Patent Grant
10407703
The present invention relates to in vitro methods for producing Neisseria meningitidis capsular polysaccharides which have a defined length. The present invention also relates to compositions comprising at least one capsule polymerase, at least one donor carbohydrate and at least one acceptor carbohydrate, wherein the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 10:1 to 400:1. Moreover, the present invention provides truncated versions of the capsule polymerases of Neisseria meningitidis serogroups A and X. Also provided herein are pharmaceuticals, in particular vaccines, comprising the synthetic capsular polysaccharides of Neisseria meningitidis which have a defined length. Furthermore, the invention provides for methods for the production of said vaccines.
Bacterial meningitidis is a serious threat to global health. It is estimated that each year bacterial meningitidis accounts for 170,000 deaths worldwide (WHO, http://www.who.int/nuvi/meningitidis/en/). Despite the availability of potent antimicrobial agents, case-fatality rates are high (10-40%) and survivors frequently suffer from sequelae such as neurologic disability or limb loss and deafness (Van Deuren et al., Clin Micriobiol Rev 2000; 13(1): 144-166; Kaper et al., Nat Rev Microbiol 2004, 2(2): 123-140). Neisseria meningitidis (Nm) is one of the most important causative agents of bacterial meningitidis because of its potential to spread in epidemic waves (Kaper et al., Nat Rev Microbiol 2004, 2(2): 123-140; Rosenstein et al., N Eng J Med 2001, 344(18): 1378-1388). Crucial virulence determinants of disease causing Nm species are their extracellular polysaccharide capsules that are essential for meningococcal survival in human serum (Vogel et al., Infect Immun 1997, 65(10): 4022-4029). Based on antigenic variation of these polysaccharides at least twelve different serogroups of Nm have been identified (A, B, C, E29, H, I, K, L, W-135, X, Y and Z), but only six (A, B, C, W-135, Y and X) account for virtually all cases of disease; see also Frosch, M., VOGEL, U. (2006) “Structure and genetics of the meningococcal capsule.” In Handbook of Meningococcal Disease. Frosch, M., Maiden, M. C. J. (eds). Weinheim: Wiley-VCH.
Serogroup A (NmA) and C (NmC) are the main causes of meningococcal meningitidis in sub-Saharan Africa, while serogroups B (NmB) and C are the major disease causing isolates in industrialized countries. However, serogroups W-135 (NmW-135) and Y (NmY) are becoming increasingly prevalent. For NmW-135, this is most explicitly evidenced by the 2002 epidemic in Burkina Faso with over 13,000 cases and more than 1,400 deaths (Connolly et al., Lancet 2004, 364(9449): 1974-1983; WHO, Epidemic and Pandemic Alert and Response (EPR) 2008). In contrast, NmY is gaining importance in the United States where its prevalence increased from 2% during 1989-1991 to 37% during 1997-2002 (Pollard et al., J Paediatr Child Health 2001, 37(5): S20-S27). However, also the previously only sporadically found serogroup X (NmX) appeared with high incidence in Niger and caused outbreaks in Kenya and Uganda (Biosier et al., Clin Infect Dis 2007, 44(5): 657-663; Lewis, WHO Health Action in Crisis 1, 6 2006).
The serogroups A, B, C, 29E, H, I, K, L, W-135, X, Y and Z are well known in the art and are described, e.g., in Frosch, M., VOGEL, U. (2006) loc. cit. The capsular polysaccharides (CPS) of all serogroups are negatively charged linear polymers. Serogroup B and C are encapsuled in homoplymeric CPS composed of sialic acid (Neu5Ac) moieties that are linked by either α-2→8 glycosidic linkages in serogroup B or by α-2→9 linkages in serogroup C (Bhattacharjee et al., J Biol Chem 1975, 250(5): 1926-1932). Serogroup W-135 and Y both are heteropolymers. They are composed of either galactose/Neu5Ac repeating units [→6)-α-D-Glcp-(1→4)-α-Neu5Ac-(2→]n in serogroup W-135 or glucose/Neu5Ac repeating units [→6)-α-D-Galp-(1→4)-α-Neu5Ac-(2→]n in serogroup Y (Bhattacharjee et al., Can J Biochem 1976, 54(1): 1-8). The CPS of NmA and NmX do not contain Neu5Ac moieties, but are instead built from N-Acetyl-mannosamine 1-phosphate [→6)-α-D-ManpNAc-(1→OPO3→]n or N-Acetyl-glucosamine 1-phosphate [→6)-α-D-GlcpNAc-(1→OPO3→]n repeating units, respectively (Bundle et al., Carbohydr Res 1973, 26(1): 268-270; Bundle et al., J Biol Chem 1974, 249(15): 4797-4801); Bundle et al., J Biol Chem 1974, 249(7): 2275-2281; Jennings et al., J Infect Dis 1977, 136 Suppl: S78-S83).
The CPS of disease causing Nm are attractive vaccine candidates and polysaccharide or polysaccharide-conjugate vaccines are available for serogroups A, C, Y, and W-135 (Broker et al., Minerva Med 2007, 98(5):575-589). Recently, also vaccines for serogroup X have been described (Micoli, P. Proc Natl Acad Sci USA. (2013) 110: 19077-82).
Key enzymes in the CPS biosynthesis are membrane associated capsule polymerases. Candidate genes have been identified for all six disease causing serogroups (Frosch et al., Proc Natl Acad Sci USA 1989, 86(5): 1669-1673; Claus et al., Mol Gen Genet 1997, 257(1): 28-34; Tzeng et al., Infect Immun 2003, 71(2): 6712-6720). However, our knowledge of enzymology or structure-function relations of those important enzymes is still very limited.
Until now, polysaccharide production for neisserial vaccines still requires fermentation of Neisseria meningitidis with subsequent multistep purification of the polysaccharides from the culture medium. In these conventional vaccine production processes, the obtained capsule polysaccharides are mostly long polymers. However, for vaccine production, the oligosaccharides ranging in size between degree of polymerization (DP) DP10 to DP60 are needed. Therefore, after purification of the capsule polysaccharides from the culture medium, the obtained polysaccharides have to be fragmented and the fragments have to be characterized and sorted by length. Foremost the biohazard associated with mass production of pathogenic Neisseria stains, but also the technological platforms needed to produce homogenous oligosaccharide fractions for vaccine production and their functionalization for coupling to carrier proteins (e.g. an inactive form of Diphteria-toxin or tetanus toxoid), make the conventional vaccine production processes cost intensive and time consuming. Moreover, due to the complexity of the vaccine production chain it can hardly be carried out in developing countries where most of the Nm infections occur. In addition, these conventional vaccine production processes are at risk for contaminations by neisserial toxins, media components or chemicals required for subsequent purification procedures.
Thus, the technical problem underlying the present invention is the provision of means and methods which lead to a more convenient production process of vaccines against Neisseria meningitidis.
The technical problem has been overcome by the methods of the present invention for producing capsular polysaccharides of Neisseria meningitidis in vitro which have a defined length and carry functional groups at the reducing end for carrier protein coupling as will be detailed below. In particular, the herein provided means and methods lead to a vaccine production process which is less expensive and less time consuming as compared to the processes of the prior art. Furthermore, these convenient processes as provided herein can also be carried out in the developing world. Accordingly, the technical problem is solved by the provision of the embodiments as defined in the claims.
Accordingly, the present invention relates to an in vitro method for producing Neisseria meningitidis capsular polysaccharides which have a defined length, said method comprising the steps:
The present invention solves the above identified technical problem since, as documented herein below and in the appended Examples, it was surprisingly found that capsule polysaccharides of Neisseria meningitidis (Nm) serogroups A and X which have a uniform and defined length can be produced in vitro.
The inventive means and methods have the advantageous property that the biohazard in association with large scale Nm cultures and the significant coasts for purifying capsular polysaccharides which have the desired length can be avoided. Moreover, the inventive methods provide the perspective that vaccine production chains can be established also in areas that are lacking advanced infrastructural conditions. In particular, the present invention provides for a pyrogen-free, enzyme catalyzed in vitro synthesis production of capsular polysaccharides which have a defined length.
More specifically, the inventors of the present invention surprisingly identified that the capsular polymerase (CP) of Nm serogroup A (CP-A) can be regulated by the ratio of donor to acceptor saccharides. More specifically, in context of the present invention it has been found that this capsular polymerase can be regulated via the donor-acceptor ratio such that it produces capsular polysaccharides with a uniform and defined length. This finding is of high relevance as for vaccine production, neisserial capsular polysaccharides with a uniform and defined length are required. In addition, regulation of the capsule polymerases of Nm serogroup A by the donor-acceptor ratio is highly unexpected as the full length capsule polymerases of Nm serogroups X, Y and W-135 cannot be easily regulated by donor-acceptor ratio.
