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. 2021 Jun 25;9(7):698.
doi: 10.3390/vaccines9070698.

Conserved Influenza Hemagglutinin, Neuraminidase and Matrix Peptides Adjuvanted with ALFQ Induce Broadly Neutralizing Antibodies

Clara J Sei  1 , Mangala Rao  2 , Richard F Schuman  3 , Luke T Daum  4 , Gary R Matyas  2 , Nimisha Rikhi  1 , Kevin Muema  1 , Alexander Anderson  2   5 , Ousman Jobe  2   6 , Kellie A Kroscher  1 , Carl R Alving  2 , Gerald W Fischer  1
Affiliations

Conserved Influenza Hemagglutinin, Neuraminidase and Matrix Peptides Adjuvanted with ALFQ Induce Broadly Neutralizing Antibodies

Clara J Sei et al. Vaccines (Basel). .

Abstract

A universal influenza candidate vaccine that targets multiple conserved influenza virus epitopes from hemagglutinin (HA), neuraminidase (NA) and matrix (M2e) proteins was combined with the potent Army liposomal adjuvant (ALFQ) to promote induction of broad immunity to seasonal and pandemic influenza strains. The unconjugated and CRM-conjugated composite peptides formulated with ALFQ were highly immunogenic and induced both humoral and cellular immune responses in mice. Broadly reactive serum antibodies were induced across various IgG isotypes. Mice immunized with the unconjugated composite peptide developed antibody responses earlier than mice immunized with conjugated peptides, and the IgG antibodies were broadly reactive and neutralizing across Groups 1 and 2 influenza viruses. Multi-epitope unconjugated influenza composite peptides formulated with ALFQ provide a novel strategy for the development of a universal influenza vaccine. These synthetic peptide vaccines avoid the pitfalls of egg-produced influenza vaccines and production can be scaled up rapidly and economically.

Keywords: ALFQ; QS21 (also known as QS-21); Th1/Th2 responses; broadly reactive antibodies; immune responses; influenza; liposomes; neutralizing antibodies; peptides; universal vaccine.

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Conflict of interest statement

Carl R. Alving is one of the two inventors on an issued ALFQ patent owned by the U.S. Army. Carl R. Alving and Gary R. Matyas are inventors of a patent application owned by the U.S. Army covering ALFQ formulated with a malaria antigen. The remaining authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The panels on the top left and right show the structural positioning of the HA residues GNLIAP (in blue) and WGVHHP (in red), while the panel on the bottom shows the position of the NA residues HVEECSCY (in green) on a three-dimensional structure of H3N2 influenza virus.
Figure 2
Figure 2
Serum antibody responses across conserved influenza epitopes in mice immunized with unconjugated or CRM-conjugated composite peptide (HA, NA and M2e) vaccine at 0.5, 1 and 2.5 µg/mouse, and formulated with ALFQ. Day 0, 21 and 42 sera samples were analyzed by ELISA. Panels (A,B), IgG1 titers (1:100 dilution) on individual peptides HA (Flu Pep3) and NA (Flu Pep10), and Panels (C,D), on composite peptides HA+NA (Flu Pep11) and M2e (Flu Pep5906), respectively. No significant differences were observed between the two groups as determined by ANOVA. Data are expressed as mean ± standard deviation (SD).
Figure 3
Figure 3
Profile of serum IgG1 responses across individual HA and NA peptides in mice immunized with 1µg of composite influenza peptide (HA, NA and M2e) vaccine, unconjugated or CRM-conjugated, and formulated with ALFQ. Day 0, 21, 35 and 42 sera samples were analyzed by ELISA. Panels (A) and (B), IgG1 titers (1:100 dilution) on Flu Pep3 and Flu Pep6 (both HA), respectively, and Panel (C), on Flu Pep10 (NA). Data are expressed as mean ± standard errors (SEM). Overall, the antibody responses on day 21 were significantly higher (ANOVA, p = 0.0435) for unconjugated peptides compared to CRM-conjugated peptides, while their differences on day 42 were not statistically significant.
Figure 4
Figure 4
Profile of serum IgG1 responses across composite HA+NA and M2e peptides in mice immunized with 1 µg of composite influenza peptide (HA, NA and M2e) vaccine, unconjugated or CRM-conjugated, and formulated with ALFQ. Day 0, 21, 35, and 42 sera samples were analyzed by ELISA. Panel (A), IgG1 titers (1:100 dilution) on Flu Pep11 (HA+NA). Panel (B), on Flu Pep5906 (M2e). Data are expressed as mean ± SEM. Overall, the antibody responses on day 21 were significantly higher (ANOVA, p = 0.0421) for unconjugated peptides compared to CRM-conjugated peptides.
Figure 5
Figure 5
Mice immunized with 1 µg of unconjugated composite influenza peptide (HA, NA and M2e) vaccine formulated with ALFQ had significantly higher (ANOVA, p = 0.0421, p = 0.0435) IgG1 antibody responses 21 days after immunization when compared to CRM-conjugated composite peptide vaccines for both the composite influenza peptides and the individual influenza HA and NA peptide epitopes (Panels (A,B)). Serum IgG1 antibody responses to individual influenza M2e peptide epitopes were also significantly higher (ANOVA, p = 0.0208) in the unconjugated vaccine group on day 100 (Panel (C)). IgG1 titers (1:100 dilution) were analyzed by ELISA and expressed as mean ± SEM.
Figure 6
Figure 6
A comparison of IgG-specific antibody responses across several live contemporary influenza A H1N1 and H3N2 viruses for Day-56 serum samples (1:100 dilution) pooled from mice immunized with 1 µg of composite influenza peptide (HA, NA and M2e) vaccine, unconjugated or CRM-conjugated, and formulated with ALFQ. Average antisera titers (blue bars) for both unconjugated and CRM-conjugated mice groups were analyzed by ELISA and expressed as mean ± SD. Serum IgG antibody responses to Group 1 and 2 influenza viruses were higher (ns) in mice immunized with the unconjugated peptides compared to CRM-conjugated peptides.
Figure 7
Figure 7
Analysis of cytokines produced by splenocytes harvested from mice injected with 1 µg of composite unconjugated or CRM-conjugated influenza peptide (HA, NA and M2e) vaccine formulated with ALFQ. Splenocytes were treated with 30 µg of each peptide, Flu Pep11 (HA+NA, Panel (A)) and Flu Pep5906 (M2e, Panel (B)), for 72 h and supernatant collected for analysis. Cytokines were analyzed using the Quansys Bioscience Q-PlexTM Arrays. No significant differences were observed between the two groups (as determined by ANOVA). Data are represented as means ± SEM.
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