Fast vaccine design and development based on correlates of protection (COPs): Influenza as a trendsetter

Figures & data

Figure 1. Nomenclature and localization of the major external and internal protein antigens of the influenza virus.

Table 1. Approved seasonal and (pre)pandemic influenza vaccines

Vaccine purpose strain	Vaccine type	Adjuvant	Route	Trade names^1 (manufacturer)
Seasonal annual strains^2	IIV whole virus	AlPO4	im	Fluval AB (Omninvest)
	IIV split	none none none none none none none none	im im im im im im id im	FluLaval^3 (ID Biomedical) Fluarix, Fluviral (GlaxoSmithKline) Enzira^4 (Pfizer) Vaxigrip, Fluzone^3 (Sanofi Pasteur) Afluria^4 (Merck) GC FLU (Green Cross Corporation) Intanza, IdFlu, Fluzone Intradermal (Sanofi Pasteur) Fluvax^4 (bioCSL)
	IIV subunit	none none none MF59C.I none virosomal lipids	im im im im im im	Influvac, Imuvac, Fluvaccinol (Abbott) Agriflu, Agrippal, Fluvirin,^4 Optaflu^5(Novartis) Mutagrip (Sanofi Pasteur) Fluad (Novartis) FluCelvax^4,5 (Novartis) Inflexal® V^4 (Berna Biotech, Crucell)
	LAIV	none	in	Fluenz, Flumist seasonal^3 (MedImmune)
	Recombinant protein	none	im	Flublok®^6 (Protein Sciences)
Pandemic 2009 H1N1 A/Cal7/2009	IIV whole virus	none AlPO4	im im	Celvapan H1N1^5 (Baxter) Fluval P (Omninvest)
	IIV split	none none none AS03	im im im im	Panvax^4 (Merck) Panenza (Sanofi Pasteur) Green Flu-S (Green Cross Corporation) Pandemrix (GlaxoSmithKline)
	IIV subunit	none MF59C.1	im im	Fluvirin-H1N1^4 (Novartis) Celtura,^5 Focetria (Novartis)
	LAIV	none none	in in	Fluenz, Flumist pandemic (Medimmune) Nasovac (Serum Institute of India)
Pre-pandemic various avian strains	IIV whole virus	AlPO4 AlOH3/PO4 none	im im im	Fluval H5N1 (Omninvest) Daronrix (GlaxoSmithKline) Celvapan H5N1,^5 Vepace^5 (Baxter)
	IIV split	AS03	im	Prepandrix, Adjupanrix (GlaxoSmithKline)
	IIV subunit	MF59C.1	im	Foclivia, Aflunov (Novartis)

¹ Vaccines are derived from processed influenza virions propagated on embryonated chicken eggs and inactivated using formaldehyde unless otherwise indicated²; seasonal vaccines are mostly trivalent (2 A strains and 1 B strain) unless otherwise indicated³; available as quadrivalant vaccine (2 A strains and 2 B strains);⁴ β propiolactone virus inactivation;⁵ cell based virus propagation;⁶ high dose trivalent uncleaved HA (rHA0) based on baculovirus expression in insect cells.

Table 2. Novel influenza vaccine approaches in clinical development categorized according to the primary mode of protection intended to be induced, vaccine type, and antigen targets