Furthermore, the inventors of the present invention astonishingly found that, when terminally truncated, also the capsule polymerase of Nm serogroup X (CP-X) can be regulated via the donor-acceptor ratio in order to produce capsular polysaccharides with a uniform and defined length. Moreover, as shown in the illustrating appended examples, this terminally truncated version of the capsule polymerase of Nm serogroup X has the additional advantage that it can also be regulated by the reaction time. Thus, as shown in the appended examples, by choosing a suitable reacting time (e.g. 3 to 45 minutes), the terminally truncated version of the capsule polymerase of Nm serogroup X can be forced to produce capsular polysaccharides with a uniform and defined length of DP10 to DP60. This length is needed for effective vaccine production. It is noted that the inventive truncated (e.g. terminally truncated) capsular polymerase of NmX has the additional advantage that it has a significantly improved activity as compared to its full length counterpart. This is a surprising finding as the prior art has shown that truncated versions of the capsule polymerase of Nm serogroup B (CP-B) have the same or even less activity as compared to the full length CP-B (Keys, Analytical Biochemistry 427, 60-68 (2012)).
Thus, the present invention paves the way for efficient, economical and fast in vitro production methods for Nm capsule polysaccharides, which are suitable for vaccine development. Furthermore, in context of the present invention two important features of the capsule polymerase of NmA (also called CP-A or CsaB) have been identified: (i) the minimal efficient acceptor is the dimer and (ii) the reducing end phosphate group can be extended with rather large chemical groups (such as decyl-ester groups). This latter finding bears the perspective that chain elongation can be primed with reagents of very high purity and functional groups that facilitate conjugation of glycans to carrier proteins in the vaccine production chain.
Moreover, in context of the present invention it was further found that an O-acetyltransferase (e.g. the CsaC of NmA) can be applied in the inventive in vitro production methods and directly effecting O-acetylation of the produced capsular polysaccharides. This is of advantage as usually for vaccine production Nm capsular polysaccharides must be O-acetylated. In addition, O-acetylation has also an effect on the immunogenicity of the produced capsular polysaccharides. Natural capsule polysaccharides of NmA are O-acetylated in position 3-O and to a minor extend in position 4-O of ManNAc (Gudlavalleti, Carbohydr. Res. (2006) 341: 557-562). It has been shown that capsular polysaccharides of NmA are immunogenic only if they are O-acetylated (Berry et al., 2002 Infection and immunity 70 (7), 3707-3713). As demonstrated in the appended examples, during in vitro synthesis of CPS an O-acetyltransferase (such as the CsaC of NmA) can be directly added to the mixture comprising the capsule polymerase, the donor carbohydrate(s) and the acceptor carbohydrate(s). Thus, in vitro production and O-acetylation can be performed in a one-pot reaction. Accordingly, the O-acetyltransferase may be used in step (a) of the herein described in vitro method for producing Nm capsular polysaccharides. In addition or alternatively, O-acetylation may be performed as an additional step after the synthesis of the Nm capsular polysaccharides. Thus, in the herein described in vitro method for producing Nm capsular polysaccharides, O-acetylation may be performed after step (a) in the additional step (a′).
Accordingly, the present invention provides for effective in vitro methods for producing exactly the type of Nm capsular polysaccharides which is required for vaccine production (i.e. capsular polysaccharides which have a defined length and which are O-acetylated). Thereby, the previously used cost- and time-intensive vaccine production processes can be avoided.
In vitro methods for producing synthetic capsular polysaccharides of Neisseria meningitidis have been described in the prior art (WO 2011/023764 A1). However, the prior art does not disclose how to generate synthetic Nm capsular polysaccharides which have a uniform and defined length and the prior art does not disclose that an O-acetyltransferase can be applied in the in vitro production of capsular polysaccharides to obtain O-acetylated capsular polysaccharides.
By contrast, in the methods of the present invention, isolated Nm capsular polymerases are regulated by the donor-acceptor ratio and/or by the reaction time to allow for the production of Nm capsular polysaccharides which have a uniform and defined length. Accordingly, the invention provides for methods for producing Nm capsular polysaccharides, wherein the capsular polymerases are regulated by the donor-acceptor ratio and/or the reaction time so as to produce capsular polysaccharides which have a uniform and defined length.
Moreover, a further advantageous property of the methods of the invention is that they can be combined with an O-acetyltransferase to generate O-acetylated capsular polysaccharides which can directly be used for vaccine production.
Thus, the present invention relates to an in vitro method for producing Neisseria meningitidis capsular polysaccharides which have a defined length, said method comprising the steps:
The appended examples demonstrate that capsular polysaccharides which have the desired length of DP10 to DP60 can be produced by using CP-A or a terminally truncated version of CP-X and a ratio of donor carbohydrate to acceptor carbohydrate which is approximately between 10:1 and 400:1. Therefore, in the herein described methods, if the length of the Nm capsular polysaccharides is regulated via the ratio of donor carbohydrate to acceptor carbohydrate, than the donor:acceptor ratio is 10:1 to 400:1 (e.g. 20:1 to 400:1). For example, the ratio may be 10:1, 20:1, 40:1, 60:1, 80:1, 100:1, 150:1, 200:1, 220:1, 240:1, 250:1, 300:1, 350:1 or 400:1. In particular aspects of the invention also other donor acceptor ratios, e.g. ratios from 2:1 to 2000:1 may be used.
If CP-A is used in the in vitro methods described herein, than the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 10:1 to 240:1, and more preferably from 10:1 to 80:1, and even more preferably from 10:1 to 75:1, and even more preferably from 20:1 to 75:1, and most preferably from 20:1 to 60:1 (e.g. 40:1 to 60:1 or 50:1). The term “CP-A” includes a polypeptide which has the amino acid sequence of SEQ ID NO: 9 or 10 and a polypeptide which has at least 80% (preferably at least 90%) sequence identity to SEQ ID NO: 9 (CP-A wildtyp/full-length) or to SEQ ID NO: 10 (ΔN69-CP-A).
If the terminally truncated CP-X is used and if it is regulated via the ratio of donor carbohydrate to acceptor carbohydrate (and not via the reaction time) then the following ratios are preferably used. For example, if the capsule polymerase is a polypeptide which has the amino acid sequence of SEQ ID NO: 28 or which has at least 80% (preferably at least 90%) sequence identity to SEQ ID NO: 28 (ΔC99-CP-X), then the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 10:1 to 80:1, more preferably from 20:1 to 60:1, even more preferably from 20:1 to 50:1, and most preferably from 33:1 to 50:1. In addition, if the capsule polymerase is a polypeptide which has the amino acid sequence of SEQ ID NO: 25 or which has at least 80% (preferably at least 90%) sequence identity to SEQ ID NO: 25 (ΔN58-CP-X), then the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 10:1 to 80:1, more preferably from 20:1 to 50:1, and most preferably from 20:1 to 33:1 (e.g. 33:1). Furthermore, if the capsule polymerase is a polypeptide which has the amino acid sequence of SEQ ID NO: 29 or which has at least 80% (preferably at least 90%) sequence identity to SEQ ID NO: 29 (ΔN58ΔC99-CP-X), then the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 50:1 to 400:1, more preferably from 80:1 to 400:1, even more preferably from 100:1 to 400:1, and most preferably from 100:1 to 200:1.
All capsule polymerases which are applied in the herein described methods are functional. For example, a polypeptide which has at least 80% (preferably at least 90%) sequence identity to any one of the sequences of SEQ ID NOs: 9, 10, 25, 28, and 29 is considered to be functional, i.e. to have the same function as the polypeptide which has the sequence of SEQ ID NO: 9, 10, 25, 28, or 29, respectively. In particular, the function of a polypeptide having the sequence of SEQ ID NO: 9 or 10 is the ability to transfer ManNAc-1P (or a derivative thereof) from UDP-ManNAc (or a derivative thereof) onto an acceptor carbohydrate. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of ≥4. In addition, the function of a polypeptide having the sequence of any one of SEQ ID NOs: 25, 28, and 29 is the ability to transfer GlcNAc-1P (or a derivative thereof) from UDP-GlcNAc (or a derivative thereof) onto an acceptor carbohydrate.
