The development of a recombinant hepatitis E vaccine HEV 239

Figures & data

Table 1. Characteristics of the 2 existing hepatitis E vaccines that have been evaluated in clinical trials

	rHEV	HEV 239 (Hecolin®)
Manufacturer	GlaxoSmithKine, Belgium	Innovax, China
Origin	HEV genotype 1	HEV genotype 1
Expression system	Baculavirus	E. coli
Gene location	ORF2 aa 112–607	ORF2 aa 368–606
Antigen content	20 μg	30 μg
Adjuvant	Aluminum hydroxide	Aluminum hydroxide
Immunization schedule	0, 1, and 6 month	0, 1, and 6 month
Sample size of the efficacy trial	2 000	112 604
Participants enrolled	Mostly male (99.6%), aged from 18–62 (mean age 25.2)	Female and male, aged from 16–65 (mean age 44.1)
Efficacy against hepatitis E in the PPS cohort	96% (95% CI, 86–99)	100% (95% CI, 72–100)
Efficacy against hepatitis E in the ITT cohort	89% (95% CI, 77–94)	96% (95% CI, 66–99)
Development stage	Phase II clinical trial finished	Licensed in China
Advantages and disadvantages	Efficacy in women and in some special population remains to be determined	Efficacy in some special population remains to be determined

PPS: per-protocol set; ITT: intention to treat.

Figure 1. Structural interpretation for HEV 239 vaccine. The structure of HEV ORF2 aa112–606 is rendering in surface mode (P2 domain) and cartoon mode (P1 and S domain), colored by key regions for several HEV historic constructs (left panel), i.e., aa112–367 in cyan, aa368–393 in green, aa394–458 in yellow and aa459–606 in orange. To elucidate the spatial configuration, 3 crystal structures were in superimposition to demonstrate major neutralization sites ( Neutralizing mAb 8C11 binding sites colored in red, by PDB (protein database) no. 3RKD), outmost protrusion domain (E2s domain in orange surface, by PDB no. 3GGQ) and P1&S domains (contributing for virus capsid assembly, by PDB no. 3HAG). The figure was prepared by the program PyMol.

Table 2. Analytical toolbox for the comprehensive characterization of a recombinant HEV 239 antigen to demonstrate the process reproducibility and product consistency during Hecolin® production.^20,26,27

Method
Biochemical methods	Characteristics	Parameters of HEV 239 or Notes (from multiple lots, n = 6 )
SDS-PAGE	Purity, integrity, molecular weight (kDa)	29.32 ± 0 .08
MALDI-TOF MS	Molecular weight (Da)	25,561.64 ± 1 .37
LC-MS	Peptide mapping (coverage, %)	100 (3 lots)
icIEF	pI, whole column detection capillary isoelectric focusing	6.55 ± 0 .03
Biophysical methods
HPSEC	Size (as reflected by retention time, min)^b	14.02 ± 0 .08
TEM	Size, morphology	20–30 nm particles with certain degree of irregularity
DLS	Hydrodynamic diameter (nm)	28.92 ± 0 .20
AUC	Sedimentation coefficient (S)	21.42 ± 0 .13
DSC	Thermal stability (℃)	75.58 ± 0 .10
Cloud point	Aggregation propensity (℃)	71.83 ± 0 .16
CD	Secondary structure	The secondary structure, including α-helices and β-sheets, is
UV	Trp/Tyr absorption	consistent with the UV spectrum of a globular protein with a maximum absorption wavelength at 280 nm
Immunochemical methods
SPR With mAbs	affinity to mAbs	The binding activity to 5 mAbs for different batches of aqueous products with the RU(Ag)/ RU(Ab)
Sandwich ELISA^d	Antigenicity	8C11 (capture) and 8H3 (detection) to analyze the consistency of the antigenicity among different batches of HEV 239 aqueous products
Immunological assessment
Mouse potency	Immunogenicity ED50 (μg)	0.025–0.060

Note: 1. For product lot release of prefilled syringe, mouse potency assay is used for lot release (spec < 1.0 μg). Lot release of HEV 239 purity (spec > 95.0%) and molecular weight (spec 27.5 ± 2.6 kDa) by SDS-PAGE was performed on aqueous product batches.

Two. The reported values were calculated from the results on 6 manufacturing lots. These values are representative on process performance since similar values were reported for sizing using DLS and antigenicity test using the results from over 20 lots.²⁰