Structural Stability and Antigenicity of Universal Equine H3N8 Hemagglutinin Trimer upon Release from Polyanhydride Nanoparticles and Pentablock Copolymer Hydrogels

Seasonal influenza A virus infections present substantial costs to both health and economic resources each year. Current seasonal influenza vaccines provide suboptimal protection and require annual reformulation to match circulating strains. In this work, a recombinant equine H3N8 hemagglutinin trimer (rH33) known to generate cross-protective antibodies and protect animals against sublethal, heterologous virus challenge was used as a candidate vaccine antigen. Nanoadjuvants such as polyanhydride nanoparticles and pentablock copolymer hydrogels have been shown to be effective adjuvants, inducing both rapid and long-lived protective immunity against influenza A virus. In this work, polyanhydride nanoparticles and pentablock copolymer hydrogels were used to provide sustained release of the novel rH33 while also facilitating the retention of its structure and antigenicity. These studies lay the groundwork for the development of a novel universal influenza A virus nanovaccine by combining the equine H3N8 rH33 and polymeric nanoadjuvant platforms

Histopathological analysis of lungs, spleens, and livers 6 weeks post-vaccination at 72 h post-challenge from mice vaccinated six weeks earlier.

<p>Lungs of S₄₀ + E₁₀ vaccinated mice (v) were free of any histopathological lesions and bacteria and were similar to lung tissue from unimmunized and uninfected control mice (i). No bacteria, necrosis or edema were present in spleens of the S₄₀ + E₁₀ vaccinated mice (x) and histology was similar to healthy spleen tissue (vi). The livers of the S₄₀ + E₁₀ vaccinated mice (xv) did not show any lesions similar to control mice (xi). Av - Alveoli, F - Follicle, Mz - Marginal zone, and RP - Red pulp. Arrow - bacteria, black arrowhead - neutrophilic infiltration, yellow arrowhead - lymphocytic infiltrations, * – edema, and + - necrotic cells. Objective lens magnification is 100X. Scale bar  = 20 µm.</p

Single-dose, intranasally administered nanovaccines induced protection against lethal <i>Y. pestis</i> challenge.

<p>C57BL/6 mice were intranasally challenged with 850 CFU (LD₁₀₀) <i>Y. pestis</i> CO92 at (<b>A</b>) 6 weeks post-vaccination (n = 5 per group) or (<b>B</b>) 23 weeks post-vaccination (n = 7 per group) with each challenge a representation of two independent experiments. *  =  p<0.007, #  =  p<0.001 and +  =  p<0.0001. (<b>C</b>) CFU of <i>Y. pestis</i> CO92 at 72 h post-infection in the lungs, livers, and spleens of mice (n = 3 per group) that were vaccinated 6 weeks prior to challenge. Treatments with different letters are significantly different from one another at p<0.05. (<b>D</b>) Photomicrographs of lung sections from mice: uninfected and unvaccinated (i) and challenged 6 (ii, iii, and iv) and 23 (v) weeks post-vaccination. S₅₀ vaccinated mice, 72 h post-challenge (ii) showed severe pathology and loss of tissue architecture due to overwhelming bacterial replication in lungs (arrows), neutrophilic infiltration (arrowhead), hemorrhage (+), edema (asterisks), and necrosis. Bronchioles had bacteria clumped with fibrin deposits and neutrophils. Absence of lung pathology was seen in S₄₀ + E₁₀ vaccinated mice at 72 h (iii), 14 days (iv), and 21 days post-challenge (v). Av - alveolus, Br - bronchiole, Pa - pulmonary artery, Pv - pulmonary vein. Objective lens magnification is 40X. Scale bar  = 50 µm.</p

Vaccination regimens.

<p>*Quantities indicate the amounts of immunogen or adjuvant delivered to each mouse in the indicated group. S  =  soluble protein; E  =  encapsulated protein. Subscripts indicate amount of soluble or encapsulated protein (in µg) administered per dose.</p

Single-dose administration of nanovaccines induced long-term antibody titers with high avidity.

<p>(<b>A</b>) Kinetics of IgG antibody titer throughout 23 weeks post-vaccination. (<b>B</b>) Antibody isotype induced by various immunization regimens. Optical density was determined by ELISA at a 1∶1000 dilution. (<b>C</b>) IgG antibody avidity throughout 23 weeks post-vaccination. Avidity was determined via ELISA at a 1∶200 dilution. Data is presented as the mean ± SEM (n = 7 per group) and is representative of two independent experiments. <b>*</b>  =  p<0.02, <b>#</b>  =  p<0.005 and <b>+</b>  =  p<0.0001 (compared to S₅₀ + MPLA).</p

Material properties of 50∶50 CPTEG:CPH nanoparticles.

