NOVEL VACCINE COMPOSITIONS
20230263877 · 2023-08-24
Inventors
Cpc classification
A61K2039/55555
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61K2039/55572
HUMAN NECESSITIES
International classification
Abstract
A Shigella flexneri O-antigen of a first serotype or subserotype are provided for use in raising an immune response against one or more Shigella flexneri O-antigen of a different serotype or subserotype, together with associated binding moieties, pharmaceutical compositions, kits, uses or methods.
Claims
1. A method for raising an immune response in a human against one or more Shigella flexneri O-antigen comprising administering to said human a Shigella flexneri O antigen of a first serotype or subserotype, wherein the immune response raised is raised against one or more Shigella flexneri O antigen of a different serotype or subserotype.
2. The method according to claim 1, wherein the different serotype or subserotype is one or more serotype or sub serotype having an SBA score in Table 2 of greater than or equal to 2.3, for example, greater than or equal to 3.0, greater than or equal to 3.6, or greater than or equal to 3.7 and/or wherein the different serotype or subserotype is not one or more serotype or subserotype having an SBA score in Table 2 of less than 3.7, for example, less than 3.6, less than 3.0, or less than 2.3.
3. The method according to claim 1, wherein the first serotype or subserotype is: 1 and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; 2 and the different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; 3 and the different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; 4 and the different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; 5 and the different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; 6 and the different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; X and the different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; and/or Y and the different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes.
4. The method according to claim 1, wherein the first serotype or subserotype is: 1a and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1b, 2a, 2b, 3a, 3b, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; 1b and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 2a, 2b, 3a, 3b, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; 2a and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2b, 3a, 3b, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; 2b and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 3a, 3b, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; 3a and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3b, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; 3b and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3a, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; 4a and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3a, 3b, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; 5b and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; 6 and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5b, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; X and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5b, 6, and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; and/or Y and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5b, 6 and X, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes.
5. The method according to claim 1, wherein the first serotype or subserotype is from another serotype to the different serotype or subserotype, for example: where the first serotype or subserotype is 1, the different serotype or subserotype is not, or is not a subserotype of, serotype 1; where the first serotype or subserotype is 2, the different serotype or subserotype is not, or is not a subserotype of, serotype 2; where the first serotype or subserotype is 3, the different serotype or subserotype is not, or is not a subserotype of, serotype 3; where the first serotype or subserotype is 4, the different serotype or subserotype is not, or is not a subserotype of, serotype 4; where the first serotype or subserotype is 5, the different serotype or subserotype is not, or is not a subserotype of, serotype 5; where the first serotype or subserotype is 6, the different serotype or subserotype is not, or is not a subserotype of, serotype 6; where the first serotype or subserotype is X, the different serotype or subserotype is not, or is not a subserotype of, serotype X; where the first serotype or subserotype is Y, the different serotype or subserotype is not, or is not a subserotype of, serotype Y;
6. The method Shigella flexneri O antigen for use according to any preceding claim 1, wherein the first serotype or subserotype is: 2a, the different serotype or subserotype is not subserotype 1b; 2a, the different serotype or subserotype is not subserotype 2b; 2a, the different serotype or subserotype is not subserotype 5b; 2a, the different serotype or subserotype is not subserotype Y; 3a, the different serotype or subserotype is not subserotype 1b; 3a, the different serotype or subserotype is not subserotype 2b; 3a, the different serotype or subserotype is not subserotype 5b; 3a, the different serotype or subserotype is not, or is not a subserotype of, serotype Y; 2a, and is provided in combination with an additional O-antigen of serotype or subserotype 3a, the different serotype or subserotype is not subserotype 1b; 2a, and is provided in combination with an additional O-antigen of serotype or subserotype 3a, the different serotype or subserotype is not subserotype 2b; 2a, and is provided in combination with an additional O-antigen of serotype or subserotype 3a, the different serotype or subserotype is not subserotype 5b; 2a, and is provided in combination with an additional O-antigen of serotype or subserotype 3a, the different serotype or subserotype is not, or is not a subserotype of, serotype Y; and/or 2a, the different serotype or subserotype is not, or is not a subserotype of, serotype 6; Y, the different serotype or subserotype is not subserotype 1b; and/or Y, the different serotype or subserotype is not subserotype 2a.
7. The method according to claim 1, wherein the first serotype or subserotype is: serotype 1 and the different serotype or subserotype is one or more serotype selected from the group consisting of 2, 5 and X, for example, 1, 2 or 3 of the different serotypes. serotype 2 and the different serotype or subserotype is one or more serotype selected from the group consisting of 4, 5, 6 and Y, for example, 1, 2 or 3 of the different serotypes. serotype 3 and the different serotype or subserotype is one or more serotype selected from the group consisting of 1, 2, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6 or 7 of the serotypes; serotype 4 and the different serotype or subserotype is one or more serotype selected from the group consisting of 1, 2, 5, X and Y, for example, 1, 2, 3, 4 or 5 of the serotypes; serotype 5 and the different serotype or subserotype is one or more serotype selected from the group consisting of 1, 2, 4, 6, X and Y, for example, 1, 2, 3, 4, 5 or 6 of the serotypes; serotype 6 and the different serotype or subserotype is one or more serotype selected from the group consisting of 5 and X, for example, 1 or 2 of the serotypes; serotype X and the different serotype or subserotype is one or more serotype selected from the group consisting of 1, 4, 6 and Y, for example, 1, 2, 3 or 4 of the serotypes; and/or serotype Y and the different serotype or subserotype is 5.
8. The method according to claim 1, wherein the first serotype or subserotype is: subserotype 1a and the different serotype or subserotype is one or more subserotype selected from the group consisting of 5b and X, for example, 1 or 2 of the subserotypes; subserotype 1b and the different serotype or subserotype is one or more subserotype selected from the group consisting of 2b, 5b and X, for example, 1, 2 or 3 of the subserotypes; subserotype 1c and the different serotype or subserotype is one or more subserotype selected from the group consisting of 2b, 5b and X, for example, 1, 2 or 3 of the serotypes; subserotype 2a and the different serotype or subserotype is subserotype 1a, 5b and Y, for example, 1, 2 or 3 of the subserotypes; subserotype 2b and the different serotype or subserotype is one or more subserotype selected from the group consisting of 4a, 6 and Y, for example, 1, 2 or 3 of the subserotypes; subserotype 3b and the different serotype or subserotype is one or more subserotype selected from the group consisting of 1a, 2a, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6 or 7 of the subserotypes; subserotype 4a and the different serotype or subserotype is one or more subserotype selected from the group consisting of 5b and X, for example, 1 or 2 of the subserotypes; subserotype 4b and the different serotype or subserotype is one or more subserotype selected from the group consisting of 1a, 2b, 5b, X and Y for example, 1, 2, 3, 4 or 5 of the subserotypes. subserotype 5a and the different serotype or subserotype is X; subserotype 5b and the different serotype or subserotype is one or more subserotype selected from the group consisting of 1a, 2a, 4a, 6 and Y, for example, 1, 2, 3, 4 or 5 of the subserotypes; serotype 6 and the different serotype or subserotype is one or more subserotype selected from the group consisting of 5b and X, for example, 1 or 2 of the subserotypes; serotype X and the different serotype or subserotype is one or more subserotype selected from the group consisting of 1a, 4a, 6 and Y, for example, 1, 2, 3 or 4 of the subserotypes; and/or serotype Y and the different serotype or subserotype is subserotype 5b.
9. The method according to claim claim 1, wherein the first serotype or subserotype is: subserotype 1b and the different serotype or subserotype is not one or more subserotype selected from the group consisting of 3a and 3b, for example, 1 or 2 of the subserotypes; subserotype 1c and the different serotype or subserotype is not 3a; subserotype 2a and the different serotype or subserotype is not one or more subserotype selected from the group consisting of 3a and 6, for example, 1 or 2 of the subserotypes; subserotype 2b and the different serotype or subserotype is not 3a; subserotype 3a and the different serotype or subserotype is not one or more subserotype selected from the group consisting of 1a, 2b and X, for example, 1, 2 or 3 of the subserotypes; subserotype 4a and the different serotype or subserotype is not one or more subserotype selected from the group consisting of 1b and 3a, for example, 1 or 2 of the subserotypes; subserotype 4b and the different serotype or subserotype is not 1b; subserotype 5a and the different serotype or subserotype is not one or more subserotype selected from the group consisting of 1b and 3a, for example, 1 or 2 of the subserotypes; the first serotype is subserotype 5b and the different serotype or subserotype is not X; serotype 6 and the different serotype or subserotype is not X; serotype X and the different serotype or subserotype is not one or more serotype selected from the group consisting of 2b and 3a, for example, 1 or 2 of the subserotypes; and/or serotype Y and the different serotype or subserotype is not one or more serotype selected from the group consisting of 1b, 2a and 3a, for example, 1, 2 or 3 of the subserotypes.
10. The method according to claim 1, wherein the O-antigen of a first serotype is provided in combination with one or more O-antigen of a further serotype or subserotype, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 O-antigen further serotypes or subserotypes, for example: wherein first and further subserotypes comprise or consist of combinations selected from the group consisting of: a. 1b and 3a; b. 1b and 3b; c. 1c and 3a; d. 1c and 3b; e. 2a and 3b; f. 3a and 4b; and g. 3b and 5b.
11. The method according to claim 1, wherein one or more of the different serotype(s) or subserotype(s) is not provided, for example, one or more of 1a, 1b , 1c (or 7a), 1d, 2a, 2b, 3a, 3b, 4a, 4av, 4b, 5a, 5b, X, Xv, Y, Yv, 6 and 7b is not provided, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 of the different serotype(s) or subserotype(s) is not provided, and (optionally), wherein the Shigella flexneri O-antigen for use is capable of raising an immune response against one or more of the different serotype(s) or subserotype(s) that is not provided, e.g.: wherein serotype 1 (for example, 1a, 1b, or 1c) is provided and serotype 6 is not provided and/or wherein serotype 3 (for example, 3a, 3b, or 3c) is provided and serotype 6 is not provided.
12. The method according to claim 1, wherein O-antigen from one or more Shigella species other than Shigella flexneri is provided in combination with the O-antigen of a first serotype, for example, wherein the one or more other Shigella species is selected from the group consisting of: a. Shigella sonnei; b. Shigella boydii (for example, serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); and c. Shigella dysenteriae (for example, serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15).
13. The method according to claim 1, wherein the first serotype or subserotype and/or other Shigella species is/are provided: a. unassociated with another macromolecule; b. as a component of lipopolysaccharide (LPS), or a fragment thereof; or c. conjugated to another macromolecule, for example, a protein (e.g., a carrier protein such as CRM197, tetanus toxoid, meningococcal outer membrane protein complex (OMPC), diphtheria toxoid, and H. influenzae protein D).
14. The method according to claim 1, wherein the Shigella O-antigen of a first serotype or subserotype is associated with a membrane component, wherein the membrane component is a component of an OMV selected from the group consisting of a detergent-extracted OMV (dOMV); or native OMV (nOMV).
15. The method according to claim 1, wherein the immune response comprises or consists of a protective immune response, e.g., an in vitro protective immune response and/or an in vivo protective immune response.
16. A binding moiety capable of specifically binding to one or more O-antigen defined in claim 1.
17. The method of claim 1, wherein the O-antigen of a first serotype of subserotype is provided as part of a pharmaceutical composition.
18. The method of claim 1, wherein the O-antigen of a first serotype of subserotype is provided as part of a kit; and the kit (optionally) further comprises instructions for use.
