Patent Application
20230263877
The field relates to novel vaccine compositions and methods thereof, wherein an O-antigen of a first serotype or subserotype is used to raise an immune response against one or more O-antigen of a different serotype or subserotype.
Shigella infections are endemic throughout the world, but the main disease burden is in developing countries. The Global Burden of Disease Study 2017 estimates that Shigella causes 15.2% (i.e. 238,000) of the 1.57 million deaths caused by diarrheal infections (1) with 98.5% of Shigella deaths occurring in low and middle income countries. Children younger than 5 years of age accounted for 33% of deaths. Consistent with these global estimates, the prospective Global Enteric Multicenter Study (GEMS) found that shigellosis is one of the top causes of moderate to severe diarrhoea (MSD) in children under 5-years-old in 7 sites in sub-Saharan Africa and South Asia (2). Of 1120 isolates typed, S. sonnei was the dominant species but a range of S. flexneri serotypes was found in the different sites. Overall, the dominant S. flexneri serotype was S. flexneri 2 but the distribution of the serotypes and subtypes varied according to location. For example, in the Bangladesh sites the order was S. flexneri 2a, 2b, 3a, 6, 1b, 4a and Y (X was not detected); in Kenya the order instead was 6, 1b, 3a, 4a, 2b and 2a (1a, X and Y, were not detected).
A comprehensive literature survey of 16,587 reported cases in low and middle-income counties extended the GEMs results to 35 countries (3). These data, even aggregated at the WHO regional level, show major differences in the serotype frequencies: S. flexneri 2 was the most common serotype in the African (AFRO), American (AMRO), South-East Asian (SEARO) and Western Pacific (WPRO) Regions; but S. flexneri 6 was the most common in the Eastern Mediterranean Region (EMRO). The second most common serotype was more variable: S. flexneri 1 in AFRO, S. flexneri 2 in EMRO, S. flexneri 3 in SEARO, both S. flexneri 3 and S. flexneri 4 in AMRO and S. flexneri 4 in WPRO. Even without considering the less frequent serotypes (that still cause a significant part of the disease burden), these data highlight the difficulties with making a broadly specific vaccine if based on the serotype or subtype specific immunogens.
Shigella vaccines under development span a spectrum of approaches and antigens (4, 5). Almost all Shigella vaccines include the O Antigen (OAg) component of the lipopolysaccharide (LPS), which is considered a protective antigen (6) but this antigen would restrict vaccine efficacy to (i) homologous protection or (ii) those cross-reactions with other serotypes capable of conferring protection, that were defined by the OAg alone.
All serotypes and subserotypes of S. flexneri, except serotype 6, share a common OAg backbone (7, 8) with repeats of:
→2)-α-L-RhapIII-(1→2)-α-L-RhapII-(1→3)- α-L-RhapI-(1→3)-β-D-GlcpNAc-(1→
The addition of glucosyl or O-acetyl residues to the backbone sugars creates the OAg structures specific to the different serotypes. The enzymes responsible for the modification of the backbone are encoded on mobile elements and new S. flexneri serotypes and subtypes could emerge by bacteriophage-mediated integration of OAg modification genes (9, 10). The serotypes S. flexneri 1, 2,3,4,5 and X are defined by type specificities (I, II, III, IV, V and X) created by glucosylation (serotypes I, II, IV, V and X). Type specificity III (S. flexneri 3) is defined by acetylation on rhamnose I and an absence of glucosylation that defines other type specificities. S. flexneri Y does not contain any of these substitutions and is defined by the absence of the serotype specificities. The polysaccharide present in serotype Y is characterized by two antigenic specificities labelled dual group O-factor 3,4. A structural domain that defines this O-factor has not been completely identified yet. In some cases, its manifestation is ambiguous as strains otherwise identical in the O-antigen structure and the presence of other immunodeterminants may express or may not express O-factor 3,4 (e.g. former serotypes 3b and 3c, which have been proposed to be combined into one serotype 3b [10]). The 3,4 factor is related to the structure, so it can be masked by other specificities. The polysaccharide can be modified by adding various chemical groups (α-D-glucopyranosyl, O-acetyl, phosphoethanolamine) to different sugars giving rise to enormously diverse O-antigen structures and, correspondingly, to serological heterogeneity, which is the basis for serotyping of S. flexneri strains (11). S. flexneri 6 does not share the common backbone. Instead it has two repetitions (11) of rhamnose, one galacturonic acid and one N-acetylgalactosamine (8).
