Abstract

Background: The human respiratory syncytial virus (hRSV) is a pathogen of global concern, causing significant morbidity and mortality, mainly in preterm infants. To date, all licenced monoclonal antibodies (mAbs) developed against hRSV have targeted its surface fusion or pre-fusion protein (F-hRSV). Methods: We developed mAbs against the hRSV nucleoprotein (N-hRSV) as a complementary prophylactic treatment. Firstly, we produced a humanised murine mAb against N-hRSV, yielding four clones. Antibody affinity assays against the N-hRSV protein, pharmacokinetic analyses, and protection assays against pulmonary and neurological pathologies associated with hRSV infection in an in vivo male and female BALB/c murine model were performed. In vitro assays were carried out to determine the mechanisms of action of the humanised anti-N-hRSV mAbs, which exhibit antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Findings: Four humanised anti-N-hRSV mAb clones (P1-04H, P1-05D, P2-01A and P2-01D) were purified and showed high affinity for N-hRSV protein, as determined by SDS-PAGE and surface plasmon resonance, respectively. Pharmacokinetic analyses showed that two clones were detectable in the animals up to 30 days post-immunisation. Furthermore, treating mice with the humanised anti-N-hRSV mAb 1 day before hRSV infection reduced weight loss (P1-05D, & lowast;& lowast;p = 0.0069; and P2-01D, p = 0.0875), clinical score (P1-05D, & lowast;& lowast;p = 0.0052; P2-01A, & lowast;p = 0.04; P2-01D, & lowast;& lowast;& lowast;& lowast;p < 0.0001), and pulmonary N-hRSV mRNA expression levels in hRSV serotype A-infected mice (P2-01A, p = 0.0612; P2-01D, p < 0.0613), while all animals infected with hRSV serotype B showed reduced levels of N-hRSV expression (P1-04H, & lowast;& lowast;p = 0.0022; P1-05D, & lowast;& lowast;p = 0.0013; P2-01A, & lowast;& lowast;p = 0.0013; and P2-01D, & lowast;& lowast;p = 0.0013). Brain viral load was also decreased (P1-04H; P1-05D; P2-01A; and P2-01D, & lowast;& lowast;& lowast;& lowast;p < 0.0001). 30 days post-infection, behavioural tests indicated protection against long-term neurological alterations, with reduced faecal boli (P1-04H; and P2-01D, & lowast;p = 0.0243), altered centre preference (P1-04H; P1-05D; and P2-01D, & lowast;& lowast;& lowast;& lowast;p < 0.0001), and restoration of marble burying behaviour (clone P2-01D, & lowast;& lowast;p = 0.0055). Finally, in vitro analyses showed that the humanised anti-N-hRSV mAb displayed ADCC and CDC mechanisms to target infected cells. Interpretation: Our findings suggest that the humanised N-hRSV-specific mAbs displayed promising preclinical profiles by protecting against the pathology caused by hRSV infection.