19. (canceled)
20. The use of a binding moiety as defined in claim 16, for detecting the presence of bacteria, for example, wherein the bacteria are one or more bacterium defined in claim 1.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0027]
[0028]
[0029]
DESCRIPTION
[0030] The present inventors examined the ability of sera raised against 14 subtypes of S. flexneri to (a) bind to a panel of 11 S. flexneri subtypes from all serotypes using fluorescence-activated cell sorting (FACS); and (b) to kill these bacteria in a complement-mediated serum bactericidal assay (SBA). The antigens were delivered as Generalized Modules for Membrane Antigens (GMMA) (Italian gemma=bud), which are outer membrane blebs of approximately 50-200 nm that bud off Gram-negative bacteria genetically modified to induce hyperblebbing (15), a technology currently in human vaccine trials for S. sonnei (16-18). GMMA contain outer-membrane components of the parent bacteria including the LPS expressing the OAg (19).
[0031] As mentioned, a broadly-protective vaccine against shigellosis needs to cover multiple S. flexneri serotypes. A challenge is to design a practical vaccine that balances coverage versus complexity and cost. Importantly, the present inventors found that a simple three-component vaccine of GMMA from S. sonnei, S. flexneri 1b and 3a would induce killing of most epidemiologically significant Shigella strains. This was not predicted based on cross-reactivity of currently-described shared serotypes and serogroups. The study presented here provides a framework for empirically designing such a vaccine.
[0032] There was strong cross-reaction within serotypes, e.g., sera raised against S. flexneri 2a reacted strongly with S. flexneri 2b. We identified some immunogens (e.g. S. flexneri 1b and 3a) that induced broadly-reactive antibodies that bound to most of the S. flexneri in the panel, while other immunogens (e.g., S. flexneri 2a) had a narrower specificity. Contrary to expectation, most cross-reactions cannot be assigned to S. flexneri serogroups e.g., sera raised with S. flexneri 1b strongly reacted with S. flexneri 6 which do not share any of the currently recognised serogroups. These results suggest that there are common group specificities not currently recognised with typing reagents and that broadly cross-reactive vaccines will be possible with limited components (e.g., just S. flexneri 1b and 3a).
[0033] Accordingly, a first aspect of the invention provides a Shigella flexneri O-antigen of a first serotype or subserotype for use in raising an immune response against one or more Shigella flexneri O-antigen of a different serotype or subserotype.
[0034] Lipopolysaccharides (LPS), also known as lipoglycans and endotoxins, are large molecules having a lipid and a polysaccharide composed of O-antigen, a core domain having an outer core and inner core joined by a covalent bond and are found in the outer membrane of Gram-negative bacteria. A repetitive glycan polymer contained within an LPS is referred to as the O antigen, O polysaccharide, or O side-chain of the bacteria. The O antigen is attached to the outer core oligosaccharide and comprises the outermost domain of the LPS molecule. The composition of the O chain varies from strain to strain. The core domain always contains an oligosaccharide component that attaches directly to lipid A and commonly contains sugars such as heptose and 3-Deoxy-D-manno-oct-2-ulosonic acid (also known as KDO, keto-deoxyoctulosonate). Lipid A is, in normal circumstances, a phosphorylated glucosamine disaccharide decorated with multiple fatty acids. These hydrophobic fatty acid chains anchor the LPS into the bacterial membrane, and the rest of the LPS projects from the cell surface. The lipid A domain is responsible for much of the toxicity of Gram-negative bacteria.
[0035] By “a Shigella flexneri O-antigen of a first serotype” we mean or include complete O-antigen, or fragments, fusions and/or derivatives thereof. The O-antigen may or may not be bound to the LPS core domain. The LPS core domain may or may not be bound to lipid A. Hence, the O-antigen may comprise part of a complete LPS molecule. By “fragment” of an O-antigen, we mean or include molecules that comprise or consist of at least 25% of the contiguous length of a reference O-antigen molecule e.g., at least 50%, at least 75%, at least 90%, at least 95%, at least 98% or at least 99% of the contiguous length of a reference O-antigen molecule. When referring to “a Shigella flexneri O-antigen of a first serotype” in the singular, as is convention in patent drafting, we mean or include pluralities of the same O-antigen. Alternatively or additionally, we mean or include single O-antigen molecules. By “first serotype” we mean or include a single subserotype within the ‘first serotype’; alternatively, we mean or include a mixture of serotypes within the ‘first serotype’, for example, 2, 3 or all of the serotypes within the ‘first serotype’.
[0036] By “different serotype or subserotype” we mean or include another serotype or subserotype to the ‘first serotype or subserotype’. For the avoidance of doubt, where there is more than one ‘different serotype or subserotype’, each ‘different serotype or subserotype’ is from a different serotype or subserotype to each other, as well as to the ‘first serotype or subserotype’.
[0037] Alternatively or additionally, the immune response is raised against one or more Shigella flexneri O-antigen of a different serotype. By “a different serotype” we mean or include another serotype to the ‘first serotype or subserotype’. Hence, the or each ‘different serotype’ is from a different serotype to the ‘first serotype or subserotype’. This does not exclude that the O-antigen of a ‘first serotype or subserotype’ induces an immune response against the ‘first serotype or subserotype’ or against other subserotypes of the same serotype as the ‘first serotype or subserotype’, only that this subject-matter does not necessarily form part of the claimed subject-matter.
[0038] The SBAs of Table 2 indicate which other S. flexneri strains a first S. flexneri strain was capable of inducing complement-mediated killing against. Table 2 shows SBA scores which reflect the strength of immune responses in an SBA assay. A respective SBA score may be determined from the experimental data. To ensure that the claimed cross-protections were sufficiently strong to be biologically relevant for vaccinology, a minimum threshold serum bactericidal activity (SBA) score was selected. The minimum threshold SBA score may be determined empirically as provided herein, and may distinguish between a baseline (no immune response) and the presence of an immune response. For the examples provided herein, a minimum threshold SBA score of 2.3 is selected, which represents a 200× increase from baseline. SBA scores of 3.0, 3.6 and 3.7 represent 400×, about 900× and 1000× increases from baseline, respectively and may be used as even more stringent SBA activity thresholds. SBA scores may be used to generate a heatmap and/or to categorize strength of responses, e.g., with higher SBA scores indicating a stronger immune response. Hence, alternatively or additionally, the different serotype or subserotype is one or more serotype or subserotype having an SBA score in Table 2 of greater than or equal to 2.3, for example, greater than or equal to 3.0, greater than or equal to 3.6, or greater than or equal to 3.7. Alternatively or additionally, the different serotype or subserotype is not one or more serotype or subserotype having an SBA score in Table 2 of less than 3.7, for example, less than 3.6, less than 3.0, or less than 2.3.
[0039] Alternatively or additionally, the minimum threshold SBA score may be 3.0, 3.6 or 3.7. Alternatively or additionally, the different serotype or subserotype is one or more serotype or subserotype having an SBA score of greater than or equal to 2.3 and/or not less than 2.3; the different serotype or subserotype is one or more serotype or subserotype having an SBA score of greater than or equal to 3.0 and/or not less than 3.0; the different serotype or subserotype is one or more serotype or subserotype having an SBA score of greater than or equal to 3.6 and/or not less than 3.6; or the different serotype or subserotype is one or more serotype or subserotype having an SBA score of greater than or equal to 3.7 and/or not less than 3.7.
[0040] Hence, the present invention relates to the use of one or more O-antigen to induce an immune response against one or more further O-antigen and so, alternatively or additionally the first serotype or subserotype is: [0041] 1 and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; [0042] 2 and the different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; [0043] 3 and the different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; [0044] 4 and the different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; [0045] 5 and the different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; [0046] 6 and the different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; [0047] X and the different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; and/or [0048] Y and the different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes.
[0049] Alternatively or additionally, the first serotype or subserotype is:
[0050] 1 and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the different serotypes or subserotypes; [0051] 2 and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the different serotypes or subserotypes; [0052] 3 and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the different serotypes or subserotypes; [0053] 4 and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the different serotypes or subserotypes; [0054] 5 and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the different serotypes or subserotypes;
[0055] 6 and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the different serotypes or subserotypes; [0056] X and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the different serotypes or subserotypes; and/or [0057] Y and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the different serotypes or subserotypes.
[0058] Alternatively or additionally, the first serotype or subserotype is: [0059] 1a and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; [0060] 1b and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; [0061] 2a and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; [0062] 2b and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; [0063] 3a and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; [0064] 3b and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; [0065] 4a and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; [0066] 5b and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; [0067] 6 and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; [0068] X and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes; and/or [0069] Y and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype selected from the group consisting of serotype or subserotype 1, 2, 3, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7 or 8 of the different serotypes.
[0070] Alternatively or additionally, the first serotype or subserotype is:
[0071] 1a and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1b, 2a, 2b, 3a, 3b, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; [0072] 1b and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 2a, 2b, 3a, 3b, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; [0073] 2a and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2b, 3a, 3b, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; [0074] 2b and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 3a, 3b, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; [0075] 3a and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3b, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; [0076] 3b and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3a, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; [0077] 4a and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or [0078] subserotype 1a, 1b, 2a, 2b, 3a, 3b, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; [0079] 5b and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 6, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; [0080] 6 and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5b, X and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; [0081] X and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5b, 6, and Y, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes; and/or [0082] Y and the one or more S. flexneri O-antigen of a different serotype or subserotype comprises or consists of a serotype or subserotype selected from the group consisting of serotype or subserotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5b, 6 and X, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the different serotypes or subserotypes.
[0083] Alternatively or additionally, the first serotype or subserotype is from another serotype to the different serotype or subserotype, for example: [0084] where the first serotype or subserotype is 1, the different serotype or subserotype is not, or is not a subserotype of, serotype 1; [0085] where the first serotype or subserotype is 2, the different serotype or subserotype is not, or is not a subserotype of, serotype 2; [0086] where the first serotype or subserotype is 3, the different serotype or subserotype is not, or is not a subserotype of, serotype 3; [0087] where the first serotype or subserotype is 4, the different serotype or subserotype is not, or is not a subserotype of, serotype 4; [0088] where the first serotype or subserotype is 5, the different serotype or subserotype is not, or is not a subserotype of, serotype 5; [0089] where the first serotype or subserotype is 6, the different serotype or subserotype is not, or is not a subserotype of, serotype 6; [0090] where the first serotype or subserotype is X, the different serotype or subserotype is not, or is not a subserotype of, serotype X; [0091] where the first serotype or subserotype is Y, the different serotype or subserotype is not, or is not a subserotype of, serotype Y; [0092] As noted in the introduction, the bivalent test vaccine of Noriega et al., (13), and infection of monkeys of
[0093] Karnell et al., 1992 (39) reported potential cross-protections between S. flexneri strains. To our knowledge, there was no teaching as to whether these potential cross-protections were O-antigen- or protein-mediated.
[0094] Alternatively or additionally, where the first serotype or subserotype is: [0095] 2, the different serotype or subserotype is not, or is not a subserotype of, serotype 1; [0096] 2, the different serotype or subserotype is not, or is not a subserotype of, serotype 2; [0097] 2, the different serotype or subserotype is not, or is not a subserotype of, serotype 5; [0098] 2, the different serotype or subserotype is not, or is not a subserotype of, serotype Y; [0099] 3, the different serotype or subserotype is not, or is not a subserotype of, serotype 1; [0100] 3, the different serotype or subserotype is not, or is not a subserotype of, serotype 2; [0101] 3, the different serotype or subserotype is not, or is not a subserotype of, serotype 5; [0102] 3, the different serotype or subserotype is not, or is not a subserotype of, serotype Y; [0103] 2, and is provided in combination with an additional O-antigen of serotype or subserotype 3, the different serotype or subserotype is not, or is not a subserotype of, serotype 1; [0104] 2, and is provided in combination with an additional O-antigen of serotype or subserotype 3, the different serotype or subserotype is not, or is not a subserotype of, serotype 2; [0105] 2, and is provided in combination with an additional O-antigen of serotype or subserotype 3, the different serotype or subserotype is not, or is not a subserotype of, serotype 5; [0106] 2, and is provided in combination with an additional O-antigen of serotype or subserotype 3, the different serotype or subserotype is not, or is not a subserotype of, serotype Y; [0107] 2, the different serotype or subserotype is not, or is not a subserotype of, serotype 6; [0108] Y, the different serotype or subserotype is not, or is not a subserotype of, serotype 1; and/or [0109] Y, the different serotype or subserotype is not, or is not a subserotype of, serotype 2.