→2)-α-L-RhapIII-(1→2)-α-L-RhapII-(1→4)-β-D-GalpA-(1→3)-β-D-GalpNAc-(1→
Although phylogenetically dissimilar (S. flexneri 6is in the S. boydii cluster) (12), S. flexneri 6 reacts with S. flexneri species-specific antisera, possibly because of the similarity of the trisaccharide→3)-β-D-GalpNAc-(1→2)-α-L-RhapIII-(1→,2)-α-L-RhapII-(1→ that crosses the junction between adjacent repeats in other S. flexneri serotypes.
All serotypes of S. flexneri (including serotype S. flexneri 6) can have additional modifications, involving substitution with glucose, acetate or phosphoethanolamine, that are common to several of the serotypes and generate the group specificities 6; 7,8; 9; 10; IV-1 or have the group specificity 3,4 that is part of the unmodified repeating unit. The O-antigens of S. flexneri non-6 serotypes are highly diverse due to various chemical modifications to the basal structure giving rise to the observed serological heterogeneity. Several genes outside the O-antigen cluster are involved in the modifications, which occur after the O-unit assembly and before the transfer of the mature O-polysaccharide to the lipid A-core region of the LPS. The O-antigen plays an important role in the pathogenesis of S. flexneri; particularly, it protects the bacteria from the lytic action of serum complement and promotes adherence and internalization of bacteria to intestinal epithelial cells. Creating antigenic diversity by O-antigen modifications is considered as an important virulence factor of S. flexneri that enhances survival of the pathogens because the host must mount a specific immune response to each serotype. Moreover, such modification as glucosylation at certain sites promotes invasion of S. flexneri into host cells mediated by the type III secretion system [11].
These type- and group-specificities are present in various combinations (at least 31 have been reported to date) (11) and generate shared epitopes that are potential targets of antibodies elicited by cross-reacting vaccines.
This was the basis of the studies by Noriega et al., (13) who tested a bivalent vaccine of S. flexneri 2a and S. flexneri 3a in challenge studies in guinea pigs, which was predicted to protect against most isolates via their group specificities. In a conjunctivitis model, this achieved protection against serotypes 1b, 2b, 5b and Y (as predicted based on shared group specificities) but not for serotypes 1a and 4b (contrary to the prediction). Noriega et al., predicted that this vaccine would have not protected against S. flexneri 6. However, subsequent work has identified epitope 9 which is common to all known S. flexneri 6 and some S. flexneri 2a isolates (11). Thus, if protection can be mediated through group specificities, and if the S. flexneri 2a isolate used in Noriega et al., had a 9 specificity, it could have been expected to be protective.
Thus, the identification of a shared group specificity between S. flexneri 6 and S. flexneri 2a suggests that group specificities are not predictive of cross-protection since, if they were, Noriega et al., would have seen cross-protection of S. flexneri 6 through vaccination with S. flexneri 2a O-antigen. Contrary to the prediction of Noriega et al., the present inventors have surprisingly found that S. flexneri 2a does not protect against serotype 1b (Table 2 herein). Further, while the hypothesis of Noriega et al., is that S. flexneri 3a would confer cross-protection against serotypes 1b, 2b, 5b and Y, Table 2 herein surprisingly shows that protection is not conferred against S. flexneri 2b. Noriega et al., were unable to detect these absences of predicted cross-protection because they did not perform control vaccinations with serotype 2a alone, and serotype 3a alone. Only vaccination with serotypes 2a and 3a in combination was performed, masking gaps in the predicted cross-protection.
To our knowledge, outside of Noriega et al., evidence for cross-protection is limited in the literature to:
While it was encouraging to see some cross-protection in Noriega et al., even so, its results suggest that the envisaged coverage of this binary combination in the field is limited since globally, S. flexneri 1b, 4b and 6 are responsible for approximately equal burden of disease, ranking behind S. flexneri 2a and 3a. To address this, Livio et al., proposed including S. flexneri 6 in a combination vaccine (14). However, this would still leave people unprotected against S. flexneri 4, the second most common S. flexneri serotypes in the Americas and the Western Pacific region (3). Based on the Noriega et al., data, S. flexneri 4b was not protected by the S. flexneri 2a/3a combination in guinea pigs, and these authors assume that S. flexneri b 6does not cross protect against S. flexneri 4b.
