Molecular and phenotypic characteristics of respiratory syncytial virus isolates recovered from medically vulnerable children: An exploratory analysis of a phase 2/3 randomized, double-blind, palivizumab-controlled trial of nirsevimab (MEDLEY).

BACKGROUND: Nirsevimab is an extended half-life monoclonal antibody (mAb) licensed for the prevention of respiratory syncytial virus (RSV)-associated lower respiratory tract disease in neonates, infants and medically vulnerable children. We characterized RSV isolates recovered from participants enrolled in MEDLEY: a randomized, palivizumab-controlled phase 2/3 trial of nirsevimab in infants born preterm and/or with congenital heart disease or chronic lung disease of prematurity. METHODS: Participants were assessed in two RSV seasons (Season 1 and 2). Season 1 participants were randomized (2:1) to receive a single dose of nirsevimab (50 mg if weight <5 kg or 100 mg if weight ≥5 kg in Season 1; 200 mg in Season 2) followed by four monthly doses of placebo, or five once-monthly doses of palivizumab (15 mg/kg weight per dose). Season 2 participants continued nirsevimab and placebo (nirsevimab/nirsevimab) or were re-randomized (1:1) to switch to nirsevimab (palivizumab/nirsevimab) or continue palivizumab (palivizumab/palivizumab). Cases of RSV infection were identified by central testing of nasal swabs from participants seeking medical attention for respiratory illnesses. Nirsevimab and palivizumab binding site substitutions were assessed via microneutralization assay. RESULTS: Twenty-five cases of confirmed RSV infection were observed during the trial and sequenced: 12 in nirsevimab recipients and 10 in palivizumab recipients during Season 1, and 1 case in each Season 2 group. Molecular sequencing of RSV A (n = 14) isolates detected no nirsevimab binding site substitutions, and 3 palivizumab neutralization-resistant substitutions (Lys272Met, Lys272Thr, Ser275Leu). The nirsevimab binding site Ile206Met:Gln209Arg and Ile206Met:Gln209Arg:Ser211Asn substitutions were the only anti-RSV mAb binding site substitutions detected among RSV B isolates (n = 11). Nirsevimab neutralized all nirsevimab and palivizumab binding site substitutions in RSV A and B isolates recovered from MEDLEY participants. CONCLUSION: No binding site substitution detected during MEDLEY affected RSV susceptibility to nirsevimab neutralization.

Fc-mediated functions of nirsevimab complement direct respiratory syncytial virus neutralization but are not required for optimal prophylactic protection.

Introduction: Nirsevimab is an extended half-life (M252Y/S254T/T256E [YTE]-modified) monoclonal antibody to the pre-fusion conformation of the respiratory syncytial virus (RSV) Fusion protein, with established efficacy in preventing RSV-associated lower respiratory tract infection in infants for the duration of a typical RSV season. Previous studies suggest that nirsevimab confers protection via direct virus neutralization. Here we use preclinical models to explore whether fragment crystallizable (Fc)-mediated effector functions contribute to nirsevimab-mediated protection. Methods: Nirsevimab, MEDI8897* (i.e., nirsevimab without the YTE modification), and MEDI8897*-TM (i.e., MEDI8897* without Fc effector functions) binding to Fc γ receptors (FcγRs) was evaluated using surface plasmon resonance. Antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent complement deposition (ADCD), and antibody-dependent cellular cytotoxicity (ADCC) were assessed through in vitro and ex vivo serological analyses. A cotton rat challenge study was performed with MEDI8897* and MEDI8897*-TM to explore whether Fc effector functions contribute to protection from RSV. Results: Nirsevimab and MEDI8897* exhibited binding to a range of FcγRs, with expected reductions in FcγR binding affinities observed for MEDI8897*-TM. Nirsevimab exhibited in vitro ADNP, ADCP, ADCD, and ADCC activity above background levels, and similar ADNP, ADCP, and ADCD activity to palivizumab. Nirsevimab administration increased ex vivo ADNP, ADCP, and ADCD activity in participant serum from the MELODY study (NCT03979313). However, ADCC levels remained similar between nirsevimab and placebo. MEDI8897* and MEDI8897*-TM exhibited similar dose-dependent reduction in lung and nasal turbinate RSV titers in the cotton rat model. Conclusion: Nirsevimab possesses Fc effector activity comparable with the current standard of care, palivizumab. However, despite possessing the capacity for Fc effector activity, data from RSV challenge experiments illustrate that nirsevimab-mediated protection is primarily dependent on direct virus neutralization.

