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

Highlights

• RSV F nanoparticle vaccine elicits protection against palivizumab-resistant RSV.

• Adjuvanted vaccine elicits anti-F IgG that binds F protein with higher affinity.

• Nanoparticle vaccine elicits antibodies targeting multiple neutralizing epitopes.

Abstract

Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infections in newborns, young children, elderly, and immune-compromised. The RSV fusion (F) glycoprotein is a major focus of vaccine development and the target of palivizumab (Synagis®) which is licensed as an immuno-prophylactic for use in newborn children at high risk of infection. However, clinical use of a narrowly targeted monoclonal antibodies leads to the generation of escape mutant strains that are fully resistant to neutralization by the antibody. Herein, we evaluated the RSV F nanoparticle vaccine (RSV F vaccine), produced as near-full-length, pre-fusogenic F trimers that form stable protein-detergent nanoparticles. The RSV F vaccine induces polyclonal antibodies that bind to antigenic site II as well as other epitopes known to be broadly neutralizing. Cotton rats immunized with the RSV F vaccine produced antibodies that were both neutralizing and protected against wild-type RSV infection, as well as against a palivizumab-resistant mutant virus. Use of aluminum phosphate adjuvant with the RSV F vaccine increased site II antibody avidity 100 to 1000-fold, which correlated with enhanced protection against challenge. The breadth of the vaccine-induced antibody response was demonstrated using competitive binding with monoclonal antibodies targeting antigenic sites Ø, II, IV, and VIII found on pre-fusion and post-fusion conformations of RSV F. In summary, we found the RSV F vaccine induced antibodies that bind to conserved epitopes including those defined as pre-fusion F specific; that use of adjuvant increased antibody avidity that correlated with enhanced protection in the cotton rat challenge model; and the polyclonal, high-avidity antibodies neutralized and protected against both wild-type and palivizumab-resistant mutant virus. These data support the ongoing clinical development of the aluminum phosphate adjuvanted RSV F nanoparticle vaccine.

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.