If (in the herein described in vitro methods for producing Nm capsular polysaccharides (Nm CPS) which have a defined length) the length of the produced capsular polysaccharides is regulated via the donor:acceptor ratio, then 50-100 nM of the capsular polymerase may be used. The amount of donor carbohydrate may be 1-100 mM, e.g. 5-10 mM. The amount of acceptor carbohydrate is adjusted to the amount of donor carbohydrate so as to result in the desired donor:acceptor ratio ratio. For example, the amount of acceptor carbohydrate may be 1-1000 μM, e.g. 10-40 μM. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of ≥4.
For example, if in the herein described methods for producing Nm polysaccharides with a defined length the CP-A is used, then 1-100 nM (preferably 70-100 nM) of CP-A, 10 mM donor carbohydrate (e.g. a dimer of ManNAc-1-phosphate), and an amount of acceptor carbohydrate (e.g. purified capsular polysaccharides of NmA) resulting in a ratio of donor carbohydrate to acceptor carbohydrate which is from 10:1 to 400:1, preferably from 10:1 to 240:1, more preferably from 10:1 to 80:1, even more preferably from 10:1 to 75:1, even more preferably from 20:1 to 75:1, and most preferably from 20:1 to 60:1 (e.g. 40:1 to 60:1 or 50:1) may be used. In a particular example of the herein described methods for producing Nm polysaccharides with a defined length, 70-100 nM of CP-A, 10 mM donor carbohydrate (e.g. UDP-ManNAc; or UDP-GlcNAc, wherein in step (a) the capsule polymerase is further incubated with the UDP-GlcNAc-epimerase), and an amount of acceptor carbohydrate (e.g. purified capsular polysaccharides of NmA of which at least 80% have a DP of ≥4) resulting in a ratio of donor carbohydrate to acceptor carbohydrate which is from 20:1 to 60:1 is used. The term “CP-A” includes a polypeptide having the amino acid sequence as shown in SEQ ID NO: 9 (CP-A wildtyp/full-length) or SEQ ID NO: 10 (ΔN69-CP-A) and a polypeptide with an amino acid sequence having at least 80% (preferably at least 90%) sequence identity to SEQ ID NO: 9 or to SEQ ID NO: 10 and being functional.
In another particular example of the herein described methods, the length of the produced Nm polysaccharides is regulated via the donor:acceptor ratio and 50-70 nM of a polypeptide having the amino acid sequence of SEQ ID NO: 25 (ΔN58-CP-X), 28 (ΔC99-CP-X), or 29 (ΔN58ΔC99-CP-X), or having an amino acid sequence which is at least 80% (preferably at least 90%) identical to SEQ ID NO: 25, 28, or 29 and being functional; 10 mM donor carbohydrate (e.g. UDP-GlcNAc), and an amount of acceptor carbohydrate (e.g. purified capsular polysaccharides of NmX of which at least 80% have a DP of ≥4) resulting in the desired ratio of donor carbohydrate to acceptor carbohydrate is used.
In another example of the invention, the length of the Nm CPS is regulated by the reaction time, and 50-70 nM capsular polymerase (e.g. ΔC99-CP-X or ΔN58ΔC99-CP-X), 10 mM donor carbohydrate (e.g. UDP-GlcNAc) and 10-30 μM acceptor carbohydrate (e.g. purified capsular polysaccharides of NmX of which at least 80% have a DP of ≥4) is used.
Step (a) of the herein provided in vitro method may be performed by incubating the capsule polymerase, the donor carbohydrate and the acceptor carbohydrate in a reaction buffer (e.g. a buffer comprising 50 mM Tris pH 8.0 and 20 mM MgCl2). The incubation in step (a) may be performed at a temperature range between 20° C. and 40° C., preferably between 25° C. and 37° C., more preferably between 30 and 37° C.
In context of the present invention it has surprisingly and unexpectedly been found that in the herein described methods, if the length of the Nm capsular polysaccharides is regulated via the ratio of donor carbohydrate to acceptor carbohydrate (and not via the reaction time) and if the CP-A or a C-terminally truncated version of the CP-X is used (e.g. ΔC99-CP-X), then the donor:acceptor ratio (i.e. the donor:acceptor quotient) reflects the avDP (i.e. the length of the major species within the produced capsular polysaccharides). For example, if the donor:acceptor ratio is 15:1, then the CP-A or the C-terminally truncated CP-X produces capsular polysaccharides which have an avDP15. Or, if the donor:acceptor ratio is 50:1, then the CP-A or the C-terminally truncated CP-X produces capsular polysaccharides which have an avDP50 (see, e.g.,
Accordingly, the present invention relates to an in vitro method for producing Neisseria meningitidis capsular polysaccharides which have a defined length, said method comprising the steps:
As indicated, in the above described method, the produced capsular polysaccharides comprise as a main component capsular polysaccharides having a DP corresponding to about the ratio of donor carbohydrate to acceptor carbohydrate. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of ≥4. Thus, the length of the produced capsular polysaccharides can easily be regulated via the donor:acceptor ratio. One aspect of the invention relates to the above described method, wherein the donor:acceptor ratio is from 15:1 to 20:1, and wherein at least 60% (preferably 80%, most preferably 90%) of the produced capsular polysaccharides have a length, or in other words, a degree of polymerisation corresponding to the ratio of donor carbohydrate to acceptor carbohydrate −20/+30 repeating units.
Herein the term “Neisseria meningitidis capsular polysaccharides which have a defined length” means that the produced capsular polysaccharides have a defined DP (degree of polymerization) or a defined avDP (average degree of polymerization), e.g. a DP between 10 and 60 or an avDP between 15 and 20. The term “Neisseria meningitidis capsular polysaccharides which have a defined length” preferably means that at least 60% (more preferably at least 80%, most preferably at least 90%) of the produced capsular polysaccharides have a length between DP10 and DP60. For example, the DP may be between DP20 and DP60. The DP indicates the number of monosaccharide-units which built up the capsular polysaccharide. For example, a DP of 10 means that the capsular polysaccharide consists of 10 monosaccharide-units and a DP of 60 means that the capsular polysaccharide consists of 60 monosaccharide-units. Accordingly, a NmX capsular polysaccharide having DP10 means that the capsular polysaccharide consists of 10 GlcNAc-1P repeating units. Acidic hydrolysis used to produce oligosaccharide fragments form long polysaccharide chains, results in fragments of different lengths, which in subsequent chromatographic steps can be separated into size classes. The average DP (avDP) describes the dispersion of chains with one class and can be calculated according to established protocols as described, e.g. in Berti (2012) Vaccine 30 (45), 6409-6415. Or, in other words, the avDP describes the average length of a polysaccharide pool. Alternative ways to determine the DP of oligosaccharides are high percentage polyacrylamide gel electrophoresis or HPLC-anion-exchange chromatography as demonstrated in the appended examples. The avDP may be determined by the protocol described in Berti (2012) 30(45): 6409-15., which includes 31P NMR and High Performance Anionic Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). For vaccine production usually capsular polysaccharides having an avDP of 15 to 20 is used. The product pool of polysaccharides with an avDP of 15 to 20 consists of polysaccharides having a DP of 10 to 60. The herein provided in vitro methods have the advantage that capsule polysaccharides with the length which is desired for vaccine production (i.e. DP10 to DP60) can directly be produced. Herein the term “Neisseria meningitidis capsular polysaccharides which have a defined length” also means that the produced capsular polysaccharides have a low dispersity (i.e. a narrow product distribution).
In the above described in vitro method, if the capsule polymerase of Nm serogroup A is used, than capsule polysaccharides of Nm serogroup A are produced. Analogously, if a truncated version of the capsule polymerase of Nm serogroup X is used, that capsule polysaccharides of Nm serogroup X are produced.
