Adjuvants

Abstract

The present disclosure provides novel adjuvants which may be used in combination with one or more antigens to augment, modulate or enhance a host immune response to the one or more antigens. The adjuvants are based on sialic acid binding molecules and may be combined with any type of antigen. The adjuvants may be formulated for mucosal and/or intranasal administration.

Claims

1. A method of improving an immune response to an antigen in a human or animal subject, said method comprising administering a composition comprising the antigen and a molecule capable of binding sialic acid to said human or animal subject, wherein the molecule capable of binding sialic acid, is an adjuvant which improves the immune response to the antigen, wherein the molecule capable of binding sialic acid comprises two or more family 40 carbohydrate binding modules.

2. The method of claim 1, wherein the immune response is a mucosal immune response.

3. The method of claim 1, wherein the composition is a vaccine.

4. The method of claim 1, wherein the composition is a mucosal vaccine.

5. The method of claim 1, wherein the molecule capable of binding sialic acid is fused, linked or conjugated to the antigen.

6. The method of claim 1, wherein the molecule capable of binding sialic acid is fused to an internal region of the antigen and/or to the N- and/or C-terminal region of the antigen.

7. The method of claim 1, wherein the antigen is one or more selected from the group consisting of: (i) (a) bacterial antigen(s); (ii) (a) viral antigen(s); (iii) (a) vaccine antigen(s); and (iv) (a) cancer/tumour antigen(s).

8. The method of claim 1, wherein the adjuvant comprises the sialic acid binding domain of Vibrio cholerae NanH sialidase and/or the sialic acid binding domain of Streptococcus pneumoniae NanA sialidase.

9. The method of claim 1, wherein the adjuvant comprises the Vibrio cholerae NanH sialidase amino acid sequence of SEQ ID NO: 1 or 2.

10. The method of claim 1, wherein the adjuvant comprises the Streptococcus pneumoniae NanA sialidase amino acid sequence of SEQ ID NO: 3 or 4.

11. The method of claim 1, wherein the adjuvant is Vc2CBMTD, wherein Vc2CBMTD is two sialic acid binding domains from Vibrio cholerae NanH sialidase (Vc2CBM) fused, bound or conjugated to Pseudomonas aeruginosa pseudaminidase trimerisation domain (TD), wherein the sequence of each sialic acid binding domain from Vibrio cholerae NanH sialidase is SEQ ID NO: 2 and the sequence of the trimerisation domain is SEQ ID NO: 6.

12. The method of claim 1, wherein the adjuvant is Sp2CBMTD, wherein Sp2CBMTD is two sialic acid binding domains from Streptococcus pneumoniae NanA sialidase (Sp2CBM) fused, bound or conjugated to Pseudomonas aeruginosa pseudaminidase trimerisation domain (TD) wherein the sequence of each sialic acid binding domain from Streptococcus pneumoniae NanA sialidase is SEQ ID NO: 4 and the sequence of the trimerisation domain is SEQ ID NO: 6.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The disclosure will now be described in detail by reference to the following figures, which show:

(2) FIG. 1: Building blocks of the multivalent CBM forms and their affinities for sialic acid. a, VcCBM, residues 25-216 of the V. cholerae sialidase (PDB:1w0p) with α-2,3-sialyllactose drawn as spheres. b, SpCBM, residues 121-305 of S. pneumoniae NanA sialidase with α-2,3-sialyllactose (PDB:4c1w). c, TD, the trimerisation domain, residues 333-438, of the P. aeruginosa pseudaminidase (PDB:2w38) in rainbow colours; the other two monomers in single colours. d, Multivalent forms: their molecular weights, valencies and binding affinities for α2,3-sialyllactose as determined by surface plasmon resonance (SPR) at 25° C. (KD values for VcCBM, Vc2CBM and Vc3CBM had been reported previously 78). Tandem repeat CBMs, and oligomeric CBMs fused to TD are linked by a 5-amino linker.

(3) FIG. 2: Titration curves of mucosal anti-mCBM40 IgA antibodies in BAL mouse samples. Mice (groups of 5) were administered either 1 μg, 10 μg, or 50 μg of mCBM40 intranasally before (Day-1) or on the day (Day 0) of an IFV-challenge. Mice were culled day 7 post infection, and BAL samples were taken and assayed for anti-mCBM40 antibodies by ELISA.

