There is evidence to suggest that the barrier to human transmissibility of some of these avian viruses is not very high, and therefore emergence in humans is possible, with most, if not all, of the population immunologically naïve. The humoral immune response to influenza is determined by the imprinting of the antibody response to immunodominant epitopes.
Introduction Part A: Influenza
WHO, Avian Influenza Weekly Update Number 7
Wang, Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Zhou, Structural basis for recognition of SARS-CoV-2 by full-length human ACE2. Barclay, The furin cleavage site in the SARS-CoV-2 spike protein is required for transmission in ferrets.
Sets, targets of T-cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals. Wilson, A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV.
Construction, characterization, and immunization of nanoparticles that display a diverse array of influenza HA trimers
115 This chapter describes the design and fabrication of mosaic nanoparticles co-displaying do.
115 This Chapter describes the design and production of mosaic nanoparticles that co-display up to
Abstract
Introduction
118 host membranes. Influenza HA is a trimer of HA1 and HA2 heterodimers, which can be subdivided
119 sequences were fused to an engineered ferritin subunit to create self-assembling particles display-
120 Catcher protein scaffolds are highly versatile, coming in different forms that range from a bacterio-
Materials and methods
The lysate was spun down at 21,000 xg for 30 min, filtered with a 0.2 µm filter, and VLPs were isolated by Ni-NTA chromatography using a prepacked HisTrapTM HP column (GE Healthcare). 123 µm filter and the NPs were isolated by Ni-NTA chromatography using a prepacked HisTrapTM HP col-µm filter and the NPs were isolated by Ni-NTA chromatography using a prepacked HisTrapTM HP column (GE Healthcare) and eluted with 2.0 M imidazole, 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% NaN3. Purified SpyCatcher-VLPs or SpyCatcher-NPs were incubated with a 1.2-fold molar excess (HA protomer to VLP or NP subunit) of SpyTagged purified HA (either single HA to make homotypic particles or an equimolar mixture of two or more HAs to create mosaic particles) at room temperature in TBS (25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% NaN3) overnight.
Conjugated VLPs or NPs were then separated from free HA trimers by SEC on a Superose 6 10/300 (GE Healthcare) column equilibrated with PBS (20 mM sodium phosphate pH 7.5, 150 mM NaCl). SEC-purified HA-VLPs and HA-NPs were prepared on grids for cryo-ET using a Mark IV Vitrobot (ThermoFisher Scientific) operated at 21°C and 100% humidity.
127 Flow cytometry. Single cell suspensions were prepared from immunized mouse spleens by me-
Statistical differences of antigen-specific B-cell populations between groups were calculated using Tukey's multiple comparison test via Graphpad Prism 8.3. Correlation between percentage of antigen-specific B cells and ELISA AUC titers was calculated using the Pearson correlation function on Graphpad Prism 8.3.
Results and discussion
129 from the supernatants of transiently-transfected mammalian cells were verified to form monodis-
130 Because VLPs conjugated with more than two different HAs tended to precipitate out of solution,
131 nm (HA-NPs) and revealed densities for individual HA trimers on VLPs and NPs. The trimers were
Thus, with regard to IgG-induced binding of HAs, it appeared that immunization with mosaic-2 VLPs containing group 1 and group 2 HAs was no better at inducing cross-reactive binding of HA- ve from divergent strains than the corresponding mixture. However, mosaic-2 and admix-2 injections induced heterologous breadth that could not be explained by overlapping immunogenicity of homotypic VLPs. For mice immunized with mosaic-2 and admix-2, neutralizing titers against homotypic infectious virus strains (CA09 and Aichi) were consistent with ELISA titers against these strains.
Against the Sh13 pseudovirus, neutralizing titers for mice immunized with mosaic-2 were higher than for the other groups, although the spread of potency was broad and overlapped with the other groups. When neutralization and ELISA results are considered together, mosaic-2 VLPs did not induce greater width than the corresponding mixture of homotypic VLPs.