As described herein and illustrated in the appended examples, one aspect of the present invention relates to the identification of terminally truncated versions of the capsule polymerase of Nm serogroup X (CP-X, also designated as CsxA). These terminally truncated proteins have the advantage that they have an increased activity as compared to the full length CP-X. A further advantageous property of these terminally truncated proteins is that they can be regulated by the ratio of donor carbohydrates to acceptor carbohydrates such that they produce capsular polysaccharides which have a uniform and defined length. This is surprising since the full length CP-X cannot be regulated by the donor-acceptor ratio. Furthermore, in context of the present invention it has astonishingly been found that these terminally truncated versions of the CP-X can also be regulated by the reaction time so as to produce capsular polysaccharides which have a uniform and defined length. More specifically, the time how long the terminally truncated CP-X is incubated with the donor carbohydrates (and, optionally, the acceptor carbohydrates) regulates the length of the produced capsule polysaccharides. Accordingly, an incubation time can be chosen which results in the production of capsule polysaccharides of NmX which have the length which is desired for vaccine production (i.e. DP10 to DP60). Accordingly, one embodiment of the invention relates to an in vitro method for producing Neisseria meningitidis capsular polysaccharides which have a defined length, said method comprising the steps:
Or, in other words, the invention relates to an in vitro method for producing Neisseria meningitidis capsular polysaccharides which have a defined length, said method comprising the steps:
Due to the distributive nature of the elongation mechanism used by the truncated CP-X, the reaction can be stopped at any time whereby the resulting avDP depends on the reaction time. Therefore, if the truncated version of the CP-X is regulated via the reaction time, then the ratio of donor carbohydrate to acceptor carbohydrate is not relevant. For example, the ratio of donor carbohydrate to acceptor carbohydrate can range from 1:1 to 50000:1, preferably from 10:1 to 10000:1 [Hier wurde entsprechend der Ansprüche 10:1 to 10000:1 gewählt] (e.g. from 200:1 to 10000:1, or more preferably from 100:1 to 8000:1).
In context of the present invention it has surprisingly been found that the herein described capsule polymerases are active even if they are coupled to a solid phase. Immobilizing a component on a solid phase has the advantage that it reduces labor in the isolating step (b). More specifically, immobilizing a component to a solid phase omits further purification steps to remove the produced capsular polysaccharides from the reaction mixture of step (a). Thus, in the herein provided in vitro methods for producing Nm capsular polysaccharides, one of the components may be immobilized (i.e. bound/coupled) on a solid phase. For example, the donor carbohydrate or the capsule polymerase may be immobilized on a solid phase. The solid phase may be column, such as a His-Trap column (i.e. a column having His-Trap beads). In a particular aspect of the inventive in vitro methods, the capsule polymerase is immobilized on a solid phase. For example, a polypeptide which has the amino acid sequence of SEQ ID NO: 29 (ΔN58ΔC99-CP-X) or which has at least 80% (preferably at least 90%) sequence identity to SEQ ID NO: 29 and being functional may be immobilized on a solid phase.
It is noted that the capsule polymerase of Nm serogroup X is capable of a de novo synthesis of capsular polysaccharides. Or, in other words, the CP-X is able to synthesize capsular polysaccharides in the absence of acceptor carbohydrates (Fiebig, Glycobiology (2014) 24:150-8). Thus, if the terminally truncated version of CP-X is regulated by the reaction time, the presence of acceptor carbohydrates is optional. Accordingly, one aspect of the invention relates to an in vitro method for producing Nm capsular polysaccharides of NmX which have a defined length, said method comprising the steps:
As described herein and illustrated in the appended examples, the inventive terminally truncated versions of the capsule polymerase of Nm serogroup X can be regulated by both, the donor-acceptor ratio and the reaction time (i.e. the time how long the terminally truncated version of the CP-X is incubated with the donor saccharides and the acceptor saccharides).
As mentioned above, if the truncated version of the CP-X is regulated via the reaction time, then the ratio of donor carbohydrate to acceptor carbohydrate is not relevant. Thus, as also demonstrated in the appended Examples, if the length of the Nm capsular polysaccharides is regulated by the reaction time, then higher donor-acceptor ratios can be used (to produce Neisseria meningitidis capsular polysaccharides which have a defined length) as compared to the method wherein the length of the Nm capsular polysaccharides is regulated via the donor-acceptor ratio (and not via the reaction time). Accordingly, one aspect of the invention relates to an in vitro method for producing Neisseria meningitidis capsular polysaccharides which have a defined length, said method comprising the steps:
Or, in other words, the invention relates to an in vitro method for producing Neisseria meningitidis capsular polysaccharides which have a defined length, said method comprising the steps:
Or, in other words, the invention relates to an in vitro method for producing Nm capsular polysaccharides of NmX which have a defined length, said method comprising the steps:
The appended examples demonstrate that capsular polysaccharides of NmX which have the desired length of DP10 to DP60 can be produced by using a terminally truncated version of CP-X and a ratio of donor carbohydrate to acceptor carbohydrate which is approximately between 10:1 and 400:1. The appended Examples also demonstrate that higher donor-acceptor ratios (e.g., 200:1 or 8000:1) can be used to produce Nm capsule polysaccharides which have a defined length, if the terminally truncated version of the CP-X is regulated via the reaction time (i.e. if the reaction time is from 3 to 45 min).
Accordingly, if the length of the Nm capsular polysaccharides is regulated by the reaction time, then the ratio of donor carbohydrate to acceptor carbohydrate is not relevant. For example, the donor:acceptor ratio can be from 10:1 to 10000:1, preferably from 100:1 to 8000:1 (e.g. from 200:1 to 1000:1)
For example, if the terminally truncated CP-X is regulated by the reaction time, then the ratio of donor carbohydrate to acceptor carbohydrate of 10:1, 20:1, 40:1, 60:1, 80:1, 100:1, 120:1, 140:1, 160:1, 180:1, 200:1, 220:1, 240:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, or 10000:1 may be used.
In the herein described and provided in vitro methods, if the length of the produced capsule polysaccharides in regulated via the reaction time, it is envisaged that the truncated version of the CP-X is used in a concentration ranging from 20 to 500 nM, preferably from 20 to 200 nM, or more preferably from 50 to 100 nM (e.g. 50 nM). In addition, it is envisaged herein that said truncated version of the CP-X is incubated with a donor concentration of 1 to 100 mM, preferably of 1 to 50 mM, more preferably of 2 to 20 mM, even more preferably of 5 to 10 mM, or most preferably of 10 mM UDP-GlcNAc; and with an acceptor concentration resulting in a donor to acceptor ratio from 10:1 to 10000:1, preferably from 100:1 to 8000:1, (e.g. from 200:1 to 1000:1) and that the reaction time ranges from 3 to 45 minutes, more preferably from 5 to 30 minutes, or most preferably from 5 to 10 minutes. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of ≥4.
Or, in other words, if the length of the Nm capsular polysaccharides is regulated by the reaction time, it is envisaged that the concentration of the truncated version of the CP-X (e.g. a capsule polymerase having at least 80% (preferably at least 90%) identity to any one of the sequences of SEQ ID NOs: 25, 28 and 29 and being functional) ranges from 20 to 500 nM, preferably from 20 to 200 nM, or more preferably from 50 to 100 nM (e.g. 50 nM). In addition, it is envisaged herein that said truncated version of the CP-X is incubated with a donor concentration of 1 to 100 mM, preferably of 1 to 50 mM, more preferably of 2 to 20 mM, even more preferably of 5 to 10 mM, or most preferably of 10 mM UDP-GlcNAc; and with an acceptor concentration resulting in a donor to acceptor ratio from 10:1 to 10000:1, preferably from 100:1 to 8000:1 (e.g. 200:1 to 1000:1); and that the reaction time ranges from 3 to 45 minutes. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of ≥4.
As indicated above, if the length of the Nm capsular polysaccharides is regulated by the reaction time, then the reaction time ranges (i.e. is) from 3 to 45 minutes. Preferably, the reaction time ranges from 5 to 30 minutes, more preferably from 5 to 10 minutes (e.g. 5 or 10 min). For example, if the capsule polymerase is a polypeptide which has at least 80% (preferably at least 90%) sequence identity to SEQ ID NO: 28 (ΔC99-CP-X) and being functional, then the reaction time ranges preferably from 5 to 10 minutes, and is most preferably 5 minutes. In addition, if the capsule polymerase is a polypeptide which has at least 80% (preferably at least 90%) sequence identity to SEQ ID NO: 25 (ΔN58-CP-X) and being functional, then the reaction time ranges preferably from 5 to 30 minutes. Furthermore, if the capsule polymerase is a polypeptide which has at least 80% (preferably at least 90%) sequence identity to SEQ ID NO: 29 (ΔN58ΔC99-CP-X) and being functional, then the reaction time ranges preferably from 5 to 30 minutes and is most preferably 10 minutes.
In the inventive in vivo methods, the temperature in the incubation step (a) may be from 20° C. to 40° C., preferably from 25° C. to 37° C., more preferably from 30° C. to 37° C., and most preferably 37° C.
