The establishment of surrogates and correlates of protection: Useful tools for the licensure of effective influenza vaccines?

Figures & data

Table 1. Characteristics of Currently Licensed Influenza Vaccines.

Vaccine Type	Origin	Characteristics and Uses
Live Attenuated (FluMist™)	Embryonated hens' eggs	Quadrivalent (QIV), cold-adapted viruses administered intra-nasally (IN). Available for children/adolescents 2-17 year olds (yo)
Inactivated split-virion (egg-based) (examples: Agrippal™, Fluzone™, Fluvirin™, Fluarix™, FluLaval™, Influvac™)	Embryonated hens' eggs	Trivalent (TIV) or QIV formulations. Viruses chemically-inactivated and detergent split. Administered intramuscularly (IM) or intra-dermally (ID). Varies with the product: available for all ages >6 months (ages 18-64yo for the ID formulation) at 15μg/strain dose. A 60μg/strain high-dose targeted for those ≥65yo (TIV only)
Inactivated split-virion and sub-unit (tissue culture-based) (examples: Flucelvax™, Preflucel™)	Madin Darby Kidney (MDCK) or Vero cells	TIV or QIV formulations. Viruses inactivated and detergent split as above. Partial purification of HA & NA proteins in subunit vaccines. 15μg/strain dose IM. Available for all ages >6 months (and 4-64yo for subunit).
Inactivated split-virion + Adjuvant (FluAd™)	Embryonated hens' eggs	TIV formulation. Viruses inactivated and detergent split as above. MF59 added as an adjuvant. 15μg/strain standard dose IM.
		Targeted for those ≥65yo
Inactivated split-virion + virosomes (Inflexal V™)	Embryonated hens' eggs	TIV inactivated and detergent split as above formulated as ‘virosomes’ with. 15μg/strain standard dose IM. Available for those ≥4yo
Recombinant hemaggulitinin	Baculovirus transfected insect cells (Spodoptera frugiperda)	QIV formulation with recombinant HA proteins only. 45μg/strain dose IM. Available for those ≥18yo
(HA) protein (FluBlok™)

Table 1 illustrates the general types of influenza vaccines licensed in different jurisdictions around the world and their most important characteristics but is not meant to be exhaustive. Not all vaccines are available in all jurisdictions. Some are available as trivalent formulations (TIV: 2xA and 1xB viruses/antigens) while others are available as quadrivalents (QIV: 2xA and 2xB viruses/antigens).

Table 2. Serologic Assays Used to Assess Influenza Vaccine Responses.

Assay	Biologic Reagents Used	Read-Out
Hemagglutination Inhibition Assay (HI)	Source of antigen	Subjective based on degree and timing of RBC agglutination in 96-well plate
	– Either live or whole inactivated virus grown in embryonated hens’ eggs or in tissue culture
	– Detergent-split antigens from virus grown in embryonated hens’ eggs or in tissue culture
	– ≥1 recombinant proteins generated in different expression systems
	– Virus-like particles bearing ≥1 viral protein generated by different platforms
	Red blood cells (RBC) from different species:
	– chicken or turkey
	– horse
	– guinea pig
	– human
	– other
	Receptor-destroying enzyme (RDE) derived from Vibrio cholerae culture supernatant
Microneutralization Assay (MN)	Live virus	Readouts vary in degree of objectivity (ie: visual inspection of plaques, immunofluorescence, etc)
	– grown in embryonated hens’ eggs
	– grown in tissue culture
	Mammalian cell lines
	– MDCK-II (ATCC CCL-34); MDCK-I; serum free MDCK; MDCK clone CB4; MDCK-Siat cells; LLC-MK2; and HepG2 cells [Meijer 2006]
Single Radial Hemolysis Assay (SRH)	RBC from different species:	Semi-objective: area of hemolysis typically read by eye using light-box and calipers (note: hemolysis not always symmetrical or clear-cut)
	– chicken or turkey
	– horse
	– guinea pig
	– other
	Source of Complement
	– typically rabbit
	– other species
	Agarose derived from seaweed
Enzyme-linked Immunosorbent Assays (ELISA) for IgG, IgG subtypes, IgA, etc.	Source of antigen	Objective: optical density (OD) or immunofluorescence read by machine
	(as per HI assay above)

Table 3. Virus Reagents for HI assay (CP-Q14VLP-009 and CP-Q14VLP-010) in Clinical Trials.^*

Selected Virus Reagents	Amino acid mutation compared to VLP sequence	Changes in N-glycosylation at RBD
A/California/7/2009 (H1N1)-like A/Brisbane/10/2010	6	None
(cell derived)
NIBSC No. 11/134
A/Switzerland/9715293/2013 (H3N2)-like A/South Australia/55/2014	1	None
(cell derived)
NIBSC No. 15/104
B/Brisbane/60/2008 (NYMCBX-35)	1	Loss of glycoyslation site
(egg-derived)
NIBSC No. 10/106
B/Phuket/3073/2013-like	None	∼ 50 % proportion of aglycosylated amino acid when evaluated by MS
B/Utah/9/2014
(cell-derived)
NIBSC No. 11/134

Table 3 illustrates the differences in HA proteins in the context of the available virus reagents selected for the HAI assay in Phase II studies that included comparator vaccines (Note: in those studies, the strains covered by the VLP vaccine were those recommended by WHO for the 2015-2016 Influenza season).

* NCTs 02768805 and 02831751

Figure 1. Post-hoc analysis of reagent impact on HAI results in phase II study of a plant-made virus-like particle (VLP) vaccine in elderly subjects ≥65 years of age. Subjects received a single intramuscular dose of a quadrivalent VLP vaccine (QIV-VLP) containing 30mg of each hemagglutinin (HA) used in the 2015-16 seasonal vaccine, a standard dose quadrivalent inactivated vaccine (SD-IIV) containing 30mg of each HA or a high-dose trivalent IIV (HD-IIV) containing 60mg of each HA. Geometric mean titres (GMT) of serum hemagglutination Inhibition (HI) titres were measured in samples from 50 subjects in each arm of the trial using either VLPs (wild-type HA sequence) or reference (egg-derived) reagents in the assay.