[0110] Alternatively or additionally, where the first serotype or subserotype is: [0111] 2a, the different serotype or subserotype is not subserotype 1b; [0112] 2a, the different serotype or subserotype is not subserotype 2b; [0113] 2a, the different serotype or subserotype is not subserotype 5b; [0114] 2a, the different serotype or subserotype is not subserotype Y; [0115] 3a, the different serotype or subserotype is not subserotype 1b; [0116] 3a, the different serotype or subserotype is not subserotype 2b; [0117] 3a, the different serotype or subserotype is not subserotype 5b; [0118] 3a, the different serotype or subserotype is not, or is not subserotype Y; [0119] 2a, and is provided in combination with an additional O-antigen of serotype or subserotype 3a, the different serotype or subserotype is not subserotype 1b; [0120] 2a, and is provided in combination with an additional O-antigen of serotype or subserotype 3a, the different serotype or subserotype is not subserotype 2b; [0121] 2a, and is provided in combination with an additional O-antigen of serotype or subserotype 3a, the different serotype or subserotype is not subserotype 5b; [0122] 2a, and is provided in combination with an additional O-antigen of serotype or subserotype 3a, the different serotype or subserotype is not, or is not subserotype Y; [0123] 2a, the different serotype or subserotype is not, or is not subserotype 6; [0124] Y, the different serotype or subserotype is not subserotype 1b; and/or [0125] Y, the different serotype or subserotype is not subserotype 2a.
[0126] Alternatively or additionally, the one or more O-antigen of a different serotype or subserotype does not share group specificities with the O-antigen(s) of the first serotype or subserotype.
[0127] By “does not share group specificities” we mean or include that the first and different O-antigens do not share group specificities as identified by typing reagents or genomic probes. Alternatively or additionally, the first and different O-antigens do not share group specificities (3,4), 6, (7,8), 9 and 10 (or the structural modifications that determine these group specificities). By “the structural modifications” we mean or include: [0128] Group Specificity 6: O-acetylation of Rhap.sup.I at position 2; [0129] Group Specificity 7,8: α-D-glucopyranosyl substitution on position 3 of Rhap.sup.III; [0130] Group Specificity 9: O-acetylation of Rhap.sup.III at position 3 or 4 (3/4-O-acetylation); and/or [0131] Group Specificity 10: O-acetylation of of GlcpNAc; [0132] and wherein [0133] Rhap.sup.I is the L-Rhap attached in a 1->3 linkage to β-D-GlcpNAc; [0134] Rhap.sup.II is the L-Rhap attached in a 1->3 linkage to α-L- Rhap.sup.I; and/or [0135] Rhap.sup.III is the L-Rhap attached in a 1->2 linkage to α-L- Rhap.sup.II.
[0136] The structural modification responsible for group 3,4 is not well defined. However, antibody typing sera is available that defines the presence or absence of the 3,4 specificity (3,4), 6, (7,8), 9 and 10, or the structural modifications that determine these group specificities (see, for example, Knirel et al., 2015, Biochemistry Moscow ‘O-Antigen Modifications Providing Antigenic Diversity of Shigella flexneri and Underlying Genetic Mechanisms’ 80(7):901-914, which is incorporated by reference herein, with particular reference to the structures listed in the table spanning pages 903-905).
[0137] As noted in the introduction, the limited cross-protection data available in the literature indicates that S. flexneri cross-protection cannot be predicted from known type- or group-specificities. Nevertheless, the present invention contemplates the inclusion of only those cross-protections that could not be predicted from the SBA scores of Table 2 that were based on shared group- and/or type-specificities. They may be defined by serotype versus serotype. Thus, alternatively or additionally, the first serotype or subserotype is: [0138] serotype 1 and the different serotype or subserotype is one or more serotype selected from the group consisting of 2, 5 and X, for example, 1, 2 or 3 of the different serotypes; [0139] serotype 2 and the different serotype or subserotype is one or more serotype selected from the group consisting of 4, 5, 6 and Y, for example, 1, 2 or 3 of the different serotypes; [0140] serotype 3 and the different serotype or subserotype is one or more serotype selected from the group consisting of 1, 2, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6 or 7 of the serotypes; [0141] serotype 4 and the different serotype or subserotype is one or more serotype selected from the group consisting of 1, 2, 5, X and Y, for example, 1, 2, 3, 4 or 5 of the serotypes; [0142] serotype 5 and the different serotype or subserotype is one or more serotype selected from the group consisting of 1, 2, 4, 6, X and Y, for example, 1, 2, 3, 4, 5 or 6 of the serotypes; [0143] serotype 6 and the different serotype or subserotype is one or more serotype selected from the group consisting of 5 and X, for example, 1 or 2 of the serotypes; [0144] serotype X and the different serotype or subserotype is one or more serotype selected from the group consisting of 1, 4, 6 and Y, for example, 1, 2, 3 or 4 of the serotypes; and/or p1 serotype Y and the different serotype or subserotype is 5.
[0145] They may also be defined by serotype versus subserotype. Hence, alternatively or additionally, the first serotype or subserotype is:
[0146] serotype 1 and the different serotype or subserotype is one or more subserotype selected from the group consisting of 2b, 5b and X, for example, 1, 2 or 3 of the different serotypes; [0147] serotype 2 and the different serotype or subserotype is one or more serotype selected from the group consisting of la, 4a, 5b, 6 and Y, for example, 1, 2, 3, 4 or 5 of the different serotypes; [0148] serotype 3 and the different serotype or subserotype is one or more serotype selected from the group consisting of la, 2a, 4a 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6 or 7 of the serotypes; [0149] serotype 4 and the different serotype or subserotype is one or more serotype selected from the group consisting of la, 2b, 5b, X and Y, for example, 1, 2, 3, 4 or 5 of the serotypes; [0150] serotype 5 and the different serotype or subserotype is one or more serotype selected from the group consisting of la, 2a, 4a, 6 and Y, for example, 1, 2, 3, 4 or 5 of the serotypes; [0151] serotype 6 and the different serotype or subserotype is one or more serotype selected from the group consisting of 5b and X, for example, 1 or 2 of the serotypes; [0152] serotype X and the different serotype or subserotype is one or more serotype selected from the group consisting of 1a, 4a, 6 and Y, for example, 1, 2, 3 or 4 of the serotypes; and/or serotype Y and the different serotype or subserotype is 5b.
[0153] They may further be defined by subserotype versus serotype. So, alternatively or additionally, the first serotype or subserotype is: [0154] subserotype 1a and the different serotype or subserotype is one or more serotype selected from the group consisting of 5 and X, for example, 1 or 2 of the serotypes; [0155] subserotype 1b and the different serotype or subserotype is one or more serotype selected from the group consisting of 2, 5 and X, for example, 1, 2 or 3 of the serotypes; [0156] subserotype 1c and the different serotype or subserotype is one or more serotype selected from the group consisting of 2, 5 or X for example, 1, 2 or 3 of the serotypes; [0157] subserotype 2a and the different serotype or subserotype is 1, 5 and Y for example, 1, 2 or 3 of the serotypes; [0158] subserotype 2b and the different serotype or subserotype is one or more serotype selected from the group consisting of 4, 6 and Y, for example, 1 or 2 of the serotypes; [0159] subserotype 3b and the different serotype or subserotype is one or more serotype selected from the group consisting of 1, 2, 4, 5, 6, X and Y, for example, 1, 2, 3, 4, 5, 6 or 7 of the serotypes; [0160] subserotype 4a and the different serotype or subserotype is one or more serotype selected from the group consisting of 5 and X, for example, 1 or 2 of the serotypes; [0161] subserotype 4b and the different serotype or subserotype is one or more serotype selected from the group consisting of 1, 2, 5, X and Y, for example, 1, 2, 3, 4 or 5 of the serotypes; [0162] subserotype 5a and the different serotype or subserotype is X; [0163] the first serotype is subserotype 5b and the different serotype or subserotype is one or more serotype selected from the group consisting of 1, 2, 4, 6, and Y, for example, 1, 2, 3, 4 or 5 of the serotypes; [0164] serotype 6 and the different serotype or subserotype is one or more serotype selected from the group consisting of 5 and X, for example, 1 or 2 of the serotypes; [0165] serotype X and the different serotype or subserotype is one or more serotype selected from the group consisting of 1, 4, 6 and Y, for example, 1, 2, 3 or 4 of the serotypes; and/or [0166] serotype Y and the different serotype or subserotype is 5.
[0167] However, they may be defined by subserotype versus subserotype. Thus, alternatively or additionally, the first serotype or subserotype is: [0168] subserotype la and the different serotype or subserotype is one or more subserotype selected from the group consisting of 5b and X, for example, 1 or 2 of the subserotypes; [0169] subserotype 1b and the different serotype or subserotype is one or more subserotype selected from the group consisting of 2b, 5b and X, for example, 1, 2 or 3 of the subserotypes; [0170] subserotype 1c and the different serotype or subserotype is one or more subserotype selected from the group consisting of 2b, 5b and X, for example, 1, 2 or 3 of the serotypes; [0171] subserotype 2a and the different serotype or subserotype is subserotype la, 5b and Y, for example, 1, 2 or 3 of the subserotypes; [0172] subserotype 2b and the different serotype or subserotype is one or more subserotype selected from the group consisting of 4a, 6 and Y, for example, 1, 2 or 3 of the subserotypes; [0173] subserotype 3b and the different serotype or subserotype is one or more subserotype selected from the group consisting of 1a, 2a, 4a, 5b, 6, X and Y, for example, 1, 2, 3, 4, 5, 6 or 7 of the subserotypes;
[0174] subserotype 4a and the different serotype or subserotype is one or more subserotype selected from the group consisting of 5b and X, for example, 1 or 2 of the subserotypes; [0175] subserotype 4b and the different serotype or subserotype is one or more subserotype selected from the group consisting of 1a, 2b, 5b, X and Y for example, 1, 2, 3, 4 or 5 of the subserotypes. [0176] subserotype 5a and the different serotype or subserotype is X; [0177] subserotype 5b and the different serotype or subserotype is one or more subserotype selected from the group consisting of 1a, 2a, 4a, 6 and Y, for example, 1, 2, 3, 4 or 5 of the subserotypes; [0178] serotype 6 and the different serotype or subserotype is one or more subserotype selected from the group consisting of 5b and X, for example, 1 or 2 of the subserotypes; serotype X and the different serotype or subserotype is one or more serotype selected from the group consisting of 1a, 4a, 6 and Y, for example, 1, 2, 3 or 4 of the subserotypes; and/or serotype Y and the different serotype or subserotype is subserotype 5b.
[0179] Alternatively or additionally, the first serotype or subserotype is serotype 1 and the one or more different serotype or subserotype comprises or consists of one or more serotype selected from the group consisting of 2, 5, 6, X, and Y, for example 1, 2, 3, 4 or 5 of these serotypes. Alternatively or additionally, the first serotype or subserotype is serotype 1 and the one or more different serotype or subserotype comprises or consists of serotype or subserotype 6. Alternatively or additionally, the first serotype or subserotype comprises or consists of 1a, 1b or 1c. Alternatively or additionally, the first serotype or subserotype is 1b.
[0180] Alternatively or additionally, the first serotype or subserotype is serotype 3 and the further serotype or subserotype is serotype 6. Alternatively or additionally, the first serotype or subserotype is one or more subserotype selected from the group consisting of 3a, 3b and 3c. Alternatively or additionally, the first serotype or subserotype is 3a.
[0181] Alternatively or additionally, the first serotype or subserotype is serotype 6 and the different serotype or subserotype is serotype 5. Alternatively or additionally, the different serotype or subserotype is one or more subserotype selected from the group consisting of 5a.
