Novel TLR4 adjuvant elicits protection against homologous and heterologous Influenza A infection

Abstract

Influenza A virus (IAV) is a leading cause of respiratory disease worldwide often resulting in hospitalization or death. In this study, TLR4 immunostimulatory molecules, Bacterial Enzymatic Combinatorial Chemistry (BECC) 438 and BECC470 were found to be superior IAV vaccine adjuvants when compared to the classic adjuvant alhydrogel (alum) and Phosphorylated Hexa-Acyl Disaccharide (PHAD), a synthetic TLR4 agonist. BECC molecules allow for antigen sparing of a recombinant HA (rHA) protein, elicit a more balanced IgG1/IgG2a response, and were protective in a prime only dosing schedule. Importantly, BECC molecules afford protection from a heterologous IAV strain demonstrating that a cross-protective influenza vaccine is possible when the antigen is effectively adjuvanted.

Keywords: Adjuvant; Cross-protection; Influenza; TLR4 agonist; Vaccine.

Fig. 1.

Determining Cal/09 HA antigen dose needed in vaccination for protection from NL/09 influenza A infection. 7-day weight loss in BALB/c mice (3 per group) after infection with 3200 PFU of NL/09 with either a) prime only (PO) vaccination schedule (Mean + SEM) or b) prime + boost (P+B) schedule (Mean + SEM). c) Pre-infection day-28 serum total IgG antibody titer in PO and P+B vaccination groups (Mean + SEM) with (d) corresponding area under the curve (AUC) (Mean ± standard error) (**p < 0.006.). e) Virus titer of lung homogenate 7-days post infection from PO and P+B groups (Mean + SEM) with (f) pathology inflammation scoring of lung histology slides (Mean + SEM). Prism 9 used to calculate Mean + SEM and AUC ± standard error.

Fig. 2.

Homologous challenge protection, a) Pre-infection day 28 serum ELISA total IgG, (b) isotype specific IgG2a and (c) IgGl in prime + boost schedule (Mean + SEM). Area under the curve (AUC) (Mean ± standard error) of above serum ELISA curves for d) total IgG (**p = 0.0016, ****p < 0.0001, ***p = 0.0002), e) IgG2a (**p < 0.006) and f) IgGl (****p < 0.0001, ***p = 0.0001). g) 7-day weight loss in BALB/c mice (5 per group) after infection with 3200 PFU of NL/09 (Mean + SEM). h) Virus titer of lung homogenate 7- days post infection (Mean + SEM) with i) pathology inflammation scoring of lung histology slides (Mean + SEM). Prism 9 used to calculate Mean + SEM and AUC ± standard error.

Fig. 3.

Adjuvant dose sparing, a) Pre-infection day-28 serum ELISA total IgG with 0.04 μg HA in combination with 0.5, 5 or 50 jig of BECC438 or BECC470 adjuvants in prime + boost schedule (Mean + SEM). b) Area under the curve (AUC) (Mean ± standard error) of ELISA curves in for total IgG antibody (****p < 0.0001, ***p = 0.0002). c) 7-day weight loss in BALB/c mice (5 per group) after infection with 3200 PFU of NL/09 (Mean + SEM). d) Virus titer of lung homogenate 7-days post infection (Mean + SEM) with e) pathology inflammation scoring of lung histology slides (Mean + SEM). Prism 9 used to calculate Mean + SEM and AUC ± standard error.

Fig. 4.

Prime only single vaccination, a) Pre-infection day 28 serum ELISA total IgG with 5, 10 or 15 μg HA protein in combination with 50 or 100 μg of BECC470 adjuvant in prime only vaccination schedule (Mean + SEM). b) Area under the curve (AUC) (Mean ± standard error) of ELISA curves for total IgG antibody (***p < 0.0008). c) 7-day weight loss in BALB/c mice (5 per group) after infection with 3200 PFU of NL/09 (Mean + SEM). d) Virus titer of lung homogenate 7-days post infection (Mean + SEM) with e) pathology inflammation scoring of lung histology slides (Mean + SEM). Prism 9 used to calculate Mean + SEM and AUC ± standard error.

Fig. 5.

Protection from heterologous Sing/2015 Influenza A challenge, a) Pre-infection day 28 serum ELISA total IgG in prime + boost schedule (Mean + SEM). b) Area under the curve (AUC) of serum ELISA curves for total IgG antibody (***p < 0.0003). c) 7-day weight loss in BALB/c mice (5 per group) after infection with 51 PFU of Sing/2015 (Mean + SEM). d) Day 28 serum antibody viral neutralization assay, e) Virus titer of lung homogenate 7-days post infection (Mean + SEM) with f) pathology inflammation scoring of lung histology slides (Mean + SEM). Prism 9 used to calculate Mean + SEM and AUC ± standard error.