133 purified HAs from homotypic and heterotypic group1 and group 2 strains (Fig 5B) and against un-
134 ELISAs were also used to evaluate recognition of CA09-miniHA, a stabilized stem-only construct
135 HAs (TX91 and WI05 strains), serum from all animals was non-neutralizing, suggesting that neu-
Interestingly, very few CA09+/Viet04+ B cells were induced in all immunized animals, except for one animal in the mosaic-4 immunized group, which also displayed a high serum IgG and neutralizing response. CA09-NP and admix-8 immunized mice also induced double-positive B cells, albeit to a lesser extent. Double-positive CA09+/Aichi+ and Viet04+/Aichi+ B cells were not detected for any of the animals (data not shown).
Interestingly, the percentage of CA09+Viet04+ double-positive B cells correlated with serum titers for Pe10-HA (Fig. S2D), a strain not represented on either particle. This suggests that animals that induced CA09+Viet04+ B cells also had cross-reactive serum antibodies.
137 Another animal experiment was conducted to compare mosaic VLPs and mosaic NPs with their
138 Somewhat surprisingly, mice immunized with either CA09-NP or CA09-VLP, which only presented
There were no statistically significant differences in the percentage of antigen-specific B cells between mosaic-4 and mosaic-8 NPs and VLPs. However, as a general trend, the higher the mosaic valence, the lower the percentage of stem-specific B cells induced, especially in the case of Viet04+ and Aichi+ B cells (Fig. 8A). The difference in the percentage of CA09+/Viet04+ B cells between mosaic, admix, and homotypic VLPs/NPs was therefore not significant.
As previously observed, the percentage of CA09+/Viet04+ B cells correlated with day 28 serum ELISA titers against Pe10, a mismatched strain not represented on any of the VLPs or NPs (S5E Fig, p=0.0234 ). Because both populations were rare, there was no significant difference in the percentage of double-positive B cells between each group of immunized mice.
Conclusions
Interestingly, mice immunized with CA09-NP and CA09-VLP induced antigen-specific B cells that were specific for Viet04, Aichi, and Sh13 ( Fig 8A ). This may explain why cross-reactive ELISA titers were observed for these animals against HAs from every strain tested. Similar to the results shown in Fig 6B, the induction of cross-reactive CA09+/Viet04+ B cells was rare (Fig 8B).
Interestingly, both CA09-NP and Ca09-VLPs were able to induce CA09+/Viet04+ B cells, suggesting that immunization with monovalent CA09-VLPs/NPs was sufficient to induce cross-reactive B- cells. Induction of Ca09 + /Aichi + B cells was observed, but infrequently, making it unclear whether they represented B cells cross-reactive with group 1 and group 2 HAs ( Fig. 8B ).
140 Attempts to develop broadly protective influenza vaccines have been challenging partly due to the
141 mixtures of homotypic particles. The finding that mosaic particles conjugated with monomeric HA
142 cles prior to immunization. Thus mosaic particles presenting HA antigens derived from multiple in-
Acknowledgements
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145 26.Hagen WJH, Wan W, Briggs JAG (2017) Implementation of a cryo-electron tomography tilt-scheme
Bottom: Surface representation of an HA trimer structure (PDB 3VUN), schematic of a SpyTagged HA and list of influenza strains from which SpyTagged HAs were derived. Bottom: EM structure of T=3 AP205 particle (PDB 5FS3) [23] with the locations of SpyCatcher fusion sites indicated by red dots (left) and schematic SpyCatcher VLP (right). Right: Reducing and non-reducing SDS-PAGE of two fractions corresponding to the VLP fractions in red on the SEC lane.
147 lated to mi3 [24] with the locations of SpyCatcher fusion sites indicated by red dots (left) and sche-
Each dot represents serum from one animal, with arithmetic means and standard deviations represented by rectangles and horizontal lines, respectively. Each dot represents serum from one animal, with geometric mean and geometric standard deviations represented by rectangles and horizontal lines, respectively. Each dot represents serum from one animal, with means and standard deviations represented by rectangles and horizontal lines, respectively.