[0182] In contrast, the present invention also contemplates the exclusion of those cross-protections that could be predicted from the SBA scores of Table 2 that were based on shared group- and/or type-specificities. Hence, alternatively or additionally, the first serotype or subserotype is: [0183] subserotype 1a and the different serotype or subserotype is not 3a; [0184] subserotype 1b and the different serotype or subserotype is not one or more subserotype selected from the group consisting of 3a and 3b, for example, 1 or 2 of the subserotypes; subserotype 1c and the different serotype or subserotype is not 3a; subserotype 2a and the different serotype or subserotype is not one or more subserotype selected from the group consisting of 3a and 6, for example, 1 or 2 of the subserotypes; subserotype 2b and the different serotype or subserotype is not 3a; [0185] subserotype 3a and the different serotype or subserotype is not one or more subserotype selected from the group consisting of 1a, 2b and X, for example, 1, 2 or 3 of the subserotypes; [0186] subserotype 4a and the different serotype or subserotype is not one or more subserotype selected from the group consisting of 1b and 3a, for example, 1 or 2 of the subserotypes; [0187] subserotype 4b and the different serotype or subserotype is not 1b; [0188] subserotype 5a and the different serotype or subserotype is not one or more subserotype selected from the group consisting of 1b and 3a, for example, 1 or 2 of the subserotypes; [0189] the first serotype is subserotype 5b and the different serotype or subserotype is not X; [0190] serotype 6 and the different serotype or subserotype is not 3a; [0191] serotype X and the different serotype or subserotype is not one or more serotype selected from the group consisting of 2b and 3a, for example, 1 or 2 of the subserotypes; and/or [0192] serotype Y and the different serotype or subserotype is not one or more serotype selected from the group consisting of 1b, 2a and 3a, for example, 1, 2 or 3 of the subserotypes.
[0193] As discussed, an object of the present invention is to provide a broadly-protective vaccine against shigellosis that balances coverage versus complexity and cost. Accordingly, alternatively or additionally, the O-antigen of a first serotype is provided in combination with one or more additional O-antigen of a further serotype or subserotype, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 further O-antigen serotypes or subserotypes. For the avoidance of doubt, the first and additional O-antigens are of different subserotypes to one-another.
[0194] Alternatively or additionally, the first and further serotypes comprise or consist of combinations selected from the group consisting of: [0195] a. serotype 1 (for example, subserotype 1a, 1b, or 1c) and serotype 3 for example, subserotype 3a, 3b, or 3c); [0196] b. serotype 2 (for example, subserotype 2a, 2b, or 2c) and serotype 3 (for example, subserotype 3a, 3b, or 3c); [0197] c. serotype 3 (for example, subserotype 3a, 3b, or 3c) and serotype 4 (for example, subserotype 4a, or 4b); and [0198] d. serotype 3 (for example, subserotype 3a, 3b, or 3c) and serotype 5 (for example, subserotype 5a, or 5b).
[0199] Alternatively or additionally, the first and further subserotypes comprise or consist of combinations selected from the group consisting of: [0200] a. 1b and 3a; [0201] b. 1b and 3h; [0202] c. 1c and 3a; [0203] d. 1c and 3h; [0204] e. 2a and 3b; [0205] f. 3a and 4b; and [0206] g. 3b and 5b.
[0207] Since the present invention seeks to provide a broadly-protective vaccine that balances coverage versus complexity and cost, where a first O-antigen serotype or subserotype protects against a further serotype or subserotype, alternatively or additionally, one or more of the different serotype(s) or subserotype(s) is not provided, for example, one or more of 1a, 1b, 1c (or 7a), 1d, 2a, 2b, 3a, 3b, 4a, 4av, 4b, 5a, 5b, X, Xv, Y, Yv, 6 and 7b is not provided, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 of the different serotype(s) or subserotype(s) is not provided.
[0208] Alternatively or additionally, the Shigella flexneri O-antigen for use is capable of raising an immune response against one or more of the different serotype(s) or subserotype(s) that is not provided. Alternatively or additionally, serotype 1 (for example, 1a, 1b, or 1c) is provided and serotype 6 is not provided. Alternatively or additionally, serotype 3 (for example, 3a, 3b, or 3c) is provided and serotype 6 is not provided.
[0209] Alternatively or additionally, serotype 6 is provided and serotype 5 (for example, 5a or 5b) is not provided.
[0210] Further combinations of serotype(s) and/or subserotype(s) would be apparent to the skilled person from Table 2 and
[0211] As mentioned, an object of the invention is to provide broad protection against shigellosis. Hence, alternatively or additionally, O-antigen from one or more Shigella species other than Shigella flexneri is provided in combination with the O-antigen of a first serotype. Alternatively or additionally, the one or more other Shigella species is selected from the group consisting of: [0212] a. Shigella sonnei; [0213] b. Shigella boydii (for example, serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); and [0214] c. Shigella dysenteriae (for example, serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15).
[0215] Hence, alternatively or additionally, the Shigella flexneri O-antigen for use may comprise (e.g., may be provided with, either separately or as a mixture) O-antigen of S. sonnei, S. boydii, and S. dysenteriae. Alternatively or additionally, the Shigella flexneri O-antigen for use may comprise O-antigen of S. sonnei and S. boydii. Alternatively or additionally, the Shigella flexneri O-antigen for use may comprise O-antigen of S. sonnei and S. dysenteriae. Alternatively or additionally, the Shigella flexneri O-antigen for use may comprise O-antigen of S. boydii, and S. dysenteriae. Alternatively or additionally, the Shigella flexneri O-antigen for use may comprise O-antigen of S. sonnei. Alternatively or additionally, the Shigella flexneri O-antigen for use may comprise O-antigen of S. boydii. Alternatively or additionally, the Shigella flexneri O-antigen for use may comprise O-antigen of S. dysenteriae.
[0216] Alternatively or additionally, the S. sonnei is selected from the group consisting of S. sonnei, S. sonnei str. Moseley, S. sonnei 08-7761, S. sonnei 08-7765, S. sonnei 09-1032, S. sonnei 09-2245, S. sonnei 09-4962, S. sonnei 1 DT-1, S. sonnei 3226-85, S. sonnei 3233-85, S. sonnei 4822-66, S. sonnei S6513 and S. sonnei Ss046.
[0217] Alternatively or additionally, two or more Shigella flexneri O-antigen types are provided in combination and comprise or consist of the group consisting of Shigella flexneri 1b, Shigella flexneri 2a, Shigella flexneri 3a, and Shigella sonnei.
[0218] Alternatively or additionally, the O-antigen is obtained or obtainable from a bacterial strain comprising an alteration that reduces lipopolysaccharide (LPS) toxicity (in particular, its pyrogenic potential). Alternatively or additionally, the lipopolysaccharide (LPS) expression modifying alteration reduces the toxicity of the Shigella flexneri, outer membrane vesicle (OMV) released by it, and/or LPS produced by it, relative to the unaltered strain. Suitable methods for reducing toxicity and measuring that reduction are known, in the art, and can be found in, for example Rossi et al., 2014. Modulation of Endotoxicity of Shigella Generalized Modules for Membrane Antigens (GMMA) by Genetic Lipid A Modifications: Relative Activation of TLR4 and TLR2 Pathways in Different Mutants. J Biol. Chem., 289:24922-24935, which is incorporated by reference herein. Alternatively or additionally, the lipopolysaccharide (LPS) expression modifying alteration is induced by down-regulation, mutation or deletion (partial or complete) of one or more gene selected from the group consisting of: [0219] msbB1 (IpxM) (lipid A biosynthesis myristoyltransferase); [0220] msbB2 (IpxM) (lipid A biosynthesis myristoyltransferase); [0221] htrB (IpxL) (lipid A biosynthesis lauroyltransferase); [0222] IpxP (lipid A palmytoleoyl trasferase) [0223] pagP (adds palmitate to the primary linked acyl chain at 2-position Outer membrane); [0224] IpxE (removes 1-phosphate group); [0225] IpxF (removes 4-phosphate group); [0226] IpxO (adds hydroxyl group to fatty acid myristate at 3 position); [0227] IpxR (removes acyl chain(s) from 3 position); [0228] pagL (removes acyl chain(s) from 3-position).
[0229] Alternatively or additionally, the O-antigen or LPS is obtained or obtainable from a bacterial strain modified to augment OMV release. Strains of Shigella flexneri , Shigella dysenteriae, Shigella boydii and Shigella sonnei can be genetically modified to exhibit a hyper-blebbing phenotype by down-regulating or abolishing expression of one or more toIR or OmpA. Suitable mutations for down-regulating or abolishing expression include point mutations, gene deletions, gene insertions, and any modification of genomic sequences that results in a change in gene expression, particularly a reduction and more particularly inactivation or silencing.
[0230] The bacterium may be further genetically engineered by one or more processes selected from the following group: (a) a process of down-regulating expression of immunodominant variable or non-protective antigens, (b) a process of up-regulating expression of protective antigens, (c) a process of down-regulating a gene involved in rendering the lipid A portion of LPS toxic, (d) a process of up-regulating a gene involved in rendering the lipid A portion of LPS less toxic, and (e) a process of genetically modifying the bacterium to express a heterologous antigen.
[0231] Alternatively or additionally, one or more of the O-antigen(s) is/are provided: [0232] a. unassociated with another macromolecule; [0233] b. as a component of lipopolysaccharide (LPS), or a fragment thereof; or [0234] c. conjugated to another macromolecule, for example, a protein (e.g., a carrier protein such as CRM197, tetanus toxoid, meningococcal outer membrane protein complex (OMPC), diphtheria toxoid, and H. influenzae protein D [see, for example, Pichichero, 2013, ‘Protein carriers of conjugate vaccines Characteristics, development, and clinical trials’ Hum. Vaccin. Immunother., 9(12):2505-2523, which is incorporated by reference herein]).
[0235] Alternatively or additionally, the protein is a carrier protein (i.e., proteins capable of increasing the potency of the immune response against polysaccharide or other polymer a conjugated to it).
[0236] Alternatively or additionally, the first serotype or subserotype, further serotype or subserotype and/or other Shigella species is/are provided as one or more membrane component, for example, a cell membrane (for example a Gram-negative bacterium cell membrane) or a vesicle membrane (for example, Gram-negative bacterium outer membrane vesicle [OMV]).
[0237] Alternatively or additionally, wherein the membrane component is obtained from a bacterial cell wherein at least 25% of the O-antigen is the same serotype as the O-antigen for use; for example, at least 35%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the O-antigen is the same serotype as the O-antigen for use.
[0238] The percentage of different O-antigen types present can be determined using any suitable means known in the art, such as the method taught in Micoli et al., 2018, ‘Comparative immunogenicity and efficacy of equivalent outer membrane vesicle and glycoconjugate vaccines against nontyphoidal Salmonella’ PNAS, 115(41): 10428-10433 which is incorporated by reference herein.
[0239] Alternatively or additionally, the bacterial cell is a strain selected from the group consisting of: S. sonnei 53G, S. flexneri 1b Stansfield, S. flexneri 2a 2457T, S. flexneri 2b 69/50, S. flexneri 3a str. 6885 and S. flexneri 6 str. 10.8537.
[0240] Alternatively or additionally, the membrane component is a component of an OMV selected from the group consisting of a detergent-extracted OMV (dOMV); or native OMV (nOMV).
[0241] Alternatively or additionally, the OMV is produced from genetically-modified bacterial strains that are mutated to enhance vesicle production and to remove or modify antigens (for example, lipid A).
[0242] Shigella bacteria used in the invention are, relative to their corresponding wild-type strains, hyperblebbing i.e. they release into their culture medium larger quantities of GM MA than the wild-type strain. These GMMA are useful as components of Shigella vaccines of the invention. The term GM MA is used to provide a clear distinction from conventional detergent-extracted outer membrane vesicles (dOMV), and native outer membrane vesicles (NOMV), which are released spontaneously from Gram-negative bacteria. GMMA differ in two crucial aspects from NOMV. First, to induce GMMA formation, the membrane structure has been modified by the deletion of genes encoding key structural components, specifically toIR. Second, as a consequence of the genetic modification, large quantities of outer membrane “bud off” (the Italian word for bud is ‘gemma’) to provide a practical source of membrane material for vaccine production, leading to increased ease of manufacturing and potential cost reduction. While NOMV have been used for immunogenicity studies, the yields are too low for practical vaccines.