As indicated above, if the length of the Nm CPS is regulated by the reaction time, the reaction time may be 3-45 min, preferably 5-30 minutes. For example, the reaction time may be 3 min, 5 min, 10 min, 15 min, 20 min, 25 min 30 min, 35 min, 40 min or 45 min. In addition, if the length of the Nm CPS is regulated by the reaction time, it is preferred that a C- and N-terminally truncated version of the CP-X (such as ΔN58ΔC99-CP-X) is used. This truncated version of the CP-X may be a polypeptide having the amino acid sequence of SEQ ID NO: 29 or a polypeptide having an amino acid sequence which has at least 80% homology to SEQ ID NO: 29 and being functional.
If the length of the Nm CPS is regulated by the ratio of donor carbohydrate to acceptor carbohydrate, then the reaction time may be, e.g., 4-7 hours.
In step (b) of the herein described in vitro methods for producing Nm CPS which have a defined length (i.e. which have low dispersity in terms of length), the resulting CPS are isolated. Isolation of the synthesized CPS may be performed, e.g., by anion exchange chromatography (AEC). For example, the produced CPS may be purified via AEC by using a MonoQ HR5/5 column (Pharmacia biotech) at a flow rate of 1 mL/min and a linear sodium chloride gradient or by High Performance Anionic Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) as described (Berti et al. 2012 Vaccine. (2012) 30:6409-15).
In accordance with the present invention, the ingredients which are used in the herein described methods for producing Nm CPS can be packed together as a composition. Accordingly, one aspect of the invention relates to a composition comprising:
The skilled person understands that, depending on the exactness of the measurements, the exact ratio can slightly deviate from 10:1 to 10000:1. Accordingly, the methods of the invention can also be performed with a ratio of about 10:1 to 10000:1. Thus, one aspect of the invention relates to a composition comprising:
For example, in the herein provided compositions, 20 nM to 500 μM (preferably 20 to 200 nM, more preferably 50 to 100 nM, e.g. 50-70 nM) of the capsular polymerase (e.g. ΔC99-CP-X, ΔN58ΔC99-CP-X or ΔN58-CP-X, or a polypeptide having at least 80% (preferably at least 90%) identity to any one of SEQ ID NOs: 25, 28 and 29 and being functional) may be used. The amount of donor carbohydrate (e.g. UDP-GlcNAc) may be 1-100 mM, preferably 1 to 50 mM, more preferably 2 to 20 mM (e.g. 5-10 mM). The acceptor carbohydrate may be purified capsular polysaccharides of NmX. Preferably, the amount of the acceptor carbohydrate is adjusted to result in a ratio of donor carbohydrate to acceptor carbohydrate of 10:1 to 10000:1, preferably of 100:1 to 8000:1 (e.g. of 200:1 to 1000:1). For example, the amount of the acceptor carbohydrate may be 1-1000 μM, e.g. 10-40 μM.
In the herein provided compositions, the ratio of donor carbohydrate to acceptor carbohydrate is 10:1 to 10000:1, e.g. 20:1, 40:1, 60:1, 80:1, 100:1, 150:1, 200:1, 220:1, 240:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1 or 10000:1. For example, the donor-acceptor-ratio may be from 2:1 to 2000:1. One aspect of the invention relates to the herein described composition, wherein the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 20:1 to 240:1.
The composition of the invention may be suitable for storage (e.g. storage under cold temperatures to prevent that the synthesis of capsular polysaccharides starts). For instance, the composition may be suitable for storage in 4° C., 0° C., −10° C., −20° C. or −80° C.
The inventive compositions and in vitro methods comprise “at least one” capsule polymerase. Therefore, the inventive compositions and in vitro methods can comprise several capsular polymerases of different types in one reaction mixture (e.g. CP-A together with the terminally truncated CP-X). However, it is preferred that the inventive compositions and in vitro methods comprise only capsule polymerases of one type (e.g. only CP-A or only the terminally truncated version of CP-X).
The in vitro methods of the invention may be realized by using an appropriate kit. Accordingly, another embodiment of the invention relates to a kit for carrying out the in vitro methods of the invention, comprising the above described composition. The embodiments disclosed in connection with the in vitro method of the present invention apply, mutatis mutandis, to the composition and the kit of the present invention.
Advantageously, the kit of the present invention further comprises, optionally (a) reaction buffer(s), storage solutions, wash solutions and/or remaining reagents or materials required for the conduction of the assays as described herein. Furthermore, parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units. These vials/bottles/containers or multicontainers may, in addition to the capsule polymerases and donor carbohydrates and acceptor carbohydrates as described herein, comprise preservatives or buffers for storage. In addition, the kit may contain instructions for use.
The composition of the present invention or the kit of the present invention may be advantageously used for carrying out the in vitro methods as described herein (i.e. for producing Nm capsular polysaccharides of NmX which have a defined length). The manufacture of the kit of the present invention follows preferably standard procedures which are known to the person skilled in the art.
In the methods and compositions of the invention, the capsule polymerase of NmA (CP-A also designated as CsaB) may be used. The DNA sequence of the wild type full length CP-A is shown herein as SEQ ID NO: 1. The DNA sequence of the full length CP-A which is optimized for expression in E. coli (i.e. a codon optimized DNA sequence) is shown herein as SEQ ID NO: 2. The polypeptide resulting from the expression of the wild type or codon optimized CP-A polynucleotide is identical. The amino acid sequence of the full length CP-A is given in SEQ ID NO: 9.
Of note, within the DNA sequence of the CP-A, the inventors of the present invention identified an additional ATG start codon and cloned the corresponding truncation ΔN69-CP-A (which starts from the alternative ATG). The designation “ΔN69-CP-A” means that 69 amino acids are lacking at the N-terminus of the CP-A protein. Herein, “ΔN69-CP-A” is also designated as “dN69-CsaB”. The polynucleotide sequence of a codon optimized version of ΔN69-CP-A is shown in SEQ ID NO: 3. The polynucleotide sequence of the wild type ΔN69-CP-A is shown in SEQ ID NO: 46. The amino acid sequence of the expressed wild type or codon optimized ΔN69-CP-A is identical and in SEQ ID NO: 10. Surprisingly, ΔN69-CP-A is advantageous over the full length CP-A. In particular, as demonstrated in the appended illustrative examples, while increased degradation and concomitantly reduced expression was seen for the codon optimized full length CP-A, the codon optimized ΔN69-CP-A is well expressed and no degradation is detectable. In addition, as shown in the appended examples, by using the radioincorporation assay as described in Fiebig, Glycobiology (2014) 24:150-8, it was demonstrated the ΔN69-CP-A protein is active (i.e. is capable of producing NmA capsular polysaccharides). Furthermore, the appended examples also show that the ΔN69-CP-A and the full length CP-A show identical activity profiles. This is surprising since several other tested truncated versions of CP-A (i.e. ΔN97-CP-A, ΔN167-CP-A, ΔN235-CP-A, ΔC45-CP-A and ΔC25-CP-A) were demonstrated to be not active. It is mentioned that herein, in particular in the appended examples, “A” is also designated as “d” or “delta”.
Accordingly, one aspect of the present invention relates to a truncated version of the CP-A (i.e. Δ69-CP-A), wherein said truncated version of the CP-A is the polypeptide of any one of (a) to (f):
As indicated above, the herein provided truncated version of the CP-A comprises the ability to transfer ManNAc-1P or a derivative thereof from UDP-ManNAc or a derivative thereof onto an acceptor carbohydrate (i.e. onto the hydroxyl group of C6 of a capsular polysaccharide from NmA or a derivative thereof). Accordingly, this function can be the ability to transfer ManNAc-1P from UDP-ManNAc onto an acceptor carbohydrate. The acceptor carbohydrate may be extended/activated at the reducing end or the reducing end phosphate. In addition, the size of the produced capsular polysaccharides can be controlled by the ratio of donor and acceptor carbohydrate when the ratio is in the range of 10:1 to 400:1, preferably from 10:1 to 240:1, more preferably from 10:1 to 80:1, even more preferably from 10:1 to 75:1, even more preferably from 20:1 to 75:1, and most preferably from 20:1 to 60:1 (e.g. 40:1 to 60:1 such as 50:1). Within this range, only one product distribution corresponding in size to the calculated donor-acceptor ratio is obtained at reaction endpoints.