<p>Representative scanning electron photomicrographs of (<b>A</b>) blank and (<b>B</b>) 2% F1-V loaded 50∶50 CPTEG:CPH nanoparticles (scale bar  =  1 µm). (<b>C</b>) Particle size distribution as determined by QELS for blank (204±62) and 2% F1-V loaded 50∶50 CPTEG:CPH nanoparticles (196±77) with n  =  3. (<b>D</b>) <i>In vitro</i> cumulative release of F1-V from 50∶50 CPTEG:CPH nanoparticles in pH 7.4 PBS analyzed by micro bicinchoninic acid assay (n  =  2, representative of two separate nanoparticle batches).</p

<i>E</i>. <i>coli</i> LF82_N mice develop severe, transmural colitis following DSS-induced inflammation.

Mice were scored at the time of necropsy for macroscopic parameters of disease including colon length (A) and macroscopic colitis score (as described in Materials and Methods) (B). Additionally, proximal colon was blindly scored for histopathologic parameters of inflammation by a pathologist (JH) (C). Representative photomicrographs of histopathological sections imaged at 40X (scale bar = 100 μm) (D). Shared letters between different groups indicate no statistical significance with an ANOVA or Kruskal-Wallis with multiple group comparisons test. Asterisk indicate statistical significance based on a direct comparison with a Mann-Whitney two-tailed test, *p ≤ 0.05, **p ≤ 0.01. p for colon length data indicates significance based on a linear regression model for 1all groups and between the 2DSS treated groups. A and B: Control n = 17, DSS n = 21, LF82A n = 21, LF82A + DSS n = 28, LF82N n = 24, LF82N + DSS n = 32; C: Control n = 13, DSS n = 12, LF82A n = 16, LF82A + DSS n = 24, LF82N n = 19, LF82N + DSS n = 29.</p

RNAscope^® analysis with HALO^® image analysis software.

RNAscope® analysis with HALO® image analysis software.</p

Evaluation of spatial distribution of the microbial community following <i>E</i>. <i>coli</i> LF82 colonization and DSS-induced inflammation.

Proximal colons with intact contents were assessed for overall microbial spatial distribution by fluorescent in situ hybridization (FISH). Spatial distribution of organisms was evaluated using a eubacteria probe (EUB338) to detect bacteria in the lumen, adherent/attaching mucus layer, and translocation into the glands (A). Representative photomicrographs (60X magnification) of E. coli LF82 detected in the microbial community by FISH by labeling the nuclei of mucosal cells with DAPI (blue), total bacteria with a EUB338 probe (FITC, green), and labeling with a E. coli-specific probe (Cy-3, orange) (B). Control n = 5, LF82A + DSS n = 8, LF82N + DSS n = 8.</p

LF82_NH LF82 mice have intermediate colitis scores compared to LF82_N and LF82_A following DSS-induced inflammation.

Mice were horizontally infected as neonates (LF82NH) to assess maternal influence on the effects observed following colonization with E. coli LF82. Mice were evaluated at the time of necropsy for parameters of disease including colon length (A) and macroscopic colitis score (B). Additionally, proximal colon was fixed, sectioned and stained with hematoxylin and eosin and blindly scored by a pathologist for histopathologic parameters of inflammation (C). The cecal load of E. coli LF82 was enumerated by bacteriological culture (D) and 16s rRNA gene amplicon sequencing was performed on DNA recovered from fecal samples to assess relative microbial abundance (E). To mimic the length of time that the neonates were colonized with E. coli LF82, a separate set of adult mice evaluated at 10 weeks post-colonization (10 wk LF82A). Mice were scored at the time of necropsy for parameters of disease including colon length (F) and macroscopic colitis score (G). Shared letters between different groups indicate no statistical significance with an ANOVA or Kruskal-Wallis with multiple group comparisons test; ns indicates no statistical significance based on a direct comparison with a t or Mann-Whitney test. A and B: Control n = 17, DSS n = 21, LF82A n = 21, LF82A + DSS n = 28, LF82NH n = 15, LF82NH + DSS n = 18, LF82N n = 24, LF82N + DSS n = 32; C: Control n = 13, DSS n = 12, LF82A n = 16, LF82A + DSS n = 24, LF82NH n = 5, LF82NH + DSS n = 5, LF82N n = 19, LF82N + DSS n = 29; D and E: LF82NH n = 6, LF82NH + DSS n = 8; A and B: Control n = 17, DSS n = 21, LF82A n = 21, LF82A + DSS n = 28, 10 wk LF82A n = 4, 10 wk LF82A + DSS n = 5, LF82N n = 24, LF82N + DSS n = 32. (TIFF)</p