Since shared group-specificity was found not to be predictive of cross-protection it was unclear which cross-reactions would result in cross-protection and, consequently, how many strains must be represented in a vaccine to obtain sufficient strain coverage to be viable. This is a critical question because vaccine cost increases with complexity and, since S. flexneri is predominantly a disease of developing countries, its treatment requires a low cost of goods to be economically viable. The lower the cost of goods, the greater the impact a vaccine can have on global health. Hence, it is important to establish which cross-reactions would result in cross-protection.
However, to our knowledge the only publications providing experimental evidence on the presence or absence of S. flexneri cross protection (as opposed to cross-reaction) largely indicate that shared group-specificity does not confer cross-protection.
Hence, there are cross-reactions identified by FACS and cross-protections identified by SBA that cannot be predicted by combination of known group- or type-specificities. This may be explained by the way serogroups and serotypes are defined. The Shigella serogroups and serotypes are identified by agglutination tests with polyclonal antibodies, where a positive interaction indicates that a strain contains antigens that specifically react with the serum antibodies.
These polyclonal antisera are obtained by hyperimmunization of healthy rabbits with heat-inactivated whole cells of S. flexneri (40). The antisera are absorbed against cells of other S. flexneri serotypes (and/or subserotypes) to remove cross-reacting agglutinins and thereby create serotype- or subserotype-specific antisera.
There are polyvalent and monovalent Shigella antisera. The polyvalent antisera are those in which their antibodies recognise antigens present in the different serotypes of Shigella (e.g., polyvalent antisera for S. flexneri, recognises all the serotypes of this group). The monovalent antisera only recognise specific epitopes of a serotype (e.g., monovalent antisera for S. flexneri 2) or of a group factor (e.g., monovalent antisera for S. flexneri group factor 7,8). There are probably a multitude of epitopes not covered by the typing scheme currently in use.
The specificity of the antisera used in typing reactions disguises that non-absorbed antisera are not serotype- or group-factor-specific and contain agglutinins for other serotypes which are removed for serotyping. Hence, although cross-reacting agglutinins are removed (or reduced to a concentration that cannot induce agglutination in the slide agglutination test), cross-reacting antibodies that do not induce agglutination are retained. Moreover, sera produced in vaccine trials are not absorbed against other strains and so retain all cross-reacting agglutinins.
However, as is clear from references 11, 13, 31 and 37-39 (supra.) these cross-reactive antibodies do not necessarily confer cross-protection and, where observed, cross-protection does not appear to correlate with serotype or sub-serotype.
Three Gtr proteins (GtrA, GtrB, and type-specific Gtr (Gtr(type)) mediate glucosylation of the O-polysaccharide backbone. A single operon on the chromosome encoding Gtr proteins (gtr cluster) is carried by a (cryptic) prophage acquired by lysogeny of the bacteria with one or two from five temperate bacteriophages (SfI, SfII, SfIV, SfV, and SfX). All bacteriophages have been isolated from the corresponding S. flexneri strains and well characterised. Lysogeny with bacteriophages SfI, SfII, SfIV, SfV, and SfX converts serotype Y to serotypes 1a, 2a, 4a, 5a, and X, respectively, whereas the potential recipient range among other serotypes is quite different. The limitation in the host recognition is evidently due to the phage immunity from a modified O-antigen, which constitutes the receptor for the phage adsorption on the cell surface, a mechanism by which lysogeny prevents subsequent infection of bacteria by homologous or related phages, providing an evolutionary advantage to phages. Similarly, genes for O-Acetylation of RhaI by an acetyltransferase and phosphorylation with PEtN groups to RhaII or/and RhaIII are/were also provided by bacteriophage.
Antigenic diversity by O-antigen modifications is considered as an important virulence factor of S. flexneri that enhances survival of the pathogens because the host must mount a specific immune response to each serotype. Moreover, such modification as glucosylation at certain sites promotes invasion of S. flexneri into host cells mediated by the type III secretion system.
