Construction, characterization, and immunization of nanoparticles that display a diverse array of influenza HA trimers

Abstract

Current influenza vaccines do not elicit broadly protective immune responses against multiple strains. New strategies to focus the humoral immune response to conserved regions on influenza antigens are therefore required for recognition by broadly neutralizing antibodies. It has been suggested that B cells with receptors that recognize conserved epitopes would be preferentially stimulated through avidity effects by mosaic particles presenting multiple forms of a variable antigen. We adapted SpyCatcher-based platforms, AP205 virus-like particles (VLPs) and mi3 nanoparticles (NPs), to covalently co-display SpyTagged hemagglutinin (HA) trimers from different influenza strains. Here we show successful homotypic and heterotypic conjugation of up to 8 different HA trimers to both VLPs and NPs, and demonstrate that conjugated particles were stable for several weeks of storage. We characterized the HA-VLPs and HA-NPs by cryo-electron tomography to derive the average number of conjugated HAs and their separation distances, and compared immunizations of mosaic and homotypic particles in wild-type mice. Both types of HA particles elicited strong antibody responses, but the mosaic particles did not consistently elicit broader immune responses. We conclude that covalent attachment of HAs from currently-circulating influenza strains represents a viable alternative to current annual influenza vaccine strategies, but in the absence of further modifications, is unlikely to represent a method for making a universal influenza vaccine.

Serological Screening of Influenza A Virus Antibodies in Cats and Dogs Indicates Frequent Infection with Different Subtypes

“…By using recombinant protein-based assays, this issue may be resolved, resulting in HI assay platforms that are easy to perform and standardize. Recently, a similar HA-nanoparticle design was used for immunization studies, and effective presentation of HA proteins was confirmed by electron microscopy (46). A comparable approach was also conducted for Middle East Respiratory Syndrome Coronavirus (MERS) serology, where the sialic acid binding MERS spike S1 A domain was presented on nanoparticles to perform HI assay (47).…”

Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice

“…SpyCatcher003-mi3 particles were prepared by purification from BL21 (DE3)-RIPL E coli (Agilent) transformed with a pET28a SpyCatcher003-mi3 gene (including an N-terminal 6x-His tag) as described ( 54 ). Briefly, cell pellets from transformed bacterial were lysed with a cell disruptor in the presence of 2.0 mM PMSF (Sigma).…”

“…cytometry. B-cell analysis using flow cytometry was carried out as described(45). Briefly, single-cell suspensions were prepared from mouse spleens using mechanical dissociation, and red blood cells were removed using ACK lysing buffer (Gibco).…”

Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines

“…We and others have recently evaluated nanoparticle immunogens that display multiple antigenic variants of viral glycoproteins as a potential route toward broadly protective vaccines (Boyoglu-Barnum et al, 2021;Cohen et al, 2021aCohen et al, , 2021bKanekiyo et al, 2019). Given the large number of coronaviruses circulating in zoonotic reservoirs, such vaccines could be important for understanding vaccine-induced neutralization/protection breadth and preventing future pandemics (Menachery et al, 2015(Menachery et al, , 2016.…”