant mutant strains.
We have developed a subunit vaccine from the near-full-length sequence of the RSV F glycoprotein. The RSV F nanoparticle vaccine has been shown to be well tolerated, immunogenic, and to reduce RSV infections in animal models and human clinical trials [33], [34], [35], [36]. In this report, we show immunization with RSV F nanoparticle vaccine elicits antibodies that are competitive with mAbs targeting multiple neutralizing epitopes on the F protein including antigenic sites Ø, II, IV, and VIII. The vaccine elicits antibodies that broadly neutralized RSV/A and B subtypes and protection against wild-type and a PR-RSV escape mutant. Adjuvanted vaccine produced high-avidity antibodies that correlates with enhanced protective immunity.
Section snippets
Cell line, viruses, RSV F nanoparticles, antibodies, and synthetic peptide
HEp-2 (ATCC, CCL-23, Manassas, VA, USA) cells were maintained in MEM with Earle’s salts and L-glutamine (Gibco Laboratories, Gaithersburg, MD, USA), 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA), and antibiotics (Life Technologies, Grand Island, NY, USA). RSV/A/Tracy, RSV/A/Long, and RSV/B/18537 reference strains were obtained from ATCC and Dr. Piedra, Baylor College of Medicine (Houston, TX, USA) [37]. Virus stocks were prepared from clarified supernatants and stored at −80 °C. RSV F
Infectivity of PR-RSV/A escape mutant (N262Y) in palivizumab and placebo treated cotton rats
To investigate the protective efficacy of RSV F nanoparticle vaccine against RSV variants, we first generated a PR-RSV/A strain by serial passage of wt-RSV/A/Tracy in cell culture with palivizumab. The infectivity of the resulting PR-RSV/A escape mutant was compared to wt-RSV/A in cotton rats. Animals were IN infected with 1.35 x 105 pfu wt-RSV/A/Tracy or 2 × 105 pfu PR-RSV/A and lung lavage and nasal samples were collected 4-days post-infection. Lung lavage collected from animals receiving PBS
Discussion
The RSV F glycoprotein is a compelling choice for vaccine development as efficacy has been demonstrated in using F-specific mAbs and it is structurally conserved between RSV subgroups. The RSV vaccine used in this study was produced from the near-full-length F protein with the p27 fragment (F-p27) in a pre-fusogenic conformation with the transmembrane region retained to facilitate incorporation of multiple F-p27 trimers into non-ionic detergent micelles which form 40–60 nm nanoparticles [38].
Conclusions
The adjuvanted recombinant RSV F nanoparticle vaccine presented in this manuscript is currently in Phase 3 clinical development (NCT02624947). The vaccine induces the generation antibodies that compete with mAbs that target pre-fusion F neutralizing epitopes as well as epitopes common to pre- and post-fusion F conformations and p27. p27 is an epitope associated with a pre-fusogenic form of the F glycoprotein, and is clearly found on the surface of an infectious virus [7]. The antibodies induced
Author contributions
Authors BEG, NP, HL, YL and MGX contributed to conceptualization of experiments, generation of data and analysis and interpretation of the data. Authors PAP, GG, GS and LE contributed to drafting, critical revisions and approval for submission.
Funding
Support for this work was provided by Novavax, Inc., Gaithersburg, MD and were supported by NIH/NIAID, United States contract HHS272201000041. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Acknowledgements
The authors thank Maggie Lewis and Sapeck Agrawal for editorial assistance in the preparation of this manuscript.
Conflicts of interests
Authors NP, HL, YL, MGX, GG, GS and LE are current or past employees of Novavax, Inc, a for-profit organizations, and these authors own stock or hold stock options. These authors have submitted and pending patent applications related to the work. These interests do not alter the author’s adherence to policies on sharing data and materials. PAP and BEG declare no competing interest.
Data availability
All relevant data are within the manuscript.
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