Accordingly, there remains a continuing need to determine which, if any, S. flexneri serotype and/or subserotype cross-reactions result in cross protection to determine the number and identity of serotypes and/or subserotypes that must be represented in a vaccine to provide acceptable coverage at an acceptable cost.
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).
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.
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).
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.
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.
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’.
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’.
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.
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.
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.
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:
Alternatively or additionally, 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 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;
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;
Alternatively or additionally, the first serotype or subserotype is:
Alternatively or additionally, 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;
Alternatively or additionally, the first serotype or subserotype is from another serotype to the different serotype or subserotype, for example:
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.
Alternatively or additionally, where the first serotype or subserotype is:
Alternatively or additionally, where the first serotype or subserotype is:
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.
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:
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).
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:
They may also be defined by serotype versus subserotype. Hence, alternatively or additionally, the first serotype or subserotype is:
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;
They may further be defined by subserotype versus serotype. So, alternatively or additionally, the first serotype or subserotype is:
However, they may be defined by subserotype versus subserotype. Thus, alternatively or additionally, the first serotype or subserotype is:
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;
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.
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.
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.
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:
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.
Alternatively or additionally, the first and further serotypes comprise or consist of combinations selected from the group consisting of:
Alternatively or additionally, the first and further subserotypes comprise or consist of combinations selected from the group consisting of:
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.
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.
Alternatively or additionally, serotype 6 is provided and serotype 5 (for example, 5a or 5b) is not provided.
Further combinations of serotype(s) and/or subserotype(s) would be apparent to the skilled person from Table 2 and
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:
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.
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.
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.
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:
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.
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.
Alternatively or additionally, one or more of the O-antigen(s) is/are provided:
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).
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]).
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.
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.
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.
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).
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).
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.
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.
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.
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.
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.
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.
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.
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.
Alternatively or additionally, the immune response comprises or consists of complement-mediated killing.
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.
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.
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.
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.
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).
Alternatively or additionally, the immune response prevents, abolishes or reduces one or more symptom of Shigella flexneri infection selected from the group consisting of:
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%.
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).
Alternatively or additionally, the immune response is raised in a mammal.
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).
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.
The terms “OMV” and “GMMA” may be used interchangeably herein.
A second aspect provides a binding moiety capable of specifically binding to one or more O-antigen defined in the first aspect.
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).
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.
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.
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.
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.
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.
Alternatively or additionally, the bacterial infection is, wholly or in part, infection with one or more bacterium defined in the first aspect.
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).
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.
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.
A tenth aspect provides an O-antigen, binding moiety, pharmaceutical composition, kit, use or method as described in the specification and figures herein.
1. Introduction
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.
2. Materials and Methods
2.1 Shigella Strains
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.
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 2×107 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.
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.
2.2. GMMA Production, Purification and Formulation
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.
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 OD600 of 0.03-0.05 and incubated at 30° C. and 200 rpm to an OD600 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).
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).
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.
2.3. Immunogenicity Studies in Mice
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.
2.4. Cross-Reactivity Measured by FACS
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.
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.
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.
2.5. High Through-Put Luminescence—Serum Bactericidal Assay (L-SBA)
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.
2.6. Modelled SBA Heat Map
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).
3. Results
3.1. Serotype and Group Specificities of the Bacteria Used in this Study
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.
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
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 (
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.
3.2.2. Binding of Sera Raised Against O Antigen Positive GMMA
3.2.2.1. Binding to O Antigen Negative S. sonnei
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.
3.2.2.2. Homologous Binding (Binding to the Parent Bacteria of the Immunizing GMMA)
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.
3.2.2.3. Heterologous Binding (Binding to Bacteria not the Parent of the Immunizing GMMA)
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.
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.
As expected, S. sonnei bacteria were not stained by any of the S. flexneri GMMA antisera.
3.3. Evaluation of Cross-Functionality by Serum Bactericidal Activity (SBA)
A heat map of SBA data containing the Log10 IC50 of the pooled sera on S. flexneri bacterial cell lines is shown in
The binding of antibodies judged by FACS and killing as judged by SBA was similar (
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 (
4. Discussion
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.
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.
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:
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.
There are two important consequences for antibodies generated by GMMA:
The observed strain specificity and cross-reactivity must predominantly be directed against epitopes in the OAg of each serotype.
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.
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).
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.
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.