Each dot represents serum from one animal, with geometric means and geometric standard deviations represented by rectangles and horizontal lines, respectively. Differences without significance are not shown, and significant differences between HA-NPs and mi3-NPs are also not shown.
Supporting Information
Pearson correlation of Day45 CA09+Viet04+ B-cell population to serum anti-Pe10 ELISA AUC titers. Serum antibody response to HA shown by ELISA binding as area under the curve (AUC) of Day 14 serum to SpyCatcher NP and VLP particles, with means and standard deviations represented by rectangles and horizontal lines, respectively. Homotypic strains that were present on the mosaic NPs and heterotypic strains that were not are indicated by the blue and red rectangles, respectively, above the ELISA data.
164 Chapter 3: Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic corona-
165 This chapter describes the development of mosaic nanoparticles that co-display RBDs from eight
166 Abstract
167 Main Text
168 In one such approach, multiple copies of an engineered protein domain called SpyCatcher fused to
169 Kenyan bat clade 3 virus (42). Mapping of the sequence conservation across selected RBDs
Anti-SARS-2 S-trimer and anti-SARS-2 RBD serum responses were similar (Fig. 3B,C), demonstrating that antibodies raised against RBDs can access their epitopes on SARS-2 S-trimer. Neutralization and ELISA titers were significantly correlated (fig. S4), suggesting that ELISAs are predictive of neutralization results when pseudotyped neutralization assays were not possible due to unknown viral entry receptor usage. Mice immunized with soluble SARS-2 S-trimer (brown bars) showed no binding or neutralization except autologous responses against SARS-2 after boosting (Fig. 3C-F).
In contrast, sera from animals immunized with RBD-nanoparticles (red, green, yellow, and blue columns) showed binding to all RBDs (Fig. 3C.F; Fig. S3A) and neutralization against all four strains after boosting (Fig. 3C). E), consistent with the increased immunogenicity of multimerized antigen on nanoparticles compared to soluble antigen (27, 28). Homotypic SARS-2 nanoparticles, but not the soluble SARS-2 trimer, induced heterologous responses to zoonotic RBDs and neutralization of heterologous coronaviruses (Figure 3D–F).
171 a mosaic-nanoparticle that included SARS-2 RBD in terms of the magnitude of immune responses
172 nanoparticle does not diminish the immune response against a particular RBD, also suggested by
173 We investigated the potential for cross-reactive recognition using flow cytometry to ask whether B-
174 2 RBD along with diverse RBDs on mosaic nanoparticles showed no disadvantages for eliciting
Kreer et al., Longitudinal isolation of potent near-lineage SARS-CoV-2-neutralizing antibodies from COVID-19 patients. Rogers et al., Rapid isolation of potent SARS-CoV-2 neutralizing antibodies and protection in a small animal model. Seydoux et al., Analysis of an individual infected with SARS-CoV-2 reveals the development of potent neutralizing antibodies with limited somatic mutation.
Walls et al., Eliciting potent neutralizing antibody responses with engineered protein nanoparticle vaccines for SARS-CoV-2. Crawford et al., Protocol and reagents for pseudotyping SARS-CoV-2 spike protein lentiviral particles for neutralization assays.
178 Vielmetter and Caltech’s Beckman Institute Protein Expression Center for protein production, An-
Epitopes for representatives of defined classes of RBD-binding antibodies (class 1-class 4) (24), indicated by dotted lines. ELISA data of serum IgG responses to SARS-2 spike trimer (B) or RBDs (C-F), shown as area under the curve (AUC). Significant differences between groups connected by horizontal lines are indicated with asterisks and p-values.
186 Fig-
188 Supplementary Materials for
189 Materials and Methods