Novel concepts: major mode of protection^1	Vaccine type (Ag)	Examples of concepts in clinical development	Ref.^2
More broadly reactive antibodies	Recombinant protein or synthetic polypeptide-based vaccines (HA, NP, M1)	Multimeric-001, purified recombinant protein (E. coli) containing 9 conserved epitopes from HA, NP, and M1, non-adjuvanted or ISA-51, i.m. (BiondVax)	140
	DNA vaccines (HA, NP, M2e, NA)	Monovalent (H5) or trivalent (H5-NP-M2) plasmid DNA, formulated with Vaxfectin® adjuvant, i.m. (Vical Inc.) VGX-3400,^3 NA, M2e, NP, and subtype specific HA regions, i.d. and i.m. electroporation (Inovio) HA expressing plasmids adjuvanted with DEI-LT (DNA encoded Immunostimulator-lanile Toxin) on gold particles, epidermal (PowderMed)	141 118, 30 142
	Particulate vaccines: virosomes, VLP,^4 BLP^5 (HA, NA, M1)	HA, NA, M1 VLP produced in insect cells by baculovirus infection, i.n. (NovaVax) Alhydrogel® formulated H5-VLP produced via the bacterial vector A. tumefaciens in tobacco leaves, i.m. (Medicago) Matrix M^TM adjuvanted H5N1 virosomes (Berna, Crucell) FluGEM®, IIV antigens mixed with self-adjuvanting BLP from inactivated L. lactis bacteria, i.n. (Mucosis)	30, 60 143 144 112 (6)
	Alpha virus-based vaccines (HA)	H3 expressed in propagation-defective, single-cycle RNA replicon α virus vector system producing virus-like replicon particles (VRP), i.m. (AlphaVax)	145
	Heterologous fusion constructs (HA cleavage site, NP, M2e)	Qbeta VLP, M2e fused to HB core particle, spontaneously packaging bacterial RNA carrying ligands for TLR7 and TLR3, i.n. (CYTOS Biotechnology) M2e and HAcleavage peptide-OMPC construct with alum or proprietary adjuvant, i.m. (Merck) M2e-Flagellin^7 fusion construct (VAX102) i.m. (VaxInnate) ACAM-FLU, recombinant M2e-HB core Ag fusion construct, non-adjuvanted, alum or QS21, i.m. (Acambis) N8295: M2e-NP fusion protein covalently linked to CpG, i.m. (Dynavax)	110 71,30 146,148 147, 30 72
Cross-reactive T cell immunity	Vector delivery of Ag MVA, adenovirus (HA, NP, M1)	MVA-NP-M1, replication defective MVA virus, i.m. or i.d. (Impfstoffwerk, Jenner Institute) Ad4-H5-Vtn, oral, also induce antibody responses (PaxVax) ChAdOx1 NP+M1, i.m, (Clinical Biomanufacturing Facility, University of Oxford)	91 95 96
	Peptide based: conserved epitopes (NP, M1, M2)	Flu-v, ISA-51 adjuvanted synthetic vaccine containing 6 conserved CTL epitopes (NP, M1, M2), s.c. (SEEK) Flunisyn™ (FP01), depot forming nanoparticles with 6 conserved T cell peptide epitopes fused to a fluorocarbon tail, i.m. (Immune Targeting Systems, ITS)	93 ITS website
Mucosal sIgA immunity	LAIV	LAIV NS1-deletion, HA altered cleavage, i.n., also induce T cell immunity (Green Hills)	30
	Mucosal recombinant viral vector delivery (HA)	Recombinant (replication defective) Ad5 delivery of HA, i.n. (Vaxin) Recombinant (replication defective) Ad5-TLR3 ds RNA adjuvanted delivery of H5, oral (Vaxart)	113 114
	Mucosal non-replicating vaccines (HA)	HA split antigens mixed with neisserial OMP (FluINsure^TM), i.n. (GSK-ID Biomedical)	111
	Mucosal delivery systems	Nasal, oral, sublingual, needle-free dermal administration, mucosal adjuvants	31

¹ In addition to the major mode of protection, several of the vaccine concepts also induce other types of immunity: all DNA vaccines, particulate vaccines, α virus vaccines, Multimeric-001, LAIV and viral vector delivery vaccines also induce T cell immunity; ²In addition to references, information is also taken from the websites of companies;³preclinical data for VGX-3400; ⁴VLP, viral like particle; ⁵BLP, bacterial like particle;⁶preclinical data for FLU-GEM®; ⁷Flagellin-platform is also used for potentiating the induction of subtype specific HI titers to H1 (VAX125, VAX128), H5, and H7.

