Correlating physicochemical and biological properties to define critical quality attributes of a rAAV vaccine candidate

Abstract

Recombinant adeno-associated viruses (rAAVs) are a preferred vector system in clinical gene transfer. A fundamental challenge to formulate and deliver rAAVs as stable and efficacious vaccines is to elucidate interrelationships between the vector's physicochemical properties and biological potency. To this end, we evaluated an rAAV-based coronavirus disease 2019 (COVID-19) vaccine candidate that encodes the Spike antigen (AC3) and is produced by a commercially viable process. First, state-of-the-art analytical techniques were employed to determine key structural attributes of AC3, including primary and higher-order structures, particle size, empty/full capsid ratios, aggregates, and multi-step thermal degradation pathway analysis. Next, several quantitative potency measures for AC3 were implemented, and data were correlated with the physicochemical analyses on thermally stressed and control samples. Results demonstrate links between decreasing AC3 physical stability profiles, in vitro transduction efficiency in a cell-based assay, and, importantly, in vivo immunogenicity in a mouse model. These findings are discussed in the general context of future development of rAAV-based vaccine candidates as well as specifically for the rAAV vaccine application under study.

Keywords: COVID-19; adeno-associated virus; analytical characterization; biological properties; formulation; immunogenicity; physicochemical properties; stability; transduction efficiency; vaccine.

Graphical abstract

Figure 1

Primary structure and PTM analysis of the AC3 capsid proteins VP1, VP2, and VP3 (A and B) MW and relative abundance of VP1, VP2, and VP3 as measured by (A) non-reducing and reducing SDS-PAGE or (B) non-reducing and reducing CE-SDS. (C and D) Intact LC-MS analysis of AC3 capsid proteins, including (C) reverse phase ultra high performance liquid chromatography (RP-UHPLC) of the non-reduced AC3 construct and (D) intact mass analysis of the major VP species comprising RP-UHPLC peaks A and B. Minor species near the VP1 or VP3 proteins were enlarged for easier visualization. (E and F) LC-MS peptide mapping analysis of the AC3 vector, including (E) representative base-peak ion chromatograms of chymotrypsin-digested (red trace) or trypsin-digested (black trace) and (F) trypsin and chymotrypsin peptide map coverage and identified post-transitional modifications. The numbers reported in panels A, B and F are the range or average ± 1SD of 3-6 samples.

Figure 2

Characterization of the size, morphology, empty-full ratios, and genome copies of the AC3 vector (A) Hydrodynamic diameter measured by DLS and fit using cumulant intensity and multimodal intensity analysis. (B) Representative TEM image showing size, morphology, and percentage of full capsid species. (C) SV-AUC profile displaying various AC3 species based on sedimentation coefficient ranges. (D) Total genome copy numbers of non-DNase-treated and DNase-treated AC3, measured using a genome titration assay. All samples were analyzed at n ≥ 3, and error bars represent 1 SD.

Figure 3

Effect of thermal stress treatment of the AC3 capsid on vector particle size, morphology, and empty-full capsid ratio (A and B) Stressed samples were incubated for 10 min at 60°C and then compared with unstressed samples by (A) SV-AUC analysis, showing sedimentation coefficient distributions, and (B) SV-AUC total peak areas of each species from triplicate analyses (n = 3 ± 1 SD). (C and D) TEM analysis showing representative unstressed (C) or thermally stressed (D) samples. A total of 250 particles per AC3 sample were used to calculate the particle size and relative abundance of full capsids.

Figure 4

Mechanistic studies on thermal degradation of the AC3 capsid as measured by a combination of fluorescence spectroscopy and light scattering studies (A–E) As the temperature of the AC3-containing solution was ramped up to 90°C, vector degradation was monitored by a combination of (A) intrinsic fluorescence, (B and C) extrinsic fluorescence in the presence of SYPRO Orange and SYBR Gold dyes, (D) DLS, and (E) SLS. Readouts from the analytical methods are grouped by temperature ranges showing “small” (35–60°C), “medium“ (55–65°C), and “major” (64–70°C) structural transitions of the AC3 vector. (F) Cartoon representation of different AC3 species in the purified sample (full, partially full, and empty capsids) and associated structural alteration events in the temperature ranges for the “small,” “medium,” and “major” transitions.

Figure 5

In vitro transduction efficiency assay design and infectious titer results with AC3 samples before and after thermal stress (A and B) Assay design and (A) linear range of the assay (B; Ct vs. Log multiplicity of particles [MOP]). (C) The relative gene expression of the AC3 vector before (unstressed) or after heat treatment (10–120 min at 60°C or 60 min at 70°C). All AC3 samples were analyzed in quadruplicate, and error bars represent 1 SD. An asterisk denotes that the relative gene expression was below the limit of quantification. Ct, threshold cycle; D, splice donor; A, splice acceptor; S1-AC3 coSpike1.

Figure 6

Analysis of unstressed and heat-stressed AC3 vector samples and correlation of results with infectious titers as measured by the in vitro transduction efficiency assay (A) AC3 genome copy numbers as measured by genome titration assay. (B) Nucleic acid accessibility of AC3, evaluated by SYBR Gold fluorescence assay. (C) Mean particle diameter obtained by DLS multimodal intensity analysis. (D–F) Correlation of results with measured in vitro transduction efficiency titers of the same samples for (D) genome copy numbers, (E) SYBR Gold fluorescence intensity at 516 nm, and (F) mean particle diameter. Thermally stressed samples were incubated from 10–120 min at 60°C or 60 min at 70°C. All AC3 samples were analyzed at n ≥ 3, and error bars represent 1 SD. An asterisk denotes that the gc/mL number was below the limit of quantification.

Figure 7

Relative genome copies and gene expression levels for unstressed vs. heat-stressed AC3 samples from temperature ramping studies (A) Relative genome copy numbers for heat-stressed AC3 samples (vs. unstressed AC3 at 4°C), as measured by genome copy assay. (B) relative gene expression levels for heat-stressed AC3 samples (vs. unstressed AC3 at 4°C), as measured by in vitro transduction efficiency assay. Samples were subjected to thermal ramping conditions identical to the SYBR Gold fluorescence assay results described in Figure 4 (shown again here in C for ease of comparison) but with no dye added, with samples taken when the temperature reached 40, 50, 60, and 70°C. All samples were measured in quadruplicate, and error bars represent 1 SD. An asterisk denotes that the gc/mL number was below the limit of quantification.

Figure 8

Results of in vivo mouse immunogenicity studies for unstressed vs. heat-stressed AC3 samples (A) Relative gene expression levels of heat-stressed AC3 samples (vs. the unstressed control) that were subsequently dosed in mouse studies, as measured by in vitro transduction efficiency. Data are represented as mean ± SD of 3 independent assay replicates. (B) In vivo mouse immunogenicity results of the AC3 samples (described in A), measured as total antibody titers (vs. RBD) in C57BL/6J mice (n ≥ 5) vaccinated with 10E+11 gc of AC3 28 days after vaccination. (C) Correlation of in vitro transduction efficiency with in vivo potency results shown in (A) and (B), respectively. (D) Longitudinal antibody responses in mice (n ≥ 5) treated with 10E+11 gc of AC3 before and after thermal stress. Data are represented as geometric mean ± SD.