Antigenicity and immunogenicity of unique prefusion-mimic F proteins presented on enveloped virus-like particles.

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.

A unique combination adjuvant modulates immune responses preventing vaccine-enhanced pulmonary histopathology after a single dose vaccination with fusion protein and challenge with respiratory syncytial virus.

Alum adjuvanted formalin-inactivated respiratory syncytial virus (RSV) vaccination resulted in enhanced respiratory disease in young children upon natural infection. Here, we investigated the adjuvant effects of monophosphoryl lipid A (MPL) and oligodeoxynucleotide CpG (CpG) on vaccine-enhanced respiratory disease after fusion (F) protein prime vaccination and RSV challenge in infant and adult mouse models. Combination CpG + MPL adjuvant in RSV F protein single dose priming of infant and adult age mice was found to promote the induction of IgG2a isotype antibodies and neutralizing activity, and lung viral clearance after challenge. CpG + MPL adjuvanted F protein (Fp) priming of infant and adult age mice was effective in avoiding lung histopathology, in reducing interleukin-4+ CD4 T cells and cellular infiltration of monocytes and neutrophils after RSV challenge. This study suggests that combination CpG and MPL adjuvant in RSV subunit vaccination might contribute to priming protective immune responses and preventing inflammatory RSV disease after infection.

A Respiratory Syncytial Virus Vaccine Vectored by a Stable Chimeric and Replication-Deficient Sendai Virus Protects Mice without Inducing Enhanced Disease.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory infections in children and elderly people, and no marketed vaccine exists. In this study, we generated and analyzed a subunit vaccine against RSV based on a novel genome replication-deficient Sendai virus (SeV) vector. We inserted the RSV F protein, known to be a genetically stable antigen, into our vector in a specific way to optimize the vaccine features. By exchanging the ectodomain of the SeV F protein for its counterpart from RSV, we created a chimeric vectored vaccine that contains the RSV F protein as an essential structural component. In this way, the antigen is actively expressed on the surfaces of vaccine particles in its prefusion conformation, and as recently reported for other vectored vaccines, the occurrence of silencing mutations of the transgene in the vaccine genome can be prevented. In addition, its active gene expression contributes to further stimulation of the immune response. In order to understand the best route of immunization, we compared vaccine efficacies after intranasal (i.n.) or intramuscular (i.m.) immunization of BALB/c mice. Via both routes, substantial RSV-specific immune responses were induced, consisting of serum IgG and neutralizing antibodies, as well as cytotoxic T cells. Moreover, i.n. immunization was also able to stimulate specific mucosal IgA in the upper and lower respiratory tract. In virus challenge experiments, animals were protected against RSV infection after both i.n. and i.m. immunization without inducing vaccine-enhanced disease. Above all, the replication-deficient SeV appeared to be safe and well tolerated.IMPORTANCE Respiratory syncytial virus (RSV) is a major cause of respiratory diseases in young children and elderly people worldwide. There is a great demand for a licensed vaccine. Promising existing vaccine approaches based on live-attenuated vaccines or viral vectors have suffered from unforeseen drawbacks related to immunogenicity and attenuation. We provide a novel RSV vaccine concept based on a genome replication-deficient Sendai vector that has many favorable vaccine characteristics. The specific vaccine design guarantees genetic stability of the transgene; furthermore, it supports a favorable presentation of the antigen, activating the adaptive response, features that other vectored vaccine approaches have often had difficulties with. Wide immunological and pathological analyses in mice confirmed the validity and efficacy of this approach after both parenteral and mucosal administration. Above all, this concept is suitable for initiating clinical studies, and it could also be applied to other infectious diseases.

An anti-G protein monoclonal antibody treats RSV disease more effectively than an anti-F monoclonal antibody in BALB/c mice.

Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. To clarify the potential for an anti-G mAb, 131-2G which has both anti-viral and anti-inflammatory effects, to effectively treat RSV disease, we determined the kinetics of its effect compared to the effect of the anti-F mAb, 143-6C on disease in mice. Treatment administered three days after RSV rA2-line19F (r19F) infection showed 131-2G decreased breathing effort, pulmonary mucin levels, weight loss, and pulmonary inflammation earlier and more effectively than treatment with mAb 143-6C. Both mAbs stopped lung virus replication at day 5 post-infection. These data show that, in mice, anti-G protein mAb is superior to treating disease during RSV infection than an anti-F protein mAb similar to Palivizumab. This combination of anti-viral and anti-inflammatory activity makes 131-2G a promising candidate for treating for active human RSV infection.