The transfer of ManNAc-1P (or a derivative thereof) from UDP-ManNAc (or a derivative thereof) onto an acceptor carbohydrate may be performed by incubating a truncated version of the CP-A (i.e. ΔN69-CP-A) with UDP-ManNAc (or a derivative thereof) and an acceptor carbohydrate (e.g. a tetramer or a dimer consisting of ManNAc-1P units linked together by phosphodiester bonds). Whether a transfer from ManNAc-1P (or a derivative thereof) from UDP-ManNAc (or a derivative thereof) onto an acceptor carbohydrate has occurred can be tested, e.g. by high percentage PAGE and developed by alcian blue/silver staining (Fiebig, Glycobiology (2014) 24:150-8). in the presence of a size marker or by 31P NMR monitoring the characteristic phosphodiester signal, or by High Performance Anionic Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) as described (Berti Vaccine (2012) 30:6409-15).
A derivative of ManNAc-1P may be ManNAc-3-O-Ac or ManNAc-4-O-Ac or ManNAc-3,4-di-O-Ac. A derivative of UDP-ManNAc may be UDP-ManNAc-3-O-Ac or UDP-ManNAc-4-O-Ac or UDP-ManNAc-3,4-di-O-Ac.
One aspect of the present invention relates to the above described truncated version of the CP-A (i.e. ΔN69-CP-A), wherein the function comprises the ability to transfer ManNAc-1P (or a derivative thereof) from UDP-ManNAc (or a derivative thereof) onto an acceptor carbohydrate, wherein the truncated version of the CP-A shows the same activity profile as the full length CP-A. An activity profile means that elongated products are of similar or identical dispersity.
The full length CP-A, the codon optimized CP-A, the ΔN69-CP-A or the codon optimized ΔN69-CP-A may be used in the herein described in vitro methods for producing Nm capsular polysaccharides or compositions. Accordingly, one embodiment of the present invention relates to the in vitro method of the invention or the composition of the invention, wherein the capsule polymerase of Neisseria meningitidis serogroup A is the polypeptide of any one of (a) to (f):
A particular embodiment of the present invention relates to the in vitro method of the invention or the composition of the invention, wherein the capsule polymerase of Neisseria meningitidis serogroup A is the polypeptide of any one of (a) to (f):
As indicated, the function of the above under items (a) to (f) described capsule polymerase of Neisseria meningitidis serogroup A comprises the ability to transfer ManNAc-1P or a derivative thereof from UDP-ManNAc or a derivative thereof onto an acceptor carbohydrate. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of ≥4. For example, the acceptor (or at least 80% of the acceptor carbohydrates) may be DP4, DP5 or DP6; preferably the acceptor (or at least 80% of the acceptor carbohydrates) is a tetramer or pentamer of ManNAc-1P with a free reducing end.
As described, the full length CP-A, the codon optimized CP-A or the codon optimized ΔN69-CP-A (i.e. the polypeptide described above under items (a) to (f)) may be used in the herein described in vitro methods for producing Nm capsular polysaccharides or in the herein described compositions. For example, if one of these capsule polymerases is used in the in vitro methods or compositions described herein, than the ratio of donor carbohydrate to acceptor carbohydrate is from 10:1 to 400:1, preferably from 10:1 to 240:1, and more preferably from 10:1 to 80:1, and even more preferably from 10:1 to 75:1, even more preferably from 20:1 to 75:1, and most preferably from 20:1 to 60:1 (e.g. 40:1 to 60:1 such as 50:1).
In a prioritized aspect of the invention, the codon optimized ΔN69-CP-A is used in the herein described in vitro methods for producing Nm CPS which have a defined length or in the herein described compositions.
The inventors of the present invention surprisingly identified truncated versions of the capsule polymerase of Nm serogroup X (CP-X). For example, the CP-X-fragment ΔN67ΔC99-CP-X (wherein 67 amino acids are lacking at the N-terminus and 99 amino acids are lacking at the C-terminus) was demonstrated to be functional (i.e. able to transfer GlcNAc-1P (or a derivative thereof) from UDP-GlcNAc (or a derivative thereof) onto an acceptor carbohydrate. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of ≥4. For example, the acceptor (or at least 80% of the acceptor carbohydrates) may be DP4, DP5, or DP6 (see, e.g.,
As demonstrated in the appended examples, the inventors of the present invention surprisingly found that, when terminally truncated, the CP-X can be regulated via the donor-acceptor ratio such that it produces CPS with a uniform and defined length. Furthermore, these terminally truncated versions of the CP-X have the additional advantage that they can also be regulated by the reaction time. Moreover, the inventive terminally truncated versions of CP-X have the superior property that they have a significantly improved activity as compared to the full length CP-X. The DNA sequence of the full length CP-X is shown herein as SEQ ID NO: 16 and the amino acid sequence of the full length CP-X is shown herein as SEQ ID NO: 24.
The illustrative appended examples demonstrate several truncated versions of the CP-X which are capable of producing NmX capsular polysaccharides. For example, the proteins ΔN58-CP-X (wherein 58 amino acids are lacking at the N-terminus of CP-X and which comprises approximately 88% of the full length CP-X), ΔC99-CP-X (wherein 98 amino acids are lacking at the C-terminus of CP-X and which comprises approximately 80% of the full length CP-X) and ΔN58ΔC99-CP-X (wherein 58 amino acids are lacking at the N-terminus and 98 amino acids are lacking at the C-terminus and which comprises approximately 68% of the full length CP-X) have been demonstrated to be functional capsular polysaccharides. Herein, in particular in the appended examples, “ΔN58-CP-X”, “ΔC99-CP-X” and “ΔN58ΔC99-CP-X” are also designated as “dN58-CsxA”, “dC99-CsxA” and “dN58dC99-CsxA”, respectively. Regarding the designation “ΔC99” it is noted that this designation has been chosen as the DNA sequence which was used to generate the ΔC99-CP-X construct ends with codon 387 of the CP-X sequence. However, this DNA sequence was cloned to have an XhoI restriction site (CTCGAG) at the 3′ end. The codon CTC within this site encodes leucine. Amino acid 388 of wild type CP-X is also a leucine (however encoded by CTT). Therefore, in the proteins ΔC99-CP-X and ΔN58ΔC99-CP-X, 98 (instead of 99) amino acids are lacking at the C-terminus. In this regard it is further noted that if the herein provided polynucleotide encoding ΔC99-CP-X (i.e. SEQ ID NO: 20) is expressed in a host cell, then the resulting polypeptide has the sequence as given herein in SEQ ID NO: 28. Analogously, if the herein provided polynucleotide encoding ΔN58ΔC99-CP-X (i.e. SEQ ID NO: 21) is expressed in a host cell, then the resulting polypeptide has the sequence as given herein in SEQ ID NO: 29. Herein, in particular in the appended examples, “ΔC99-CP-X” and “ΔN58ΔC99-CP-X” are also designated as “dC99-CsxA” and “dN58dC99-CsxA”, respectively.
As mentioned, the inventors of the present invention surprisingly found that truncated versions of the CP-X have a considerably higher activity as compared to the full length CP-X. More specifically, by using a radioincorporation assay, the appended illustrative examples demonstrate that truncated versions of the CP-X (e.g. ΔN58-CP-X, ΔC99-CP-X or ΔN58ΔC99-CP-X) are more active (i.e. assemble more saccharide monomers to capsule polysaccharides) as compared to the full length CP-X. In the herein described and provided in vitro methods and compositions, the truncated version of the CP-X may preferably be a terminally truncated version of the CP-X. Accordingly, a further embodiment of the present invention relates to a truncated version of the CP-X, which is a polypeptide comprising:
The function of the terminally truncated version of the CP-X comprises the ability to transfer GlcNAc-1P from UDP-GlcNAc onto an acceptor carbohydrate. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of ≥4. For example, the acceptor (or at least 80% of the acceptor carbohydrates) may be DP4, DP5, or DP6; preferably, the acceptor (or at least 80% of the acceptor carbohydrates) is a tetramer or pentamer of GlcNAc-1P with a free reducing end as shown in
As described above, the herein provided terminally truncated version of the CP-X comprises maximal 50-95% of the full length CP-X. It is envisaged, that the terminally truncated version of the CP-X comprises minimal 50% of the full length CP-X and maximal 95% of the full length CP-X. For example, the terminally truncated CP-X of the invention may comprise 50-95%, 55-90%, 60-85% or 65-80% of the full length CP-X. For example, the terminally truncated CP-X may comprise 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or the full length CP-X.