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 (
Therefore, we conclude that most of the cross-reactivity cannot be explained by group specificities.
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 (
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.
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).
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 (
1. Institute for Health Metrics and Evaluation (IHME) (2017) GBD Compare Data Visualization.
2. Kotloff K L, et al. (2013) Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet 382(9888):209-222.
3. Pozzi C, Martin L B, & Saul A (2019) A systematic review of the distribution of serotypes causing Shigella disease in low and middle-income countries from 2006 to 2018 to inform vaccine development PLoS neglected tropical diseases.
4. Barry E M, et al. (2013) Progress and pitfalls in Shigella vaccine research. Nat Rev Gastroenterol Hepatol 10(4):245-255.
5. Camacho A I, Irache J M, & Gamazo C (2013) Recent progress towards development of a Shigella vaccine. Expert Rev Vaccines 12(1):43-55.
6. Robbins J B, Chu C, & Schneerson R (1992) Hypothesis for vaccine development: protective immunity to enteric diseases caused by nontyphoidal salmonellae and shigellae may be conferred by serum IgG antibodies to the O-specific polysaccharide of their lipopolysaccharides. Clin Infect Dis 15(2):346-361.
7. Liu B, et al. (2008) Structure and genetics of Shigella O antigens. FEMS Microbiol Rev 32(4):627-653.
8. Perepelov A V, et al. (2012) Shigella flexneri O-antigens revisited: final elucidation of the O-acetylation profiles and a survey of the O-antigen structure diversity. FEMS Immunol Med Microbiol 66(2):201-210.
9. Allison G E & Verma N K (2000) Serotype-converting bacteriophages and O-antigen modification in Shigella flexneri. Trends Microbiol 8(1):17-23.
10. Sun Q, et al. (2014) Serotype-converting bacteriophage SfII encodes an acyltransferase protein that mediates 6-O-acetylation of GlcNAc in Shigella flexneri O-antigens, conferring on the host a novel O-antigen epitope. J Bacteriol 196(20):3656-3666.
11. Knirel Y A, et al. (2015) O-antigen modifications providing antigenic diversity of Shigella flexneri and underlying genetic mechanisms. Biochemistry (Mosc) 80(7):901-914.
12. Pupo G M, Lan R, & Reeves P R (2000) Multiple independent origins of Shigella clones of Escherichia coli and convergent evolution of many of their characteristics. Proc Natl Acad Sci U S A 97(19):10567-10572.
13. Noriega F R, et al. (1999) Strategy for cross-protection among Shigella flexneri serotypes. Infect Immun 67(2):782-788.
14. Livio S, et al. (2014) Shigella isolates from the global enteric multicenter study inform vaccine development. Clin Infect Dis 59(7):933-941.
15. Gerke C, et al. (2015) Production of a Shigella sonnei Vaccine Based on Generalized Modules for Membrane Antigens (GMMA), 1790GAHB. PLoS One 10(8):e0134478.
16. Launay O, et al. (2017) Safety Profile and Immunologic Responses of a Novel Vaccine Against Shigella sonnei Administered Intramuscularly, Intradermally and Intranasally: Results From Two Parallel Randomized Phase 1 Clinical Studies in Healthy Adult Volunteers in Europe. EBioMedicine 22:164-172.
17. Launay O, et al. (2019) Booster Vaccination With GVGH Shigella sonnei 1790GAHB GMMA Vaccine Compared to Single Vaccination in Unvaccinated Healthy European Adults: Results From a Phase 1 Clinical Trial. Frontiers in immunology 10:335.
18. Obiero C W, et al. (2017) A Phase 2a Randomized Study to Evaluate the Safety and Immunogenicity of the 1790GAHB Generalized Modules for Membrane Antigen Vaccine against Shigella sonnei Administered Intramuscularly to Adults from a Shigellosis-Endemic Country. Frontiers in immunology 8:1884.
19. Maggiore L, et al. (2016) Quantitative proteomic analysis of Shigella flexneri and Shigella sonnei Generalized Modules for Membrane Antigens (GMMA) reveals highly pure preparations. Int .1 Med Microbiol 306(2):99-108.
20. Van De Verg L L, et al. (1996) Cross-reactivity of Shigella flexneri serotype 2a O antigen antibodies following immunization or infection. Vaccine 14(11):1062-1068.
