Comparative Immunogenicity of Bacterially Expressed Soluble Trimers and Nanoparticle Displayed Influenza Hemagglutinin Stem Immunogens

Abstract

Current influenza vaccines need to be updated annually due to mutations in the globular head of the viral surface protein, hemagglutinin (HA). To address this, vaccine candidates have been designed based on the relatively conserved HA stem domain and have shown protective efficacy in animal models. Oligomerization of the antigens either by fusion to oligomerization motifs or display on self-assembling nanoparticle scaffolds, can induce more potent immune responses compared to the corresponding monomeric antigen due to multivalent engagement of B-cells. Since nanoparticle display can increase manufacturing complexity, and often involves one or more mammalian cell expressed components, it is important to characterize and compare various display and oligomerization scaffolds. Using a structure guided approach, we successfully displayed multiple copies of a previously designed soluble, trimeric influenza stem domain immunogen, pH1HA10, on the ferritin like protein, MsDps2 (12 copies), Ferritin (24 copies) and Encapsulin (180 copies). All proteins were expressed in Escherichia coli. The nanoparticle fusion immunogens were found to be well folded and bound to the influenza stem directed broadly neutralizing antibodies with high affinity. An 8.5 Å Cryo-EM map of Msdps2-pH1HA10 confirmed the successful design of the nanoparticle fusion immunogen. Mice immunization studies with the soluble trimeric stem and nanoparticle fusion constructs revealed that all of them were immunogenic, and protected mice against homologous (A/Belgium/145-MA/2009) and heterologous (A/Puerto Rico/8/1934) challenge with 10MLD50 mouse adapted virus. Although nanoparticle display conferred a small but statistically significant improvement in protection relative to the soluble trimer in a homologous challenge, heterologous protection was similar in both nanoparticle-stem immunized and trimeric stem immunized groups. Such rapidly producible, bacterially expressed antigens and nanoparticle scaffolds are useful modalities to tackle future influenza pandemics.

Neutralizing Efficacy of Encapsulin Nanoparticles against SARS-CoV2 Variants of Concern

“…Each protomer of the Encapsulin can be fused to a desired antigen for surface presentation [ 42 , 43 ]. We have characterized Encapsulin for the display of Influenza hemagglutinin stem derived vaccine antigens, which provided complete protection against lethal challenge in mice [ 26 ]. In this case, the antigen of interest was a mammalian expressed, monomeric derivative of the SARS-CoV-2 spike receptor binding domain, mRBD (residues 332–532) [ 44 ].…”

“…Serum antibody-binding titers were measured as described previously [ 26 ]. Briefly, mRBD or spike-2P (4 µg/mL, in 1× PBS, 50 µL/well) was coated on 96-well plates which were incubated at 25 °C for 2 h. Four washes with 1× PBST (200 µL/well) were given, following which wells were blocked with blocking solution (100 µL, 3% skimmed milk in 1× PBST) for 1 h at 25 °C.…”

“…We have previously displayed influenza hemagglutinin derived stem antigens on diverse self-assembling proteins such as 12 mer-MsDps2, 24-mer ferritin, and 180-mer encapsulin. Our nanoparticle antigens induced complete protection against homologous and heterologous challenge in mice [ 26 ]. Several SARS-CoV-2 nanoparticle vaccines have also been described recently, which display either a full-length spike or spike receptor-binding domain (RBD) [ 27 , 28 , 29 ].…”

Cryo-EM reveals the mechanism of DNA compaction byMycobacterium smegmatisDps2

“…Purified MsDps2 and its variants were incubated with various types of DNA and visualized by negative staining TEM to observe the interaction of protein and DNA. All the samples were prepared by conventional negative staining methods (Kar et al ., 2022). A carbon-coated copper grid (EM grid, 300 mesh; TedPella) was glow-discharged (GloQube glow discharge system, Quorum) for 30 s at 20 mA.…”

Heterologous Prime-Boost with Immunologically Orthogonal Protein Nanoparticles for Peptide Immunofocusing

“…20 The former particle was administered to mice with Freund’s adjuvant and the latter with the Sigma adjuvant system. Encapsulins from Myxococcus xanthus were recently engineered to display receptor binding domains form SARS-CoV-2 virus 24 or influenza hemagglutinin, 23 and were tested immunologically with a squalene-in-water emulsion adjuvant. Lastly, only one report has appeared of an encapsulin-peptide conjugate vaccine, in this case using the prototypical Ova2 peptide (SIINFEKL) antigen attached by chemical means to T. maritima encapsulins and administered with poly (I:C) adjuvant.…”