The herein provided terminally truncated version of the CP-X comprises the ability to transfer GlcNAc-1P (or a derivative thereof) from UDP-GlcNAc (or a derivative thereof) onto an acceptor carbohydrate (i.e. onto the hydroxyl group of C4 of a capsular polysaccharide from NmX (or a derivative thereof)). The acceptor carbohydrate may be extended/activated at the reducing end or the reducing end phosphate. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of ≥4. For example, the acceptor (or at least 80% of the acceptor carbohydrates) may be DP4, DP5, or DP6; preferably, the acceptor (or at least 80% of the acceptor carbohydrates) is a tetramer or pentamer of GlcNAc-1P with a free reducing end as shown in
The transfer GlcNAc-1P (or a derivative thereof) from UDP-GlcNAc (or a derivative thereof) onto an acceptor carbohydrate may be performed by incubating a truncated version of the CP-X (e.g. ΔC99-CP-X, ΔN58-CP-X or ΔN58ΔC99-CP-X) with UDP-GlcNAc (or a derivative thereof) and an acceptor carbohydrate and in reaction buffer (50 mM Tris pH 8.0, and 20 mM MgCl2, and optionally 1-2 mM (e.g. 1 mM) DTT) and incubated for 1 h—over night (preferably for 4-7 hours) at 25-37° C. (preferably between 30 and 37° C.) (Fiebig et al., 2014; Glycobiology. 2014 February; 24(2):150-8). Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of ≥4. For example, the acceptor (or at least 80% of the acceptor carbohydrates) may be DP4, DP5, or DP6; preferably, the acceptor (or at least 80% of the acceptor carbohydrates) is a tetramer or pentamer of GlcNAc-1P with a free reducing end as shown in
Whether a transfer from GlcNAc-1P (or a derivative thereof) from UDP-GlcNAc (or a derivative thereof) onto an acceptor carbohydrate has occurred can be tested, e.g. by high percentage PAGE and developed by alcian blue/silver staining (Fiebig et al., Glycobiology. 2014 February; 24(2):150-8) in the presence of a size marker or by 31P NMR monitoring the characteristic phosphodiester signal (Fiebig et al., Glycobiology. 2014 February; 24(2):150-8), or by High Performance Anionic Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) as described (Berti et al. Vaccine. 2012 Oct. 5; 30(45):6409-15; Fiebig et al., Glycobiology. 2014 February; 24(2):150-8).
In context of the present invention it was surprisingly found that the C-terminally truncated CP-X as well as the C- and N-terminally truncated CP-X can be regulated via the donor-acceptor ratio and/or via the reaction time so as to produce capsular polysaccharides that have a uniform and defined length. In particular, the appended examples show that the truncated proteins ΔC99-CP-X and ΔN58ΔC99-CP-X can be regulated via the donor-acceptor ratio or via the reaction time such that they produce capsular polysaccharides which have a uniform and defined length. Accordingly, one aspect the invention relates to a terminally truncated version of the CP-X, which is a C-terminally or a C- and N-terminally truncated version of the full length CP-X.
Thus, the invention relates to the herein provided terminally truncated version of the CP-X, comprising:
In one aspect of the present invention, the terminally truncated version of the CP-X which is described and provided herein is the construct ΔN58-CP-X, ΔC99-CP-X or ΔN58ΔC99-CP-X.
Thus, the invention relates to the herein provided terminally truncated version of the CP-X, comprising
Accordingly, in one aspect the invention relates to the terminally truncated version of the CP-X comprising:
A particular embodiment of the invention relates to the above described terminally truncated version of the CP-X, wherein the function comprises the ability to transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate; wherein the produced polymers have a DP between 10 and 60 or an avDP between 15 and 20, when the ratio of donor carbohydrate to acceptor carbohydrate is in the range of 10:1 to 400:1. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of ≥4. For example, the acceptor (or at least 80% of the acceptor carbohydrates) may be DP4, DP5, or DP6; preferably, the acceptor (or at least 80% of the acceptor carbohydrates) is a tetramer or pentamer of GlcNAc-1P with a free reducing end as shown in
As described, the terminally truncated version of the CP-X (i.e. the polypeptide described above under items (i) and (ii)) may be used in the herein described in vitro methods for producing Nm capsular polysaccharides or in the herein described compositions.
For example, if the terminally truncated CP-X is regulated via the ratio of donor carbohydrate to acceptor carbohydrate (and not via the reaction time) then the following preferred ratios can produce a product distribution with a low dispersity. Or, in other words, if the terminally truncated version of the CP-X is regulated via the ratio of donor carbohydrate to acceptor carbohydrate, then the following preferred ratios can produce capsular polysaccharides of which at least 60%, (preferably at least 80%) have a defined length between DP10 and DP60 or between avDP15 and avDP20. Moreover, the following capsule polymerases and ratios are preferably used in the compositions described herein.
For example, if the capsule polymerase is a polypeptide which has at least 80% (preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99%) sequence identity to a polypeptide encoded by a nucleic acid molecule which comprises the nucleic acid sequence of SEQ ID NO: 20 (ΔC99-CP-X) and being functional, then the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 10:1 to 80:1, more preferably from 20:1 to 60:1, even more preferably from 20:1 to 50:1, and most preferably from 33:1 to 50:1. In addition, if the capsule polymerase is a polypeptide which has at least 80% (preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99%) sequence identity to a polypeptide encoded by a nucleic acid molecule which comprises the nucleic acid sequence of SEQ ID NO: 17 (ΔN58-CP-X) and being functional, then the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 10:1 to 80:1, more preferably from 20:1 to 50:1, and most preferably from 20:1 to 33:1 (e.g. 33:1). Furthermore, if the capsule polymerase is a polypeptide which has at least 80% (preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99%) sequence identity to a polypeptide encoded by a nucleic acid molecule which comprises the nucleic acid sequence of SEQ ID NO: 21 (ΔN58ΔC99-CP-X) and being functional, then the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 50:1 to 400:1, more preferably from 80:1 to 400:1, even more preferably from 100:1 to 400:1, and most preferably from 100:1 to 200:1. In addition, if the capsule polymerase is a polypeptide which comprises an amino acid sequence which has at least 80% (preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99%) sequence identity to SEQ ID NO: 28 (ΔC99-CP-X) and being functional, then the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 10:1 to 80:1, more preferably from 20:1 to 60:1, even more preferably from 20:1 to 50:1, and most preferably from 33:1 to 50:1. Furthermore, if the capsule polymerase is a polypeptide which comprises an amino acid sequence which has at least 80% (preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99%) sequence identity to SEQ ID NO: 25 (ΔN58-CP-X) and being functional, then the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 10:1 to 80:1, more preferably from 20:1 to 50:1, and most preferably from 20:1 to 33:1 (e.g. 33:1). Moreover, if the capsule polymerase is a polypeptide which comprises an amino acid sequence which has at least 80% (preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99%) sequence identity to SEQ ID NO: 29 (ΔN58ΔC99-CP-X) and being functional, then the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 50:1 to 400:1, more preferably from 80:1 to 400:1, even more preferably from 100:1 to 400:1, and most preferably from 100:1 to 200:1.
In context of the invention the terminally truncated version of the CP-X may also be a polypeptide comprising the construct ΔN65ΔC10-CP-X (which is shown in SEQ ID NOs: 22 and 30) or ΔN67ΔC99-CP-X (which is shown in SEQ ID NOs: 23 and 31) which have been demonstrated to be active (data not shown) and which can be regulated via the donor-acceptor ratio or via the reaction time such that they produce capsular polysaccharides with a uniform and defined length.
Accordingly, the terminally truncated version of the CP-X of the present invention may be a polypeptide comprising the amino acid of any one of SEQ ID NOs: 25, 28, 29, 30 or 31. Also encompassed by the present invention are terminally truncated versions of the CP-X comprising the amino acid sequence of any one of SEQ ID NOs: 25, 28, 29, 30 or 31 wherein one, two, three or more amino acid residues are added, deleted or substituted. The polypeptides may have the function of the terminally truncated version of the CP-X (i.e. the ability to transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate). Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of ≥4. The amino acid sequence of the polypeptides may be at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identical to SEQ ID NO: 25, 28, 29, 30 or 31. The polypeptides may have the function of a truncated version of the CP-X. Preferably, the function comprises the ability to transfer GlcNAc-1P (or a derivative thereof) from UDP-GlcNAc (or a derivative thereof) onto an acceptor carbohydrate; wherein at least 60% (preferably at least 80%) of the produced polymers have a DP between 10 and 60 or an avDP between 15 and 20, when the ratio of donor carbohydrate to acceptor carbohydrate is in the range of 10:1 to 400:1. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of ≥4. For example, the acceptor (or at least 80% of the acceptor carbohydrates) may be DP4, DP5, or DP6; preferably, the acceptor (or at least 80% of the acceptor carbohydrates) is a tetramer or pentamer of GlcNAc-1P with a free reducing end as shown in
The herein described terminally truncated versions of the CP-X may be linked (e.g. operatively linked) to additional, heterologous sequences. E.g. the nucleic acid molecule encoding a truncated version of the CP-X may be linked to additional, heterologous nucleic acid sequences in a recombinant nucleic acid molecule. For example, said additional nucleic acid sequence may be a coding gene.