(4) FIG. 3: Detection of IgA and IgG antibodies to IFV A/WSN/33 HA (a), Vc2CBMTD (b), and Sp2CBMTD (c), in mouse tissue. Tissue sample were obtained from a mouse study where mice were administered 1 μg of mCBM40 intranasally on Day 0, 14 and 27 before a viral challenge with 5000 PFU of IFV A/WSN/33 (H1N1) on Day 28. Mice were culled on Day 35 (i.e. day 7 post infection). BAL (1:10 diluted), lung extract (1:4) and sera (1:30) samples were assayed for the presence of both anti-mCBM40, and antiviral HA antibodies by ELISA. Brackets indicate ELISA coat antigen. Data represent the mean±SD. Asterisks indicate P values of *<0.05, **0.01, ***<0.001, ****<0.0001 of infected mice compared to treated, infected groups, using Bonferroni's t-test.

(5) FIG. 4: Detection of IgA and IgG antibodies to IFV A/California/pdm09 HA (a), and Vc2CBMTD (b) in mouse tissue. Tissue sample were obtained from a mouse study where mice were administered 10 μg of mCBM40 intranasally on Day 0, 4 and 6 before a viral challenge with 150 PFU of IFV A/California/pdm09 (H1N1) on Day 7. Mice were culled around Day 21 (i.e. ˜day 15 post infection), unless otherwise stated (see text). BAL (1:1 diluted), lung extract (1:5) and sera (1:30) samples were assayed for the presence of both antiviral HA antibodies and anti-mCBM40 by ELISA. Brackets indicate ELISA coat antigen. Data represent the mean±SD. Asterisks indicate P values of *<0.05, **0.01, ***<0.001, ****<0.0001 of infected mice compared to treated, infected groups, using Bonferroni's t-test.

(6) FIG. 5: Intranasal administration protocol. (A) Timeline of prime and boost intranasal inoculations of antigens in mice including sera sampling. (B) Table of dosing amounts of antigens. All mouse groups, with the exception of Group 1 (control), were treated with molar equivalent amounts of protein as represented by different amounts of protein given in 40 μL endotoxin-free PBS.

(7) FIG. 6: Detection of sera IgG to GFP, Vc2CBMTD and Sp2CBMTD following intranasal administration in mice. Mice were intranasally administered up to 2 μg (40 μL) of antigen on day 0 and day 14. Sera were taken on days 7 and 21 to assess anti-GFP (A), anti-Vc2CBMTD (B) and anti-Sp2CBMTD (C) IgG antibodies by ELISA, as described in Methods. Bars indicate the mean absorbance change ±SD for IgG from five mice per group. All values are presented as mean±SD, with statistical results presented as: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

(8) FIG. 7. Detection of IgA and IgG to GFP, Vc2CBMTD and Sp2CBMTD from various tissues after intranasal treatment in mice. Mice were intranasally administered up to 2 μg (40 μL) of antigen on day 0, and day 14. Tissue samples (BAL, lung and sera) were taken on day 35 to assess anti-GFP (A), anti-Vc2CBMTD (B) and anti-Sp2CBMTD (C) IgG and IgA antibodies by ELISA, as described in Methods. Bars indicate the mean absorbance change ±SD for IgG or IgA from five mice per group. All values are presented as mean±SD, with statistical results presented as: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

EXAMPLE 1

(9) Materials and Methods

(10) Standard ELISAs using immobilised biologics were used to analyse bronchiolar alveolar lavage (BAL) samples collected from a murine study where mCBM40s were given intranasally prior to a lethal influenza challenge. The mice were culled 7 days later.

(11) In one study, mCBM40s were shown to elicit a strong mucosal IgA response in a dose-dependent manner (see FIG. 2). This suggests that mCBM40 are potent mucosal immunogens, similar to other bacterially-derived immunogens such as cholera toxin and LTB.

(12) In another mouse study a low (1 μg) repeated dose of mCBM40 was administered up to a month before a IFV-challenge, with mice being culled 7 days later. Mouse tissues were analysed for evidence of IFV HA-specific, and mCBM40-specific antibodies from BAL, lung extracts and sera. We observed both anti-IFV HA IgA and IgG antibodies in BAL and lung extracts from mice that were treated with mCBM40s and that the levels of antibodies were increased in some of these mice compared to mice that were given virus only (see FIG. 3).

