Treatment and/or prevention of sepsis

Abstract

The disclosure provides molecules with an affinity for (or an ability to bind to), sialic acid, for use in compositions, medicaments and methods for the treatment of sepsis, its symptoms and sepsis associated pathologies and immune responses.

Claims

1. A method of treating and/or preventing sepsis and/or one or more symptoms thereof, said method comprising administering a sialic acid binding molecule to a subject who has sepsis or an infection which could lead to sepsis or a subject diagnosed as suffering from one or more sepsis associated pathologies, wherein the sialic acid binding molecule comprises two or more family 40 carbohydrate binding molecules.

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

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

4. The method of claim 2, wherein the Streptococcus pneumoniae NanA sialidase comprises the amino acid sequence of SEQ ID NO: 4.

5. The method of claim 1, wherein the sialic acid binding molecule comprises (i) four Vibrio cholerae NanH sialidase CBM units linked, bound, or conjugated together (Vc4CBM); and/or (ii) two Vibrio cholerae NanH sialidase CBM units fused, bound, or conjugated to a Pseudomonas aeruginosa pseudaminidase trimerisation domain (Vc2CBMTD).

6. The method of claim 1, wherein the sialic acid binding molecule is formulated for oral, mucosal or parenteral administration.

7. The method of claim 1, wherein the sialic acid binding molecule is formulated for intranasal administration.

8. The method of claim 1, wherein the sialic acid binding molecule is formulated for intravenous administration.

Description

DETAILED DESCRIPTION

(1) The present invention will now be described in detail with 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 -2,3-sialyllactose drawn as spheres. b, SpCBM, residues 121-305 of S. pneumoniae NanA sialidase with -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 colors. d, Multivalent forms: their molecular weights, valencies and binding affinities for 2,3-sialyllactose as determined by surface plasmon resonance (SPR) at 25 C. (K.sub.D values for VcCBM, Vc2CBM and Vc3CBM had been reported previously.sup.7). Tandem repeat CBMs, and oligomeric CBMs fused to TD are linked by a 5-amino linker (details in Full Methods).

(3) FIG. 2: Survival time of mice infected intravenously only with 8.510.sup.5 CFU/mouse (control) or with same number of pneumococci plus 12.5 g/mouse Vc4CBM. The protein was administered with bacterial inoculums and mice were observed for 168 h. The survival time of each animal is shown with a dot. The median survival time is given with a horizontal line and was 59 h (SD: 53) for control and 84 h (SD:55) for Vc4CBM group.

(4) FIG. 3: Survival time of mice infected intravenously only with approximately 6.3-7.510.sup.5 CFU/mouse (control) or with same number of pneumococci plus 13-15 g/mouse Vc4CBM. The protein was administered with bacterial inoculums and mice were observed for 168 h. The survival time of each animal is shown with a dot. The median survival time is given with a horizontal line and was 59 h (SD: 48.7) for control and 168 h (SD:48.5) for Vc4CBM-treated group.

(5) FIG. 4: The effect of intranasal administration of two mCBM40s on survival of Streptococcus pneumoniae D39-infected mice. CD1 outbred mice (n=20) were administered intranasally with either a single dose of 500 g Vc4CBM (CBM1) or 100 g Vc2CBMTD (CBM2) a day before (A), at the time of (B) or 1 day after intranasal infection (C) with S. pneumoniae D39. Control group received PBS only. Difference in percent median survival was calculated using Wilcoxon test. *p<0.05, ***p<0.001 relative to the control that received PBS.

(6) FIG. 5: The impact of mCBM40s on disease sign scores in pneumococcal infection. CD1 mice that received either 500 g Vc4CBM (CBM1) or 100 g Vc2CBMTD (CBM2) a day before (1), at the time of (0) or 1 day after (+1) intranasal infection when administered with a lethal dose of S. pneumoniae D39, were monitored for signs of disease at 24, 36 and 48 h post infection. A score of 2, 4, or 6 was given if the animals were piloerect, hunched, or lethargic, respectively. Each column represents the mean disease sign scores at different time points for 8 to 14 animals that eventually died. The vertical lines are for standard error of mean. Data was analysed by two-way ANOVA followed by Dunnett's multiple comparisons test. *p<0.05, ***p<0.001

(7) FIG. 6: The effect of mCBM40s on mortality time of Streptococcus pneumoniae infected mice after intranasal administration. CD1 mice were administered intranasally with either 500 Vc4CBM (CBM1) or 100 g Vc2CBMTD (CBM2) a day before (1), at the time of (0) or 1 day after intranasal infection (+1) with S. pneumoniae D39. Difference in time to mortality was calculated using Mann-Whitney U test. *p<0.05, ****p<0.0001 relative to the control that received PBS.

(8) FIG. 7: The impact of CBM protein on pneumococcal bacteremia: CD1 mice received either 500 g Vc4CBM (CBM1) or 100 g Vc2CBMTD (CBM2) a day before (1), at the time of (0) or 1 day after (+1) intranasal infection with lethal dose of S. pneumoniae D39. A blood sample was taken from the tail vein 24 h post infection and CFU/ml was determined by serial dilution and plating. Each column represents the mean CFU/ml for 8 to 14 animals. Vertical lines are for standard error of mean. Data was analysed by two-way ANOVA followed by Dunnett's multiple comparisons test. *p<0.05 relative.

