Nanovaccines Displaying the Influenza Virus Hemagglutinin in an Inverted Orientation Elicit an Enhanced Stalk‐Directed Antibody Response

Abstract

Despite the availability of licensed vaccines, influenza causes considerable morbidity and mortality worldwide. Current influenza vaccines elicit an immune response that primarily targets the head domain of the viral glycoprotein hemagglutinin (HA). Influenza viruses, however, readily evade this response by acquiring mutations in the head domain. While vaccines that target the more conserved HA stalk may circumvent this problem, low levels of antistalk antibodies are elicited by vaccination, possibly due to the poor accessibility of the stalk domain to B cell receptors. In this work, it is demonstrated that nanoparticles presenting HA in an inverted orientation generate tenfold higher antistalk antibody titers after a prime immunization and fivefold higher antistalk titers after a boost than nanoparticles displaying HA in its regular orientation. Moreover, nanoparticles presenting HA in an inverted orientation elicit a broader antistalk response that reduces mouse weight loss and improves survival after challenge to a greater extent than nanoparticles displaying HA in a regular orientation. Refocusing the antibody response toward conserved epitopes by controlling antigen orientation may enable the design of broadly protective nanovaccines targeting influenza viruses and other pathogens with pandemic potential.

Chemical and biological conjugation strategies for the development of multivalent protein vaccine nanoparticles

“…The process involves genetic modification of an antigen for site‐specific conjugation via a biological interaction, which leads to proper orientation of the antigen. Antigen orientation on particles has been shown to be an important factor for humoral immune responses, [ 76–78 ] and site‐specific biological conjugation can enhance vaccine effectiveness of protein nanoparticles. Biotag coupling requires the addition of a tag, usually at a terminus of an antigen.…”

“…The process involves genetic modification of an antigen for site-specific conjugation via a biological interaction, which leads to proper orientation of the antigen. Antigen orientation on particles has been shown to be an important factor for humoral immune responses, [76][77][78] and sitespecific biological conjugation can enhance vaccine effectiveness F I G U R E 6 Formulation of M2e nanoparticles via desolvation with organic solvent is followed by adsorption of HA stalk on the surface and stabilization with DTSSP crosslinker, which links accessible lysines and terminal amines through NHS-ester conjugation. [ 69 ] (Illustrated by BioRender).…”

Multivalent S2-subunit Vaccines Provide Broad Protection Against Clade 1 Sarbecoviruses

“…We have previously developed streptavidin-coated VLPs and multivalently presented numerous biotinylated antigens on them as vaccine constructs, including the Zika virus envelope protein domain III 23 , In uenza virus hemagglutinin 24 , various sarbecovirus S proteins 12,25 , and the SARS-CoV-2 S2 subunit. 14 The core of the VLP is the bacteriophage MS2 coat protein, which self-assembles from 90 MS2 homodimers.…”

“…A 5 mL 2xYT media starter culture was made using the transformed E. coli, which were incubated at 37°C overnight in a rotating incubator. 12,14,24,25 The starter culture was then added to 1 L of 2xYT and further grown at 37°C in a shaking incubator at 225 rpm until it reached an optical density of 0.6-0.8, after which the culture was induced by adding 1 mL of of 1 M IPTG (Fisher BioReagents). Simultaneously, 50 mM D-biotin was added to the culture, and the incubation temperature was reduced to 30°C.…”

“…The lysate was sonicated on ice at 35% amplitude with 3-second pulses for 3 minutes (Soni er S-450, Branson Ultrasonics). 12,14,24,25 The lysate was sonicated again after being allowed to rest on ice for 5 minutes. The lysate was centrifuged at 19,000xg for 30 minutes, the supernatant was collected and centrifuged again.…”