(13) Despite mice being exposed to virus for only a week before culling, the data suggests that the mCBM40s, even at low doses, appear to enhance the level of antiviral antibodies in lung extracts. A similar murine study where mCBM40, Vc2CBMTD, was given to mice up to 1 week before a lethal IFV A/California/pdm09 challenge, and with mice culled approximately 2 weeks later, also showed the presence of both mCBM40 and antiviral IgA and IgG antibodies in BAL and lung homogenates (see FIG. 4).

(14) The difference in antiviral HA antibodies between treated and untreated infected mice were statistically significant; however, some mice from the untreated, infected group were culled just before the designated cull date due to greater than 25% weight loss (around Days 9-11 post infection). Despite this, mCBM40s have the potential to be potent non-toxic, mucosal adjuvants for antigens or vaccines of interest, particularly for respiratory pathogens.

EXAMPLE 2

(15) Introduction

(16) When mCBM40s are administered in mice, they have been shown to be non-toxic, and capable of generating protective immune responses against respiratory pathogens, such as an influenza virus (IFV), that target cell surface sialic acid-receptors during infection (PNAS (2014) 111, 6401; AAC (2015) 59, 1495). Analyses of mucosal tissue from mice infected with IFV indicate that mCBM40s are also potent immunogens and appear to enhance an IFV antigen-specific antibody response when mCBM40s are directed to the same tissue. Based on these findings, we investigated whether the immune response of a test antigen is enhanced through the use of a CBM-based adjuvant.

(17) Methods

(18) Intranasal immunization of mice. All CBM40 proteins were prepared as described in PNAS (2014) 111, 6401. Female BALB/c mice (6-week old) were used for the study. All intranasal inoculations and mice bleeds were performed under isofluorane anaesthesia. The immunization schedule and bleeds were performed as outlined in FIG. 5. Eight groups of mice (n=5) were pre-bled (via tail) one week before an initial dose of purified recombinant protein (up to 2 μg purified proteins in 40 μl sterile endotoxin free PBS), was given intranasally. At day 14, mice were given a further intranasal dose of antigens. Mice were weighed on day 0 and day 14 and on days 1 & 2 after dosing to monitor any effects of dosing. Mice were also monitored throughout for clinical signs. On day 35, mice were culled by rising CO.sub.2, and BAL fluid, lungs and serum, harvested for analysis.

(19) Antibody Analysis.

(20) Immune samples (BAL, lungs and sera) were analysed for the presence of anti-GFP antibodies and anti-mCBM40 antibodies using the ELISA technique as described in PNAS (2014) 111, 6401. For this, antigens (1 μg per well) were immobilized on 96-well plates (Corning). Tissue samples were diluted in blocking buffer as followed: BAL (1:4), homogenized lung tissue (1:4) and sera (1:50). Samples were added to wells followed by goat anti-mouse IgG, IgA, or IgM HRP-conjugate antibodies (Santa Cruz, 1:5000 dilution), and the presence of antibodies was detected using TMB (Sigma). Absorbance was measured at the 450-nm wavelength (620-nm wavelength used as reference).

(21) Statistical Analysis.

(22) Pairwise comparisons were made using one-way ANOVA and Tukey's multiple comparisons test. GraphPad Prism 7 (GraphPad Software) was used for all analysis. Tests with p<0.05 were deemed statistically significant. Unless otherwise stated, all values are presented as mean±SD, with statistical results presented as: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

SUMMARY

(23) 1. Conjugation of the test antigen (GFP) to Sp2CBMTD, generated both local and systemic anti-GFP antibodies from 7 days post intranasal administration.

(24) 2. The conjugation method using Sp2CBMTD was more efficient in generating anti-GFP antibodies compared to either GFP alone or co-administration with Sp2CBMTD or Vc2CBMTD (FIGS. 6 and 7).

(25) 3. Mucosal IgA and IgG responses of both GFP and mCBM40 (Sp2CBMTD, Vc2CBMTD) in lung tissue and BAL were also observed after 35 days.