[0243] S. sonnei GMMA used in the invention typically have a diameter of from 25 nm to 140 nm by electron microscopy, for example from 25 nm to 40 nm. GMMA may also have a bimodal size distribution. For example, the majority of GMMA having an average size from 25 nm to 40 nm in diameter (by EM) and a fraction of the particles having an average size from 65 nm to 140 nm. Particularly, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 80%, at least 85%, at least 90% of the GMMA will have a diameter of from 25 nm to 140 nm.
[0244] GMMA are released spontaneously during bacterial growth and can be purified from the culture medium. The purification ideally involves separating the GMMA from living and/or intact Shigella bacteria, for example, by size-based filtration using a filter, such as a 0.2 μm filter, which allows the GMMA to pass through but which does not allow intact bacteria to pass through, or by using low speed centrifugation to pellet cells while leaving GMMA in suspension. Suitable purification methods are known in the art. A preferred two-step filtration purification process is described in WO2011/036562 herein incorporated by reference. Particularly the two-step filtration process is used to separate GMMA from cell culture biomass without using centrifugation.
[0245] GMMA containing compositions of the invention will generally be substantially free from whole bacteria, whether living or dead. The size of the GMMA means that they can readily be separated from whole bacteria by filtration e.g. as typically used for filter sterilisation. Although GMMA will pass through a standard 0.22 μm filters, these can rapidly become clogged by other material, and so it may be useful to perform sequential steps of filter sterilisation through a series of filters of decreasing pore size before using a 0.22 μm filter. Examples of preceding filters would be those with pore size of 0.8 μm, 0.45 μm, etc. GMMA are spontaneously-released from bacteria and separation from the culture medium, for example, using filtration, is convenient. Outer membrane vesicles formed by methods which involve deliberate disruption of the outer membrane (e.g. by detergent treatment, such as deoxycholate-extraction, or sonication) to cause outer membrane vesicles to form are excluded from the scope of the invention. GMMA used in the invention are substantially free from inner membrane and cytoplasmic contamination and contain lipids and proteins.
[0246] Shigella strains for use in the invention include one or more further changes relative to a wild-type strain. Particularly, strains for use with the invention include one or more mutations resulting in inactivation of htrB, msbB1 and/or msbB2. By way of non-limiting example, suitable mutations may be selected from the group consisting of ΔhtrB, ΔmsbB1 and ΔmsbB2.
[0247] Alternatively or additionally, the immune response is an immune activating response. As used herein “immune activating response” includes or means an immune response that increases inflammation, antibody-directed cell death and/or dormancy, and/or complement-mediated cell death and/or dormancy.
[0248] Alternatively or additionally, the immune response is antibody-directed. As used herein “antibody-directed” includes or means the induction of cell death and/or dormancy by an antibody-dependent mechanism.
[0249] Alternatively or additionally, the immune response comprises or consists of a protective immune response, e.g., an in vitro protective immune response and/or an in vivo protective immune response.
[0250] Alternatively or additionally, the immune response comprises or consists of complement-mediated killing.
[0251] As used herein, “complement-mediated killing” includes or means the induction of cell death and/or dormancy by a complement-dependent mechanism. Complement-mediated killing can be measured by any suitable means known to the skilled person, in particular, serum bactericidal assay (SBA) as described in the Examples section below.
[0252] Alternatively or additionally, the immune response comprises or consists of prevention or reduction of entry of Shigella flexneri cells into host macrophages and/or epithelial cells.
[0253] Measurement of S. flexneri interaction with and/or entry into host macrophages and/or epithelial cells can be determined using any suitable means known in the art, such as the methods taught in Raygoza-Anaya et al., 1990 ‘In vitro model for the analysis of the interaction between Shigella flexneri and the intestinal epithelium’ Arch. Invest. Med. (Mex), 21(4):305-9; Willer Eda et al., 2004, ‘In vitro adhesion and invasion inhibition of Shigella dysenteriae, Shigella flexneri and Shigella sonnei clinical strains by human milk proteins’ BMC Microbiol., 28; 4:18; Guhathakurta et al., 1999, ‘Adhesion and invasion of a mutant Shigella flexneri to an eukaryotic cell line in absence of the 220-kb virulence plasmid’ FEMS Microbiol. Lett., 181(2):267-75; or Bando et al., 2010, ‘Expression of bacterial virulence factors and cytokines during in vitro macrophage infection by enteroinvasive Escherichia coli and Shigella flexneri: a comparative study’ Mem. Inst. Oswaldo Cruz., 105(6):786-91, which are each incorporated by reference herein.
[0254] Since this organism is unable to invade epithelial cells through the apical route, Shigella exploits M cells, the specialized epithelial cells in the follicular associated epithelium (FAE) that overlie lymphoid tissue, to gain entry into the colonic epithelium (Wassef et al. 1989). M cells allow intact Shigella to traverse into the underlying subepithelial pocket where macrophages reside. Macrophages engulf Shigella, but instead of successfully destroying the bacteria in the phagosome, the macrophage succumbs to apoptotic death (Zychlinsky et al. 1992). Prior to cell death, infected macrophages release IL-1b through the direct activation of caspase-1 by Shigella (Zychlinsky et al. 1994). The pro-inflammatory nature of this cytokine results in the recruitment of polymorphonuclear cells (PMNs) that infiltrate the infected site and destabilize the epithelium (Perdomo et al. 1994a,b). Loss of integrity of the epithelial barrier allows more bacteria to traverse into subepithelial space and gives these organisms access to the basolateral pole of the epithelial cells (Mounier et al. 1992). Shigella can then invade the epithelial cells lining the colon, spread from cell to cell and disseminate throughout the tissue. Cytokines released by infected epithelial cells attract increased numbers of immune cells to the infected site, thus compounding and exacerbating the inflammation.
[0255] Shigellosis produces a spectrum of clinical outcomes ranging from watery diarrhoea to classic dysentery characterized by fever, violent intestinal cramps and discharge of mucopurulent and bloody stools. Inflammation of the infected tissue is a key feature of shigellosis. Histopathological studies of colonic biopsies from infected patients reveal inflammatory cell infiltration into the epithelial layer, tissue oedema and eroded regions of the colonic epithelium (Mathan & Mathan 1991).
[0256] Alternatively or additionally, the immune response prevents, abolishes or reduces one or more symptom of Shigella flexneri infection selected from the group consisting of: [0257] a. watery diarrhea; [0258] b. fever; [0259] c. intestinal cramps; [0260] d. abdominal pain; [0261] e. tenesmus; [0262] f. mucopurulent stools; [0263] g. bloody stools; [0264] h. inflammation of infected tissue (e.g., colon tissue [e.g., inflammatory cell infiltration into the epithelial layer]); [0265] i. oedema of infected tissue (e.g., colon tissue); [0266] j. faecal haemoglobin; [0267] k. bacterial shedding; [0268] l. erosion of colonic epithelium (number of eroded regions, diameter, depth); and [0269] m. macrophage apoptotic cell death.
[0270] By “prevents, abolishes or reduces” we include or mean reduction in the symptom by at least 25%, at least 50%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or at least 100%. Alternatively or additionally, the one or more symptom is reduced by at least 10%, for example, reduced by at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.
[0271] By “raising an immune response” we mean or include that the immune system is activated in a host following exposure to an antigen (e.g., the Shigella flexneri O-antigen).
[0272] Alternatively or additionally, the immune response is raised in a mammal.
[0273] Alternatively or additionally, the mammal is selected from the group consisting of armadillo (Dasypus novemcinctus), baboon (Papio anubis; Papio cynocephalus), camel (Camelus bactrianus, Camelus dromedarius, Camelus ferus), cat (felis catus), dog (canis lupus familiaris), horse (Equus ferus caboilus), ferret (Mustela putorius furo), goat (Capra aegagrus hircus), guinea pig (cavia porcellus), golden hamster (Mesocricetus auratus), kangaroo (macropus rufus), llama (Lama glama), mouse (Mus musculus), pig (Sus scrofa domesticus), rabbit (Oryctolagus cuniculus), rat (Rattus norvegicus), Rhesus macaque (Macaca mulatta), sheep (Ovis aries) and human (Homo sapiens).
[0274] Alternatively or additionally, the protective immune response is protective against a disease or condition caused by an organism selected from the group consisting of: Shigella sonnei, Shigella flexneri, Shigella and Shigella dysenteriae.
[0275] The terms “OMV” and “GMMA” may be used interchangeably herein.
[0276] A second aspect provides a binding moiety capable of specifically binding to one or more O-antigen defined in the first aspect.
[0277] By “specifically binding” we mean or include that the binding moiety binds at least 10-fold more strongly to its target antigen or epitope than to any other antigen or epitope (in particular, any other Shigella [in particular, S. flexneri]) O-antigen or fragment thereof); preferably at least 50-fold more strongly and more preferably at least 100-fold more strongly. Preferably, the binding moiety of the invention specifically binds to the antigen or epitope under physiological conditions (for example, in vivo; and for example, during S. flexneri infection). Binding strength can be measured by surface plasmon resonance analysis using, for example, a Biacore™ surface plasmon resonance system and Biacore™ kinetic evaluation software (e.g., version 2.1).
[0278] Alternatively or additionally, the binding moiety is selected from the group consisting of: antibodies; antigen-binding fragments; and antibody mimetics. Alternatively or additionally, the binding moiety is an antibody. Alternatively or additionally, the antibody is polyclonal or monoclonal. Alternatively or additionally, the binding moiety is an antigen-binding fragment selected from the group consisting of: Fab (fragment antigen binding); F(ab′)2; Fab′; scFv (single chain variable fragment); di-scFv; sdAb (single domain antibody/domain antibody); trifunctional antibody; chemically-linked F(ab′)2; and BiTE (bi-specific T-cell engager). Alternatively or additionally, the antibody or antigen binding fragment thereof is an antigen binding fragment selected from the group consisting of affibodies molecules; affilins; affimers; affitins; alphabodies; anticalins; avimers; DARPins; fynomers; kunitz domain peptides; monobodies and nanoCLAMPs.
[0279] A third aspect provides a pharmaceutical composition comprising an O-antigen for use defined in the first aspect and/or a binding moiety as defined in the second aspect. Alternatively or additionally, the composition comprises an adjuvant. Yet more particularly, the adjuvant is an adsorbent. Still yet more particularly, the adjuvant is an adsorbent that does not enhance immunogenicity of GMMA, for example, as measured by anti- LPS antibody response. Particular adjuvants include, for example, aluminium adjuvants including aluminium hydroxide, ALHYDROGEL®, aluminium phosphate, potassium aluminium sulphate and alum.
[0280] A fourth aspect provides a kit comprising or consisting of an O-antigen for use defined in the first aspect, a binding moiety as defined in the second aspect and/or a pharmaceutical composition as defined in the third aspect; and (optionally) instructions for use.
[0281] A fifth aspect provides an O-antigen for use defined in the first aspect, a binding moiety as defined in the second aspect, a pharmaceutical composition as defined in the third aspect and/or a kit as defined in the fourth aspect, for use in medicine.
[0282] A sixth aspect provides an O-antigen for use defined in the first aspect, a binding moiety as defined in the second aspect, a pharmaceutical composition as defined in the third aspect and/or a kit as defined in the fourth aspect, for use in preventing or treating bacterial infection and/or symptoms thereof.
[0283] Alternatively or additionally, the bacterial infection is, wholly or in part, infection with one or more bacterium defined in the first aspect.
