H5N1 influenza vaccine formulated with AS03 A induces strong cross-reactive and polyfunctional CD4 T-cell responses

Abstract

Objective: Adjuvantation of an H5N1 split-virion influenza vaccine with AS03(A) substantially reduces the antigen dose required to produce a putatively protective humoral response and promotes cross-clade neutralizing responses. We determined the effect of adjuvantation on antibody persistence and B- and T-cell-mediated immune responses.

Methods: Two vaccinations with a split-virion A/Vietnam/1194/2004 (H5N1, clade 1) vaccine containing 3.75-30 μg hemagglutinin and formulated with or without adjuvant were administered to groups of 50 volunteers aged 18-60 years.

Results: Adjuvantation of the vaccine led to better persistence of neutralizing and hemagglutination-inhibiting antibodies and higher frequencies of antigen-specific memory B cells. Cross-reactive and polyfunctional H5N1-specific CD4 T cells were detected at baseline and were amplified by vaccination. Expansion of CD4 T cells was enhanced by adjuvantation.

Conclusion: Formulation of the H5N1 vaccine with AS03(A) enhances antibody persistence and induces stronger T- and B-cell responses. The cross-clade T-cell immunity indicates that the adjuvanted vaccine primes individuals to respond to either infection and/or subsequent vaccination with strains drifted from the primary vaccine strain.

Fig. 1

Schematic representation of the study design

Fig. 2

Overlapping peptides for T cell stimulation. Sequences of the A/Vietnam and A/Indonesia HA proteins are shown with overlapping peptides indicated in black (conserved) or red (non-conserved). Note that the last conserved A/Indonesia peptide was included in the non-conserved rather than in the conserved pool

Fig. 3

a Neutralizing antibody GMTs against the homologous A/Vietnam/1194/2004 NIBRG-14 vaccine strain after the first and second vaccine dose with 95% confidence intervals. Data shown for all groups at the different time points. b SCRs of HI antibodies to the homologous A/Vietnam/1194/2004 NIBRG-14 vaccine strain after the first and second vaccine dose with 95% confidence intervals

Fig. 4

Frequencies of memory B cells specific to the split-virion A/Vietnam/1194/2004 NIBRG-14 strain obtained before vaccination (Pre), at 21 days after the first dose (D21), and at 21 or 159 days after the second dose (D42 and D180). Data shown are for the study groups administered with 3.75 μg HA (all time points) or 7.5 μg HA (Pre and D21) of recombinant H5N1 (A/Vietnam/1194/2004 NIBRG-14) split-virion vaccine adjuvanted with AS03_A or unadjuvanted (AS03 vs Plain)

Fig. 5

a Frequencies of CD4 T cells specific to the split-virion A/Vietnam/1194/2004 NIBRG-14 strain obtained before vaccination (Pre), 21 days after the first dose (D21), and 21 or 159 days after the second dose (D42 and D180) in study groups administered with 3.75 or 7.5 μg HA of recombinant H5N1 (A/Vietnam/1194/2004 NIBRG-14, clade 1) split-virion vaccine adjuvanted with AS03_A or unadjuvanted (AS03 vs plain). b Functional characterization of the split-virion A/Vietnam/1194/2004-specific CD4 T cells obtained at 21 days after the second vaccination in study groups administered with 3.75 or 7.5 μg HA of recombinant H5N1 (A/Vietnam/1194/2004 NIBRG-14, clade 1) split-virion vaccine adjuvanted with AS03_A or not (AS03 vs plain). The frequencies of specific CD4 T cells are represented as expressing (after in vitro stimulation) two or more immune markers among CD-40 L, IL-2, TNF-α, or IFN-γ (‘all double’) or expressing CD40L, IL-2, TNF-α, or IFN-γ and at least one of the other immune markers evaluated within CD-40 L, IL-2, TNF-α, or IFN-γ (indicated as +CD40L⁺, +IL-2⁺, +TNF-α⁺, +IFN-γ⁺)

Fig. 6

Frequencies of CD4 T cells specific to the split-virion A/Vietnam/1194/2004 NIBRG-14 strain expressing IFN-γ (IFNγ ⁺) or IL-13 (IL-13 ⁺) obtained before (Pre) and 21 days after the second dose (D42) in study groups administered with 3.75 or 7.5 μg HA of recombinant H5N1 (A/Vietnam/1194/2004 NIBRG-14, clade 1) split-virion vaccine adjuvanted with AS03_A or not (AS03 vs plain)

Fig. 7

a Expression of CCR7 and CD27 on H5N1-specific CD4 T cells obtained after stimulation of PBMC with a pool of peptides spanning the A/Vietnam/1194/2004 H5 sequence. Antigen-specific CD4 T cells were identified by intracellular cytokine staining (left panel) and analyzed for the expression of the CCR7 and CD27 memory markers (right panel). b Distribution of the different CCR7/CD27 expression patterns at different time points (D0, D42, and D180) and after vaccination with 3.75 μg HA AS03_A-adjuvanted (‘adj’) or non-unadjuvanted vaccine

Fig. 8

a Schematic representation of the peptide pool design covering conserved and unconserved amino acid sequences between H5N1 A/Vietnam/1194/2004 and A/Indonesia/5/2005 strains. b Frequencies of CD4 T cells specific to the HA proteins of A/Vietnam, A/Indonesia, or A/Anhui or specific to the conserved and unconserved sequences among these three strains (Vietnam–Indonesia conserved, Vietnam–Indonesia non-conserved, Vietnam–Indonesia–Anhui conserved). CD4 T-cell responses were obtained before (Pre) and 21 days after the second dose (D42) in study groups administered with 3.75 or 7.5 μg HA of recombinant H5N1 (A/Vietnam/1194/2004 NIBRG-14, clade 1) split-virion vaccine adjuvanted with AS03_A or unadjuvanted (AS03 vs unadjuvanted)