Preclinical characterisation of the protective capacity of an anti-nucleoprotein hRSV monoclonal antibody.

Abstract

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). 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). 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, ∗∗p = 0.0069; and P2-01D, p = 0.0875), clinical score (P1-05D, ∗∗p = 0.0052; P2-01A, ∗p = 0.04; P2-01D, ∗∗∗∗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, ∗∗p = 0.0022; P1-05D, ∗∗p = 0.0013; P2-01A, ∗∗p = 0.0013; and P2-01D, ∗∗p = 0.0013). Brain viral load was also decreased (P1-04H; P1-05D; P2-01A; and P2-01D, ∗∗∗∗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, ∗p = 0.0243), altered centre preference (P1-04H; P1-05D; and P2-01D, ∗∗∗∗p < 0.0001), and restoration of marble burying behaviour (clone P2-01D, ∗∗p = 0.0055). Finally, in vitro analyses showed that the humanised anti-N-hRSV mAb displayed ADCC and CDC mechanisms to target infected cells. Our findings suggest that the humanised N-hRSV-specific mAbs displayed promising preclinical profiles by protecting against the pathology caused by hRSV infection. This study was supported by funding from the Millennium Institute on Immunology and Immunotherapy ANID ACE 210015 (CN09_016/ICN 2021_045; former P09/016-F, AMK); Biomedical Research Consortium CTU06 (AMK); FONDEF ID22I10252 (AMK and SMB); Fundación COPEC-UC 2019.R.1169 (AMK); FONDECYT Regular grant #1231866 (JAS), #1240971 (PAG), #1231905 (SMB), #1190830 and #1231851 (AMK). FONDECYT Iniciación grant #11221280 (KB). ANID FONDECYT Postdoctoral grant #3240624 (MAF). ANID Scholarship#21210662 (CAP), #21251811 (RAL), #21230429 (LRG), #21242058 (JTM), and #21221163 (BDV).