[0284] A seventh aspect provides an effective amount of an O-antigen in the first aspect, a binding moiety as defined in the second aspect, a pharmaceutical composition as defined in the third aspect and/or a kit as defined in the fourth aspect for use in the manufacture of a medicament for treating for the prevention or treatment of bacterial infection and/or symptoms thereof (e.g., where the bacterial infection is, wholly or in part, infection with one or more bacterium defined in the first aspect).
[0285] An eighth aspect provides a method of treating or preventing bacterial infection and/or symptoms thereof comprising administering a suitable amount of an O-antigen for use defined in the first aspect, a binding moiety as defined in the second aspect, a pharmaceutical composition as defined in the third aspect and/or a kit as defined in the fourth aspect.
[0286] A ninth aspect provides a binding moiety as defined in the second aspect for detecting the presence of bacteria, for example, wherein the bacteria are one or more bacterium defined in the first aspect. Alternatively or additionally, the detection is in vitro and/or in vivo.
[0287] A tenth aspect provides an O-antigen, binding moiety, pharmaceutical composition, kit, use or method as described in the specification and figures herein.
EXAMPLES
[0288] 1. Introduction
[0289] A broadly-protective vaccine against shigellosis needs to cover multiple S. flexneri serotypes. A challenge is to design a practical vaccine that balances coverage versus complexity and cost. Importantly, we found, based on immunogenicity in mice, that a simple three-component vaccine of GMMA from S. sonnei, S. flexneri 1b and 3a would induce killing of most epidemiologically significant Shigella strains. This was not predicted based on cross-reactivity of currently described shared serotypes and serogroups. We don't know how these results translate to human immunogenicity there are data that show humans recognized some Shigella serospecificities differently to mice. However, the study presented herein provides a framework for empirically designing such a vaccine for upcoming human vaccine trials.
[0290] 2. Materials and Methods
[0291] 2.1 Shigella Strains
[0292] S. sonnei 53G (32) was obtained from Walter Reed Army Institute of Research, Washington, D.C., USA. The S. sonnei ΔvirG::cat strain used in FACS and SBA was generated by Caboni et al. (33) to ensure a stable expression of OAg during growth by stabilization of the pSS virulence plasmid that contains the OAg cluster genes by culturing the bacteria in presence of chloramphenicol.
[0293] S. flexneri lines of the 14 subtypes were purchased from the Public Health England, London, UK. Working cell banks were prepared and typed using both agglutination and surface staining by FACS typing with the commercial Shigella typing antisera from Denka Seiken Co., Ltd; the type specific serum I, II, III, IV, V, VI and grouping sera 3,4; 6; 7,8; 9; 10. Manufacturer's recommendations were followed for the agglutination. For FACS typing, bacteria were grown in LB medium, diluted to 2×10.sup.7 CFU/mL in PBS, then 50 μl were transferred in 96 well plate on ice, incubated with 1:400 dilution of typing and grouping antisera from Denka Seiken Co. Ltd., washed, then incubated with 1:1,000 dilution of fluorescein-conjugated F(ab′)2 fragment goat anti-rabbit IgG specific (Jackson Immuno Research Europe Ltd.). The cells were then fixed for 3 h with BD Cytofix® (containing 4.2% formaldehyde), washed and then resuspended in 130 μl PBS. Samples were measured with a BD FACS Canto equipped with a high throughput sample reader using BD FACS DIVA version 8.0.1 software. Cells were gated on FSC-A versus SSC-A. The signal was then measured (FITC/fluorescein channel). Analyses were performed with FlowJo version 10.3 (FlowJo, LLC, Ashland, Oreg.). The Mean Fluorescence Intensity (MFI) was used as the measure of strength of the staining. All lines gave the expected typing pattern. For S. flexneri X the reaction with group 7,8 antisera was weak; this weak reaction was not confirmed in the clone selected for GMMA production. By FACS analysis, an instability of the S. flexneri 5b cell line was identified; the population had a mixture of cells that were positive or negative for group 7,8 and thus a mixed S. flexneri 5a/5b phenotype, presumably due to variable expression of the gtrXgene encoding the glycosyl-transferase that distinguishes S. flexneri 5a from 5b. This was also true of the GMMA producing line derived from this line and thus the GMMA used for vaccination were probably a mixture of S. flexneri 5a and 5b. For use in the FACS and SBA assays, a new working cell line was selected from the S. flexneri 5b bacterial cells that uniformly reacted strongly with the group 7,8 antisera.
[0294] In addition to the serological typing, the lines used for the GMMA production and the target panel were genotyped by PCR for the genes that encode the group specific 9 (oacB or oacC) and 10 (oacD) phenotypes. The PCR reaction mixtures contained 12.5 μL DreamTaq Green PCR Master Mix (2×), 9.5 μL sterile water, 1 μL 10 mM forward primer, 1 μL 10 mM reverse primer and 1 μL template (bacteria suspended in water to an OD600 of 5). After amplification, the presence of the amplified gene was detected following electrophoresis on ethidium bromide stained agarose gels.
[0295] 2.2. GMMA Production, Purification and Formulation
[0296] To generate the GMMA producing lines, the toIR gene was deleted as described for the generation of the S. sonnei ΔtoIR mutant (34). The resulting clonal lines were re-typed to assure that the cloning process had not changed serotype and serogroup specificities.
[0297] These GMMA were used to immunize mice, but the resulting sera did not react with OAg positive homologous bacteria and the results are not included in this study. As for the parent line, most, but not all, of the S. flexneri 5b GMMA producing bacteria were typed by FACS as S. flexneri 5a (i.e. negative for group 7,8). These GMMA were used to immunize mice and the resulting sera were included in the cross-reaction panel testing. Bacterial strains were grown at 30° C. on LB agar or in liquid chemically defined medium (SDM), as described (34, 35). When required, kanamycin (30 μg/mL), was added for selection of the GMMA producing strains. For GMMA production, overnight cultures were used to inoculate the SDM at an OD.sub.600 of 0.03-0.05 and incubated at 30° C. and 200 rpm to an OD.sub.600 8-10. Culture supernatants were collected by centrifugation followed by a 0.22-μm filtration, ultracentrifuged and the resulting pellet containing GMMA was resuspended in PBS as described (35).
[0298] GMMA quantities were expressed as total protein present using the micro-BCA protein assay (Bio-Rad) kit according to the manufacturer's instructions, using Bovine serum albumin (Pierce) for the standard curve. The amount of OAg in the GMMA was determined by HPAEC-PAD analysis by measuring rhamnose content, (3 rhamnose residues per repeating units (RU) for all S. flexneri serotypes except S. flexneri 6 for which there are 2). The OAg to protein ratio in the GMMA varied from 0.39 to 0.8 (Table S2). GMMA from S. flexneri X contained lower amount of OAg (the OAg/protein ratio was 0.12 for S. flexneri X).
[0299] The GMMA were adsorbed onto aluminum hydroxide (Alhydrogel 2%, Brenntag Biosector, Denmark). GMMA were added to Alhydrogel to give 4 μg/mL GMMA protein and 0.7 mg Al3+/mL in 10 mM Tris, pH 7.4 and 9 g/L NaCl, then stirred for 2h. Preparations were tested to show they had no bacterial contamination and were stored at 2-8° C. for one week prior to use.
[0300] 2.3. Immunogenicity Studies in Mice
[0301] Animal studies were performed as part of the Italian Ministry of Health Animal Ethics Committee project number 201309. Four CD1 mice per group (female, 4 to 6 weeks old) were immunized intraperitoneally (500 μL each mouse) with 2 μg of GMMA (protein) on days 0 and 21; sera were collected on day 21 and 35 (bleed out). The day 35 sera were pooled and used for the studies reported in this paper.
[0302] 2.4. Cross-Reactivity Measured by FACS
[0303] Prior to assessment of cross-reactivity, all the S. flexneri bacteria from the different serotypes used in the study were tested for binding of sera raised against OAg negative S. flexneri 2a GM MA using the methodology described below.
[0304] Surface staining of the panel of 11 OAg positive S. flexneri lines was carried out with the pooled day 35 sera 10 from the 14 immunization groups and pooled sera similarly raised against an OAg negative S. flexneri 2a GMMA. The sera were also tested on OAg positive and negative S. sonnei and the sera raised against OAg negative S. flexneri 2a were also tested on OAg negative S. flexneri 2a bacteria.
[0305] The pooled day 35 sera, were added to the bacterial suspensions, incubated for 1 h, washed, then APC-conjugated anti-mouse IgG (1:400 dilution) was added and incubated for 1 h. The signal was then measured in the allophycocyanin (APC) channel. The baseline was set by S. flexneri 1b, 2a, 3a and 6 controls incubated only with the secondary antibodies and without any mouse serum. A matrix showing the mean fluorescence intensities (MFI) of surface staining of S. flexneri wild type bacteria lines of the different serotypes is reported in Table S3.
[0306] 2.5. High Through-Put Luminescence—Serum Bactericidal Assay (L-SBA)
[0307] SBA were performed as described (36). Briefly, S. sonnei and S. flexneri bacteria derived from the same working cell banks used for the FACS were grown to log-phase (OD: 0.2), diluted 1:1,000 in PBS and distributed in 96-well plates. To each well, dilutions of heat-inactivated pooled mouse sera and active Baby Rabbit Complement (BRC; 7-20% of the final volume) were added. As control, bacteria were incubated with sera plus heat-inactivated BRC, sera alone (no BRC), SBA buffer or active BRC. After 3 h incubation, surviving bacteria were determined by measuring ATP. SBA is reported in serum titers, defined as serum dilutions giving 50% inhibition of the ATP level in the positive control. Titers below the minimum measurable titer of 100 was assigned titer of 10. A matrix showing serum titers on S. flexneri wild type cell lines of the different serotypes is reported in Table S4.
[0308] 2.6. Modelled SBA Heat Map
[0309] The observed average log (SBA titer) for sera tested on the homologous serotypes (i.e. anti-S. flexneri 2a antisera tested on S. flexneri 2a or on S. flexneri 2b) was 4.7. Therefore, in constructing a theoretical SBA heat map, the SBA log titer) for sera tested on homologous serotypes was assigned a value of 4.7. The observed average SBA log titer tested on heterologous serotypes where the SBA was measurable was 3.9. Where a vaccinating GMMA shared a single strongly typing group specificity we assigned a value of 3.9 to this interaction. As shown in Table S1, typing of the target bacteria with the standard group-specific reagents showed several strains that gave positive but weak interaction with typing reagents. On average these had log MFI that were 0.9 (group 3,4) or 0.7 (group 7,8) log units lower than the high responders. In this case we assigned a value of 3.1 (i.e. 0.8 log units lower than the high responders) to the modelled SBA value (we assumed that a weakly typing positive GMMA producing strain still had sufficient group specific antigen to elicit a full group specific antibody response). Where the immunizing GMMA and the target bacteria shared two group specificities we assigned an SBA log titer as the log of the sum of the titers. Thus, the modelled titer of anti-S. flexneri la GMMA on S. flexneri 2a that share both the 3,4 and the 9 group specificities is assigned an SBA log titer of 4.2=log (10{circumflex over ( )}3.9+10{circumflex over ( )}3.9). For both the observed SBA titers and the modelled SBA titers, a calculated SBA log titer that could be obtained by immunizing with a mixture of S. flexneri 1a and 3a was calculated similarly: e.g. the estimated SBA log titer of a mixture of anti-S. flexneri 1b and 3a GMMA on S. flexneri 2a was 4.0=log (10{circumflex over ( )}3.9+10{circumflex over ( )}3.1).
[0310] 3. Results
[0311] 3.1. Serotype and Group Specificities of the Bacteria Used in this Study
[0312] A summary of the serotype and group specificities of the bacteria used in this study based on typing with specific antisera or inferred by the presence of genes encoding O-acetylases are shown in Table 1. The presence of O-acetylation was demonstrated by NMR for S. flexneri 1b, 2a and 3a. The details of the typing are included in Table S1.