(9) FIG. 8: A schematic referred to hereinafter as General Formula 1. An exemplary Vc4CBM may take the form of General Formula 1.

EXAMPLE 1

(10) When identifying different routes of mCBM40 delivery in vivo, it was noted that a tetrameric Vc-based CBM40 (Vc4CBM), protected mice when given intravenously with a lethal dose of Streptococcus pneumoniae. This suggests a role for CBMs (including multivalent molecules comprising the same) in the treatment and/or prevention of sepsis, its symptoms and/or associated pathologies.

(11) Multivalent forms of CBM40s target sialylated cell surfaces and therefore it was expected that, at certain concentrations, agglutination of red (and white) blood cells in vivo would occur as a result of cross-linking protein-ligand interactions between cells. This could potentially lead to a number of blood clotting symptoms such as thrombosis or stroke.

(12) The results of initial dose-response intravenous Vc4CBM dosing in mice and clinical activity show that an intravenous Vc4CBM dose of 12.5 g/mouse was tolerated.

(13) Production of the Vc4CBM in E. coli meant that it was necessary to eliminate contamination of the CBM preparation with endotoxin as the cause of any observed adverse eventsthis would ensure that the results represented a proper assessment of the effect the Vc4CBM molecule against sepsis. As stated an intravenous dose of 12.5 g Vc4CBM was tolerated by mice and a pneumococcal challenge was attempted.

(14) Groups of mice (n=5) were infected intravenously with a lethal dose of pneumococcus (8.510.sup.5 CFU/mouse) in the presence or absence of a single dose of Vc4CBM (12.5 g/mouse). Survival times of mice are shown in FIG. 2. There appeared to be a slight, increase in survival of mice in the CBM-treated group (median survival time 84 h) compared to the control group (59 h). To determine whether the survival rate in mice could be improved, a further study was performed whereby the pneumococcal CFU dose was slightly reduced to correspond with a slight increase in the amount of Vc4CBM given intravenously. The results of this experiment are shown in FIG. 3. Here it appears that the survival rate of mice improved significantly under the conditions tested, with a combined survival rate of 70% after 7 days in the CBM-treated groups, displaying a median survival time of 168 h, compared to 20% survival of mice in the untreated, infected groups (median survival time of 59 h).

(15) These results indicate that mCBM40s have the potential to alleviate symptoms of sepsis in a bacterially-infected host. In the case of Vc4CBM, it is likely that this biologic modulates the immune response by dampening down the pro-inflammatory cascade of a S. pneumoniae infection that leads to sepsis. Further, while there was a concern that the multivalent CBMs might (through the cross-linking of protein-ligand interactions between cells) induce agglutination of cells (including red (and white) blood cells) in vivo (which could potentially lead to the induction of blood clotting cascades and a number of blood clotting symptoms such as thrombosis or stroke), this did not occur.

EXAMPLE 2

(16) Novel, bacterially-derived, proteins (mCBM40s) that target and bind host cell surface sialic acid-receptors with high affinity (JBC (2009), 284, 7339) have been engineered. When mCBM40s are administered in mice, they are shown to be non-toxic and can protect mice from respiratory pathogens (PNAS (2014) 111, 6401; AAC 59(3): 1495-1504). Using a pneumococcal-challenged mouse model, it has been shown that mCBM40, Vc4CBM can provide significant survival rates in mice against a lethal pneumococcal infection. To further evaluate the protective utility of mCBM40 proteins in reducing bacteraemia in a pneumococcal mouse model, mCBM40s were also given intranasally (as described below).

(17) mCBM40 Dosing Via the Intranasal Route

(18) Method: All CBM40 proteins (Vc4CBM and Vc2CBMTD, endotoxin-free) were prepared as described in Connaris et al (2014).sup.10. Groups of female mice (CD1 outbred strain, n=10 to 20), weighing 28-35 g, were intranasally administered with a single dose of either Vc4CBM (up to 500 g) or Vc2CBMTD (up to 100 g) in 50 l sterile PBS, either one day before (D1), on the day (D0) or one day after (D+1) a lethal intranasal challenge with approximately 110.sup.6 CFU of S. pneumoniae D39 (in 50 l of PBS)/mouse. The control group received PBS only. Clinical signs of disease were monitored and recorded over 7 days (168 h), where an ascending score from 0 to 6 (0 being no clinical symptoms) was given to all animals. At the end of experiment, the lungs, and blood will be collected and bacterial counts determined. Bacteraemia was monitored by sampling blood at specific time-points during infection, with the number of colony forming units from blood determined using the technique as described by Miles and Misra.sup.11.

(19) TABLE-US-00005 TABLE 1 The impact of mCBM40 dosing on survival of CD1 mice challenged with a lethal pneumococcal dose. *Survival of treated groups was evaluated after 168 h. Treatments Dead Survived % Survival* CBM1/1 8 12 60 CBM1/0 12 8 40 CBM1/+1 11 9 45 CBM2/1 9 11 55 CBM2/0 14 6 30 CBM2/+1 8 12 60 Control (PBS) 14 6 30

SUMMARY

(20) 1. mCBM40s demonstrated reduction of bacteraemia in mice when intranasally given in a lethal pneumococcal infection model.

REFERENCES

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