Table 3. Established and emerging COPs for influenza vaccine development

COP assay immune parameter Ag specificity	Mode of protection for immune response	Examples of relevant vaccine concepts	Status of COP assay and comments
HI assay Serum IgG HA globular head	Preventing intracellular infection to be established: Inhibition of binding to cell surface receptor	WIV, split, subunit Virosomes Recombinant Ag DNA vaccines	Well established as COP (EMA, FDA)^12^,^13 Limit for protection defined (HI titer > 40) Variation with virus subtype and target age group Standardized but still large variability between labs.
HA ELISA Serum IgG HA stem region	Preventing intracellular infection to be established: Virus neutralization Inhibition of fusion with the endosomal membrane	Recombinant protein or polypeptide based Ag with relevant HA sequences	Experimentally demonstrated^47^-^49 More data needed Conserved among subtypes Limit for protection not defined Relevant for universal vaccines
NA ELISA and NAI assay Serum IgG NA	Preventing mature virus to be released after intracellular infection: Inhibition of NA enzyme activity	LAIV WIV, split, subunit, Virosomes	Experimentally established^25^,^42^,^43^,^56^,^57 Broadly cross-reactive non-neutralizing antibodies Limit for protection not defined
VN assay Serum IgG HA, NA	Neutralizing virus: Preventing intracellular infection to be established	LAIV WIV, split, subunit, Virosomes	Experimentally demonstrated^25^,^42^,^43 More data needed Limit for protection not defined
M2e ELISA Serum IgG M2e	Not virus neutralizing but reducing virus spread after intracellular infection (ADDC)	Recombinant fusion proteins: M2e sequence coupled to TLR ligands or in immunogenic particles	Demonstrated in animal models^73 More data needed Conserved among subtypes Limit for protection not defined Relevant for universal vaccines
IgA ELISA Mucosal sIgA HA, NA	Preventing virus to enter the body by neutralization at mucosal surfaces	Mucosally delivered vaccines: LAIV, WIV, and recombinant viral vector based delivery	Experimentally demonstrated^45^,^99 More data needed Limit for protection not defined
Cytotoxicity assays^1 CD8+ and CD4+ T cell cytotoxicity M1, M2, NP, PA, PB	Preventing severe clinical disease and death when infection is established by lysing infected cells	LAIV, WIV Recombinant fusion protein, synthetic (poly)peptide or vector-based vaccines covering T cell epitopes	Experimentally established^45^,^99 Granzyme B assay: standardized, validated^79^,^97 Flow based CD107a assay and Cr51 release assayused in a human challenge model^83 Lower limit for protection not defined Relevant for universal vaccines
INFγ ELISPOT^1 INFγ pos. cells M1, M2, NP, PA, PB	Preventing severe clinical disease and death when infection is established	LAIV, WIV Recombinant fusion protein, synthetic (poly)peptide or vector-based vaccines covering T cell epitopes	Experimentally demonstrated in humans^22 Frequency of INFγ producing cells IFNγ^+IL2^- CD8^+ phenotype^77 Standardization possible by commercial kits Proposed limit for protection: 100 SFC/mill. PBMC Relevant for universal vaccines
Cytokine assays^1 Cytokine profiles M1, M2, NP, PA, PB	Preventing severe clinical disease and death when infection is established by helping (other) T- and B cell functions	LAIV, WIV Recombinant fusion protein, synthetic (poly)peptide or vector-based vaccines covering T cell epitopes	Experimentally established in humans^23^,^97 IFNγ/IL10 ratio: standardized, validated assay^23^,^97 IL-21 production^81 Lower limit for protection not defined Relevant for universal vaccines

¹ Require cell isolation and (depending on study design) cryopreservation.

Figure 2. Connectograms showing both type and strength of a spectrum of immune responses induced by principally different influenza vaccine concepts: (A) Classical split or subunit vaccines mostly inducing vaccine specific HA and NA antibodies, (B) Vaccines specifically targeting a more broad antibody response with potential for cross-protection, (C) Vaccines specifically targeting conserved antigens for cross-reactive T cell responses, and (D) LAIV covering a broad range of both humoral and cellular immune responses.

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