[0313] 3.2. Evaluation of Cross-Reactivity and Cross Functionality of Antibodies Raised in Mice Against GMMA from One Subtype of S. flexneri on heterologous S. flexneri Subtypes 3.2.1. Evaluation of Cross-Reactivity by FACS
[0314] A heat map was generated with the Logio of the Mean Fluorescence Intensities (Log MFI) of surface staining of a panel of S. flexneri bacteria to visualize the cross-reactivity patterns (
[0315] Binding of sera raised against OAg negative GMMA: Antisera raised against OAg negative S. flexneri 2a GMMA (GMMA from S. flexneri 2a ΔtoIR ΔrfbG) gave strong fluorescence on OAg negative S. flexneri 2a bacteria (MFI 5000) and OAg negative S. sonnei (MFI 6300); binding was undetectable on all tested OAg positive bacteria, including OAg positive S. flexneri 2a.
[0316] 3.2.2. Binding of Sera Raised Against O Antigen Positive GMMA
[0317] 3.2.2.1. Binding to O Antigen Negative S. sonnei
[0318] All the antisera raised with OAg positive GMMA gave detectable binding to OAg negative S. sonnei. Anti-S. flexneri 4b had the weakest binding (MFI 40). All including anti-S. flexneri 4b, gave an MFI that was more intense on the S. sonnei OAg negative GMMA than to at least one of the OAg positive S. flexneri tested.
[0319] 3.2.2.2. Homologous Binding (Binding to the Parent Bacteria of the Immunizing GMMA)
[0320] All homologous sera gave strong binding, ranging from MFI of 4,508 (Log MFI 3.7) for S. flexneri 5b to 98,520 (Log MFI 5.0) for S. flexneri 2b, except for S. flexneri X that gave relatively weak binding to S. flexneri X bacteria (MFI 541, log MFI 2.7). S. flexneri 4b pooled serum gave generally weak binding but was not tested for binding to the parent S. flexneri 4b.
[0321] 3.2.2.3. Heterologous Binding (Binding to Bacteria not the Parent of the Immunizing GMMA)
[0322] For most of the antisera tested, the highest level of cross-reaction was identified among homologous serotypes (S. flexneri serotypes having a common glucosyl or acetyl modification at the same position on the OAg backbone, e.g. S. flexneri 1c antisera binding to S. flexneri 1a and 1b bacteria). The level of cross-reactivity varied: antisera from S. flexneri 2a GMMA strongly reacted only with the homologous serotypes and only weakly with two other serotypes S. flexneri 4a and Y (i.e. with an MFI>130 for 2/9 heterologous serotypes tested). By contrast, antisera against S. flexneri 1b GMMA elicited broad cross-reactions to homologous serotypes and most heterologous serotypes giving an MFI>130 to 7/9 subtypes from heterologous serotypes. Thus S. flexneri 1b, 1c, 3b, 4a, 5a and 5b GMMA are broad-specificity immunogens by FACS (MFI>130 on ≥60% heterologous serotypes/subtypes); S. flexneri 1a, 2b, 3a and X, medium-specificity immunogens (MFI>130 on 50% to <60% heterologous serotypes/subtypes) and S. flexneri 2a, 6 and Y, narrow-specificity immunogens (MFI>130 on <50% heterologous serotypes/subtypes). S. flexneri 4b GMMA had an indeterminate breadth of specificity. As the 4b GMMA failed to generate strong binding to homologous serotypes (i.e. S. flexneri 4a) and to OAg negative bacteria, the lack of binding to other serotypes may be indicative of a poor immunogenicity of these GMMA.
[0323] The subtypes varied considerably in their ability to be recognized by heterologous sera. Some of the subtypes were widely recognized by many different antisera, specifically S. flexneri 1a, 4a, 5b, 6, X and Y. Thus, these are broad-specificity targets. By contrast, some subtypes were only recognized by a few antisera. S. flexneri 3b was the most restricted target, only recognized strongly by sera raised against S. flexneri 3a or 3b and weakly by sera raised against S. flexneri 4b. S. flexneri 3a was the next most restrictedly recognized subtype with binding only by anti-S. flexneri 3b and 5b antisera. By these criteria, S. flexneri 1b, 2a, 2b, 3a and 3b are narrow-specificity targets.
[0324] As expected, S. sonnei bacteria were not stained by any of the S. flexneri GMMA antisera.
[0325] 3.3. Evaluation of Cross-Functionality by Serum Bactericidal Activity (SBA)
[0326] A heat map of SBA data containing the Log.sub.10 IC50 of the pooled sera on S. flexneri bacterial cell lines is shown in
[0327] The binding of antibodies judged by FACS and killing as judged by SBA was similar (
[0328] On the other hand, the heat map of SBA data poorly correlated with a heat map predicted from the reactivity expected from serotype and group antigens (
[0329] 4. Discussion
[0330] There are only a few reports of cross-reactivity among S. flexneri serotypes and subtypes in the literature. An extensive screening using preclinical animal models to identify cross-reactive antibodies and the structural basis of cross-reactivity has not been carried out.
[0331] In this study we used FACS and SBA, the two techniques that give a direct measure of interactions between host antibody response and infective bacteria. The SBA assay is the method of choice to evaluate the complement-mediated functional activity of antibodies induced by a bacterium during infection; additionally, for Neisseria meningitidis, SBA is the accepted correlate of protection on which the vaccine for N. meningitidis is registered.
[0332] GMMA contain all the outer membrane components of their parent bacteria (19) and thus could elicit antibodies that bind to many bacterial surface components. Indeed, as measured by FACS, OAg negative GMMA (i.e. S. flexneri 2a ΔtoIR ΔrfbG GMMA) elicit antibodies that strongly bind to bacteria without OAg, suggesting that the GMMA can induce a broad range of antibody responses. However, three observations from this study show that the antibody induced by OAg positive GMMA measured by FACS and by SBA on OAg positive bacteria are dominantly directed against the OAg: [0333] 1. The observed FACS and SBA responses are predominantly serotype or subtype specific and no S. flexneri GMMA induced immune responses that recognized S. sonnei which has a similar LPS core oligosaccharide (21) and most of the outer membrane proteins (19). Furthermore, for each pool of anti-S. flexneri antisera there was at least one subtype of S. flexneri bacteria to which the antisera failed to give a binding stronger than that seen on S. sonnei, again despite sharing most outer membrane components other than the OAg. The negative subtypes differed depending on the specificity of the pool, e.g. anti-S. flexneri 1a gave no detectable binding to S. flexneri 3a (
[0336] Although all the sera have antibodies capable of significant binding to the surface of bacteria, they are unable to do so if the bacteria have an OAg coat. This is in agreement with earlier findings from immunization studies with intact bacteria (22) suggesting that the OAg shields the bacteria from binding to antigen on the surface of the outer membrane and that the observed binding is to dominant surface components that do differ from one serotype to another—i.e. the OAg.
[0337] There are two important consequences for antibodies generated by GMMA:
[0338] The observed strain specificity and cross-reactivity must predominantly be directed against epitopes in the OAg of each serotype.
[0339] The OAg specificities induced by GMMA will be important for inducing broad protection from a vaccine by binding of antibody to the surface of bacteria.
[0340] This is consistent with the observation that the immunity in humans elicited by attenuated Shigella strains is dominantly OAg specific and with the results of earlier animal studies with immunization by killed or attenuated bacteria (23-25). Given the complex mechanism by which Shigella invades the intestinal lumen and the infection is established, this does not rule out protection via other mechanism not involving OAg, e.g. T cell response against macrophages or other cells containing intracellular Shigella (26-29).
[0341] The data from both FACS and SBA showed that, as expected, the different GMMA generated substantial cross-reactivity on strains of S. flexneri that shared the same serotype specificities. For example, antisera to S. flexneri 2a GMMA bound strongly to S. flexneri 2b bacteria and vice versa. These two serotypes only share the Type II epitopes and no group specificities. Importantly there was also substantial binding to strains that did not share the same type specificities. For example, antisera to S. flexneri 1a, 1b and 1c GMMA bound strongly to S. flexneri 2a bacteria.
[0342] It has been generally assumed that for immunizing and target pairs that do not share the same type specificity, cross-reactivity will be mediated by the group specificities (i.e. epitopes 3,4; 6; 7,8; 9 and 10). This was the basis of the experimental cross-protecting vaccine developed by Noriega et al., (13) based on attenuated S. flexneri 2a and S. flexneri 3a to deliver Type II and III and group 3,4; 6 and 7,8 specificities.
[0343] However, the pattern of cross-reactivity observed with the larger panel in this GMMA study was unexpected: detailed comparison of the cross-protection modelled on shared group specificities (
[0344] Therefore, we conclude that most of the cross-reactivity cannot be explained by group specificities.
[0345] A feature was the lack of reciprocity between immunogen and antigen. For example, S. flexneri 3b GMMA generated substantial SBA titers and to a lesser extent FACS MFI against all 9 of the 10 non-homologous OAg positive S. flexneri strains tested. By contrast, other than a weak reactivity generated by S. flexneri 4b, the only other strain to generate detectable SBA/FACS activity against S. flexneri 3b, was S. flexneri 3a. Similarly, S. flexneri 1b GMMA generated substantial SBA/FACS activity against 8/10 heterologous S. flexneri OAg positive S. flexneri strains (except S. flexneri 3a and 3b). In fact, the cross-reactivity was so broad that a bivalent vaccine consisting only of S. flexneri 1b and 3a could give antibodies in the mouse that react strongly with all isolates tested (
[0346] The opposite was also observed. GMMA from S. flexneri 2a, 4b, X and Y and, to a lesser extent, S. flexneri 6 generated antibody that reacted with relatively few other isolates. All, except S. flexneri 4b, generated significant reaction by FACS to OAg negative S. sonnei, suggesting that they were intrinsically immunogenic, at least for non OAg components. In contrast to the poor immunogenicity observed, S. flexneri 2a, 4b, 5a, 6, X and Y were commonly recognized by antisera from other serotypes suggesting that inclusion of these serotypes in a vaccine would be less critical since there would be a high likelihood of being covered through cross-reactions.
[0347] Finding that the cross-reactivities do not match the known group specificities reflects older data on the generation of type and group specific typing sera. Initially sera raised against a strain of bacteria have extensive cross-reactions and it is only after exhaustive adsorption to remove the cross-reactions that the sera are useful as mono-specific typing reagents (30).
[0348] This lack of correlation of cross-reactivity and serotype/group specificities limits the rational design of combination vaccines based only on these serotype and group specificities. Despite that, the observation of extensive cross-reactivity in this mouse system and the observation of broadly specific immunogens such as S. flexneri 1b and 3a is encouraging, suggesting that practical Shigella vaccines may be possible that cover multiple serotypes with limited components due to currently undescribed specificities. There is an important caveat: these data are generated from mouse studies and there is at least one set of data from humans that show that the mouse results may not always be translatable to humans (31). As found in this study with mice immunized with S. flexneri 2a GMMA (
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[0387] 39. Karnell et al., 1992. Auxotrophic live oral Shigeila flexneri vaccine protects monkeys against challenge with S. flexneri of different serotypes. Vaccine , 10,167.
TABLES
[0388]
TABLE-US-00001 TABLE 1 Type and group specificities of the S. flexneri bacteria used in this study Type Specificities Group Specificities*** I II III IV V VI 3, 4 6 7, 8 9 10 1a + + + 1b + + + + 1c* + (+/−).sup.† 2a + + + + 2b + + 3a + (+/−) + + 3b + + 4a + + 4b + + 5a + + 5b** + + 6 + (+/−) + X (+/−) Y (+/−) *S. flexneri 1c is also classified as S. flexneri 7a **S. flexneri 5b bacteria used in this study typed 5b. However, the S. flexneri 5b GMMA, by FACS had a mixed expression of group 7,8 and thus types as a mixture of S. flexneri 5a and 5b ***Groups 9 and 10 are defined by the PCR genotyping, not by phenotyping with type specific sera .sup.†Specificities that were positive by agglutination or by FACS but gave a titre approximately an order lower than other positive reactions. See Text and Table S1 for details.