21. Knirel Y A, et al. (2011) Lipopolysaccharide core structures and their correlation with genetic groupings of Shigella strains. A novel core variant in Shigella boydii type 16. Glycobiology 21(10):1362-1372.
22. Kim MJ, et al. (2018) Cross-Protective Shigella Whole-Cell Vaccine With a Truncated O-Polysaccharide Chain. Front Microbiol 9:2609.
23. Kotloff K L, et al. (1996) Safety, immunogenicity, and transmissibility in humans of CVD 1203, a live oral Shigella flexneri 2a vaccine candidate attenuated by deletions in aroA and virG. Infect Immun 64(11):4542-4548.
24. Karnell A, Sweiha H, & Lindberg A A (1992) Auxotrophic live oral Shigella flexneri vaccine protects monkeys against challenge with S. flexneri of different serotypes. Vaccine 10(3):167-174.
25. Vecino W H, et al. (2004) Mucosal immunization with attenuated Shigella flexneri harboring an influenza hemagglutinin DNA vaccine protects mice against a lethal influenza challenge. Virology 325(2):192-199.
26. Sansonetti P J, Arondel J, CanteyJ R, Prevost M C, & Huerre M (1996) Infection of rabbit Peyer's patches by Shigella flexneri: effect of adhesive or invasive bacterial phenotypes on follicle-associated epithelium. Infect Immun 64(7):2752-2764.
27. Wassef J S, Keren D F, & Mailloux J L (1989) Role of M cells in initial antigen uptake and in ulcer formation in the rabbit intestinal loop model of shigellosis. Infect Immun 57(3):858-863.
28. Zhang Z, Jin L, Champion G, Seydel K B, & Stanley S L, Jr. (2001) Shigella infection in a SCID mouse-human intestinal xenograft model: role for neutrophils in containing bacterial dissemination in human intestine. Infect Immun 69(5):3240-3247.
29. Zychlinsky A, et al. (1996) In vivo apoptosis in Shigella flexneri infections. Infect Immun 64(12):5357-5365.
30. Gray S B, Jr., Green J H, Harrell W K, Britt L, & Bolin RC (1974) Reuse of Salmonella and Shigella absorbing cells for preparing monospecific Salmonella O and Shigella antisera. Appl Microbio) 28(2):320-322.
31. Farzam N, et al. (2017) Vaccination with Shigella flexneri 2a conjugate induces type 2a and cross-reactive type 6 antibodies in humans but not in mice. Vaccine 35(37):4990-4996.
32. Formal S B, et al. (1966) Protection of monkeys against experimental shigellosis with a living attenuated oral polyvalent dysentery vaccine. J Bacteriol 92(1):17-22.
33. Caboni M, et al. (2015) An O antigen capsule modulates bacterial pathogenesis in Shigella sonnei. PLoS Pathog 11(3):e1004749.
34. Berlanda Scorza F, et al. (2012) High yield production process for Shigella outer membrane particles. PLoS One 7(6):e35616.
35. Rossi O, 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(36):24922-24935.
36. Necchi F, Saul A, & Rondini S (2018) Setup of luminescence-based serum bactericidal assay against Salmonella Paratyphi A. J. Immunol Methods.
37. Ferrecio et al., 1991. Epidemiologic patterns of acute diarrhea and endemic Shigella infections in children in a poor periurban setting in Santiago, Chile. Am. J. Epidemiol. 134:614-627.
38. Mel et al., 1971. Studies on vaccination against bacillary dysentery 6. protection of children by oral immunization with streptomycin-dependent Shigella strains. Bull. WHO, 45:457.
39. Karnell et al., 1992. Auxotrophic live oral Shigeila flexneri vaccine protects monkeys against challenge with S. flexneri of different serotypes. Vaccine , 10,167.
†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.
12500
7250
6840
8240
9850
6500
15530
12750
45430
98520
76520
25540
45840
58390
17997
133
9958
4508
20700
540
3250
12500
61730
62779
5844
4984
5554
1176
62313
104306
334660
511451
106335
125249
276484
243798
152145
112804
457295
75871
131323
4294
4989
2850
| Number | Date | Country | Kind |
|---|---|---|---|
| 19203834.7 | Oct 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/079140 | 10/15/2020 | WO |