TABLE-US-00002 ALTERNATE TABLE 1 Type and group specificities of the S. flexneri bacteria used in this study Type Specificities Group Specificities*** I II III IV V VI 3, 4 6 7, 8 9 10 1a 7,500 30 50 60 60 40 7,450 30 40 + 1b 8,100 50 60 60 50 60 7,200 8,950 + 1c* 7,900 60 150 50 60 40 2a 50 9,200 90 60 60 40 5,200 30 25 + + 2b 70 8,700 60 60 30 40 1,530 3a 30 40 8,500 60 30 60 1,200 6,250 3,540 3b 50 90 7,200 50 40 80 30 4,500 45 4a 80 60 30 7,500 30 40 2,350 25 30 4b 90 80 60 7,600 30 60 20 1,250 30 5a 30 60 40 80 5,800 80 6,500 40 30 5b** 250 120 170 30 6,600 60 40 30 620 6 150 190 120 90 60 2,400 250 30 30 + X 80 90 110 60 90 60 20 30 450 Y 150 180 90 90 90 80 540 20 20 *S. flexneri 1c is also classified as S. flexneri 7a **S. flexneri 5b bacteria used in this study typed 5b. However, the S. flexneri 5b GMMA, by FACS had a mixed expression of group 7,8 and thus types as a mixture of S. flexneri 5a and 5b ***Groups 9 and 10 are defined by the PCR genotyping, not by phenotyping with type specific sera and thus are only typed a positive or negative
TABLE-US-00003 TABLE 2 Comparison of FIG. 1A, B and C 1a 1b 2a 2b 3a 3b 4a 5b 6 X Y 1a FACS 4.1 3.9 2.3 1 1.6 1.3 4.1 3.7 2.7 1.7 3.3 Observed 4.8 4.8 3.4 1 1 1 5.2 4.6 3.7 2.8 4 Predicted 4.7 4.7 4.2 1 3.1 1 3.9 1 4 1 3.1 1b FACS 3.8 3.9 2.9 3.7 1.3 1 3.5 3.3 3.4 3.7 3.2 Observed 3.8 3.7 3.9 4.7 1 1 6 3.8 3.9 4.6 4.1 Predicted 4.7 4.7 4.2 1 4 3.9 3.9 1 4 1 3.1 1c FACS 4 3. .8 2.9 3.1 1 1 3.8 3.5 3.1 3 3.4 Observed 3.7 3.1 3.9 4.7 1 1 6.3 4 3.9 4 4.3 Predicted 4.7 4.7 3.9 1 3.1 1 3.9 1 3.1 1 3.1 2a FACS 2 1.3 4.2 4.1 1.5 1 2.6 2.1 1 1 2.2 Observed 3 1 4.8 5 1 1 5.2 3.2 1 1 3 Predicted 4.2 3.9 4.7 4.7 3.1 1 3.9 1 4 1 3.1 2b FACS 1.5 1 4.7 5 1 1.5 2.8 2.3 2.2 3.1 2.7 Observed 1 1 5.5 5.7 1 1 4.8 3.5 3 3.9 3.4 Predicted 1 1 4.7 4.7 3.9 1 1 3.9 1 3.1 1 3a FACS 1.8 3.7 2.1 1 4.9 4.4 3.1 2.6 2.2 1. 2.5 Observed 1 4.7 3 1 5 5.1 5.8 4.3 3.1 1 3.4 Predicted 3.1 4 3.1 3.9 4.7 4.7 3.9 3.9 3.1 3.1 3.1 3b FACS 2.7 4.2 2 1 4.7 4.8 2.9 2 2.3 2.8 2.4 Observed 3.4 4.6 3 1 5.4 5.4 6.2 3.6 3.3 3.6 3.4 Predicted 1 3.9 1 1 4.7 4.7 1 1 1 1 1 4a FACS 3 2.7 2.7 1 1 1 4.3 3.6 2.7 1.9 3.4 Observed 3.8 1 3.5 1 1 1 5.2 3.9 3.5 2.9 4.2 Predicted 3.9 3.9 3.9 1 3.1 1 4.7 1 3.1 1 3.1 4b FACS 1.7 1.3 1 1.8 2.2 2.5 2.1 1.9 1 1.9 2 Observed 2.7 1 1 2.8 3 2.3 5.1 3.2 1 2.9 2.9 Predicted 1 3.9 1 1 3.9 3.9 4.7 1 1 1 1 5a FACS 3.6 1.6 2.2 1.5 1 1 4 4 2.7 2.4 3 Observed 4.1 1 3.1 1 1 1 5.9 5.7 3.9 3.4 3.9 Predicted 3.9 3.9 3.9 1 3.1 1 3.9 4.7 3.1 1 3.1 5b FACS 2.4 1.3 1.3 3.9 4.5 1 3.9 3.7 2.4 3.1 2.7 Observed 3.4 1 3 4.9 4.8 1 5.5 4.9 3.4 1 3.7 Predicted 1 1 1 3.9 3.9 1 1 4.7 1 3.1 1 6 FACS 2.3 1.8 1.8 1.3 1.6 1.6 3.2 3.1 4.3 1.6 1.8 Observed 5.6 3.6 2.3 1 1 1 5.1 4 5.1 4.3 3.5 Predicted 4 4 4 1 3.1 1 3.9 1 4.7 1 3.1 X FACS 2.4 1.5 1 1 1.3 1 3.6 3.7 2.3 2.7 2.5 Observed 3.3 1 1 1 1 1 4.7 3.7 2.9 3.6 3.3 Predicted 1 1 1 3.9 3.9 1 1 3.9 1 4.7 1 Y FACS 3.1 1.3 1.6 1 1 1.7 3.9 3.9 2.3 1 3.5 Observed 3.8 1 1 1 1 1 5 4.5 3.1 1 3.7 Predicted 3.9 3.9 3.9 1 3.1 1 3.9 1 3.1 1 4.7 1b + 3a FACS 3.8 4.1 3.0 3.7 4.9 4.4 3.6 3.4 3.4 3.7 3.3 Observed 3.8 4.7 4.0 4.7 5.0 5.1 6.2 4.4 4.0 4.6 4.2 Predicted 4.7 4.8 4.2 3.9 4.8 4.8 4.2 3.9 4.1 3.1 3.4
TABLE-US-00004 TABLE S1 Characterization of S. flexneri strains used in the study by slide agglutination and FACS typing FACS typing and agglutination of S. flexneri cell lines received from Public Health England with Denka monovalent rabbit typing and grouping sera (only agglutination) to confirm identity of the serotypes. Mean Fluorescence Intensities from 10 to 100 were considered indicative of absence of signal (no binding of indicated antisera to the surface of S. flexneri serotypes) and correlated with negative agglutination (−) Type specific serum Grouping sera Mean Fluorescent Intensity Mean Fluorescent Intensity Agglutination strength Agglutination strength Serotype I II III IV V VI 3, 4 6 7, 8 1a 7500 30 50 60 60 40 7450 30 40 +++ − − − − − +++ − − 1b 8100 50 60 60 50 60 7200 8950 30 +++ − − − − − ++ ++ − 1 7900 60 150 50 60 40 1550 40 30 +++ − − − − − + − − 2a 50 9200 90 60 60 40 5200 30 25 − +++ − − − − +++ − − 2b 70 8700 60 60 30 40 30 25 1530 − +++ − − − − − − ++ 3a 30 40 8500 60 30 60 1200 6250 3540 − − +++ − − − +/− ++ ++ 3b 50 90 7200 50 40 80 30 4500 45 − − +++ − − − − + − 4a 80 60 30 7500 30 40 2350 25 30 − − − +++ − − +++ − − 4b 90 80 60 7600 30 60 20 1250 30 − − − +++ − − − − − 5a 30 60 40 80 5800 80 6500 40 30 − − − − +++ − +++ − − 5b 250 120 170 30 6600 60 40 30 620 − − − − +++ − − − +/− 6 150 190 120 90 60 2400 250 30 30 − − − − − ++ +/− − − X 80 90 110 60 90 60 20 30 450 − − − − − − − − +/− Y 150 180 90 90 90 80 540 20 20 − − − − − − +/− − −
TABLE-US-00005 TABLE S2 GMMA OAg to Protein ratios ratio w/w Material OAg/protein GMMA-1a 0.39 GMMA-1b 0.58 GMMA-1c 0.53 GMMA-2a 0.42 GMMA-2b 0.8 GMMA-3a 0.48 GMMA-3b 0.39 GMMA-4a 0.39 GMMA-4b 0.48 GMMA-5a 0.38 GMMA-5b 0.58 GMMA-6 0.39 GMMA-X 0.12 GMMA-Y 0.46
TABLE-US-00006 TABLE S3 Surface Staining Mean Fluorescence Intensities Matrix showing the mean fluorescence intensities of surface staining of S. flexneri and S. sonnei with pooled sera raised against GMMA. Binding to homologous serotypes is shown in bold. S.s.: S. sonnei Target Bacteria 1a 1b 2a 2b 3a 3b 4a 5b 6 x y S.s Vaccinating 1a 12500 7250 185 10 40 20 11974 5062 550 50 2050 10 GMMA 1b 6840 8240 840 5520 20 10 3196 1956 2500 5300 1560 10 1c 9850 6500 850 1250 10 10 6310 3239 1300 1000 2530 10 2a 100 20 15530 12750 30 10 425 137 10 10 150 10 2b 30 10 45430 98520 10 30 570 197 150 1250 450 10 3a 60 4650 130 10 76520 25540 1372 394 150 50 350 10 3b 450 15540 100 10 45840 58390 812 97 220 590 250 10 4a 1050 450 450 10 10 10 17997 3923 480 85 2450 10 4b 50 20 10 60 150 300 133 77 10 90 10 5a 3650 40 150 30 10 10 9440 9958 560 250 950 10 5b 240 20 20 7820 28530 10 8667 4508 250 1220 550 10 6 180 70 70 20 40 40 1777 1125 20700 40 70 10 X 250 30 10 10 20 10 4211 4546 180 540 350 10 Y 1200 20 40 10 10 50 8614 8852 220 10 3250 10 S.s. 12500
TABLE-US-00007 TABLE S4 Serum Bactericidal Activity (SBA) Titers. Matrix showing SBA titers on S. flexneri and S. sonnei cell lines with the pooled sera raised against GMMA. Titers on homologous serotypes is shown in bold. BRC %: Percent of baby rabbit complement in the SBA. S.s. = S. sonnei Target Bacteria 1a 1b 2a 2b 3a 3b 4a 5b 6 x y S.s. BRC % 10 15 15 7.5 15 15 7.0 7.0 25 7.5 7.5 20 Vaccinating 1a 61730 62779 2270 10 10 10 173154 39576 5411 618 10472 10 GMMA 1b 5844 4984 7560 51582 10 10 1003208 6914 8814 42844 13094 10 1c 5554 1176 8271 51272 10 10 2020060 9653 8323 10958 20995 10 2a 1009 10 62313 104306 10 10 159426 1650 10 10 948 10 2b 10 10 334660 511451 10 10 59787 2900 1001 8100 2696 10 3a 10 53119 1033 10 106335 125249 627983 20820 1194 10 2715 10 3b 2739 40955 1014 10 276484 243798 1676371 3903 1945 4085 2547 10 4a 6658 10 3399 10 10 10 152145 7475 3302 732 16870 10 4b 555 10 10 697 1035 185 112804 1513 10 755 820 10 5a 13966 10 1141 10 10 10 750632 457295 7401 2276 8303 10 5b 2647 10 942 81134 66789 10 312004 75871 2457 10 5412 10 6 372900 4243 178 10 10 10 116782 10461 131323 21149 3257 10 X 1835 10 10 10 10 10 48647 4705 728 4294 2122 10 Y 6077 10 10 10 10 10 96609 30544 1262 10 4989 10 S.s. 2850