The term “recombinant nucleic acid molecule” relates to nucleic acid molecules originating from a different genetic context and combined by molecular biological methods. Here, the term “different genetic context” relates to genomes from different species, varieties or individuals or different positions within a genome. Recombinant nucleic acid molecules can contain not only natural sequences but also sequences, which, compared to the natural ones are mutated or chemically modified or else, the sequences are altogether newly synthesized sequences.
As indicated above, the terminally truncated version of the CP-X may be linked to an additional heterologous molecule. Said additional heterologous molecule may be a nucleic acid molecule which is preferably operatively linked to a truncated version of the CP-X. Said additional heterologous nucleic acid molecule may originate from a different genetic context than the truncated version of the CP-X. Non-limiting examples of additional heterologous molecules comprise in particular marker molecules, like luciferase, galactosidase, GFP, EGFP, DsRed, etc. or tag-molecules, like Flag-tags, CBP and others. In a particular aspect of the invention, said additional heterologous molecule is a tag, such as a StrepII-tag, a thrombin-tag, a his-tag, a MBP-tag or a S3N10-tag. In a further aspect of the invention, said additional heterologous molecule is a nucleic acid molecule encoding a restriction site (e.g. a restriction site of the enzymes BamHI/BgIII, XhoI or NdeI). It is also envisaged that the truncated CP-X of the invention is linked to several additional sequences (e.g. several tags or several restriction sites). Yet, as detailed below, also sequences which optimize (e.g. enhance) the expression of the terminally truncated CP-X may be operatively linked to the nucleic acid sequence of the truncated CP-X. Such a sequence may be a particular promoter sequence. A suitable promoter for the expression of the terminally truncated version of the CP-X may be, e.g., Tac-, T7-, or Lacpromoter.
One embodiment of the present invention relates to a nucleic acid molecule encoding the herein described truncated version of the CP-X. The invention also relates to a vector comprising said nucleic acid molecule. The present invention further relates to vectors containing a nucleic acid molecule of the present invention encoding a terminally truncated version of the CP-X. The present invention relates also to a vector comprising the nucleic acid construct encoding the herein described truncated version of the CP-X.
The term “vector” relates to circular or linear nucleic acid molecules which can autonomously replicate in host cells into which they are introduced. The “vector” as used herein particularly refers to plasmids, cosmids, viruses, bacteriophages and other vectors commonly used in genetic engineering. In a preferred embodiment, the vectors of the invention are suitable for the transformation of cells, like fungal cells, cells of microorganisms such as yeast or prokaryotic cells. In a particularly preferred embodiment such vectors are suitable for stable transformation of bacterial cells, for example to express the truncated version of the CP-X of the present invention.
Accordingly, in one aspect of the invention, the vector as provided is an expression vector. Generally, expression vectors have been widely described in the literature. As a rule, they may not only contain a selection marker gene and a replication-origin ensuring replication in the host selected, but also a promoter, and in most cases a termination signal for transcription. Between the promoter and the termination signal there is preferably at least one restriction site or a polylinker which enables the insertion of a nucleic acid sequence/molecule desired to be expressed.
It is to be understood that when the vector provided herein is generated by taking advantage of an expression vector known in the prior art that already comprises a promoter suitable to be employed in context of this invention (for example expression of a truncated version of the CP-X as described herein above) the nucleic acid construct is inserted into that vector in a manner that the resulting vector comprises only one promoter suitable to be employed in context of this invention. The skilled person knows how such insertion can be put into practice. For example, the promoter can be excised either from the nucleic acid construct or from the expression vector prior to ligation.
A non-limiting example of the vector of the present invention is the plasmid vector pET22b comprising a nucleic acid construct of the present invention. Further examples of vectors suitable to comprise a nucleic acid construct of the present invention to form the vector of the present invention are known in the art and are, for example other vectors for bacterial expression systems such as vectors of the pET series (Novagen) or pQE vectors (Qiagen).
In an additional embodiment, the present invention relates to a host cell comprising the herein described vector. In particular, the invention relates to a host cell comprising a nucleic acid construct encoding a terminally truncated version of the CP-X and/or the vector of the present invention. Preferably, the host cell of the present invention may be a prokaryotic cell, for example, a bacterial cell. As a non limiting example, the host cell of the present invention may be Escherichia coli. The host cell provided herein is intended to be particularly useful for generating the terminally truncated version of the CP-X of the present invention.
Generally, the host cell of the present invention may be a prokaryotic or eukaryotic cell, comprising a nucleic acid construct of the invention or the vector of the invention or a cell derived from such a cell and containing a nucleic acid construct of the invention or the vector of the invention. In a preferred embodiment, the host cell is genetically modified with a nucleic acid construct of the invention or the vector of the invention in such a way that it contains the nucleic acid construct of the present invention integrated into the genome. For example, such host cell of the invention, but also the host cell of the invention in general, may be a bacterial, yeast, or fungus cell.
In one particular aspect, the host cell of the present invention is capable to express or expresses a terminally truncated version of the CP-X as defined herein and as illustrative characterized in SEQ ID NOs: 17, 20, 21, 22 or 23, for example in SEQ ID NO: 21. An overview of examples of different corresponding expression systems to be used for generating the host cell of the present invention is for instance contained in Methods in Enzymology 153 (1987), 385-516, in Bitter et al. (Methods in Enzymology 153 (1987), 516-544), in Sawers et al. (Applied Microbiology and Biotechnology 46 (1996), 1-9), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12 (1994), 456-463), and in Griffiths et al., (Methods in Molecular Biology 75 (1997), 427-440).
The transformation or genetically engineering of the host cell with a nucleic acid construct of the invention or the vector according to the invention can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990.
The nucleic acid molecules, vectors, host cells and polypeptides described herein may be used for producing (i.e. synthesizing) Nm capsular polysaccharides which have a defined length.
As mentioned, in the herein provided in vitro methods for producing Nm CPS which have a defined length or in the compositions of the invention, the terminally truncated version of the CP-X as described herein may be used.
As for vaccine production the Nm CPS have preferably a DP of 10 to 60 (i.e. an avDP of 15 to 20), it is preferred that the Nm CPS which are produced by the herein described in vitro methods have this size. Advantageously, if in the herein described in vitro methods the CP-A or a terminally truncated version of the CP-X is used, then the produced Nm CPS have a DP or 10 to 60 (i.e. an avDP of 15 to 20), if the donor-to-acceptor-ratio is in the range from 10:1 to 400:1. In addition, if in herein described in vitro methods the truncated version of the CP-X is used, then the produced Nm CPS have a DP of 10 to 60 (i.e. an avDP of 15 to 20) if the incubation time ranges from 3 to 45 minutes. Thus, one aspect of the invention relates to the herein provided in vitro methods, wherein the capsular polysaccharides have a DP of 10 to 60 or an avDP of 15 to 20. In a preferred aspect of the herein provided in vitro methods, at least 60% (more preferably at least 80%) of the capsular polysaccharides have a DP of 10 to 60 or an avDP of 15 to 20. In addition, a further embodiment of the invention relates to the in vitro methods of the invention or the composition of the invention, wherein
In item (i) of the above described embodiment, the ratio of donor carbohydrate to acceptor carbohydrate may be 50:1.
According to the inventive in vitro method, the donor carbohydrate which is contacted with at least one purified capsule polymerase (CP) may further be activated during step (a). Thus, one embodiment of the invention relates to the in vitro methods of the invention, wherein said donor carbohydrate is activated or wherein said donor carbohydrate is activated during step (a). Said activation during step (a) may be performed by contacting said donor carbohydrate with an activating enzyme. In addition, during this in step (a) of the inventive in vitro methods, said donor carbohydrate may be further contacted with PEP and/or at least one activating nucleotide. Analogously, in the herein provided composition said donor carbohydrate may be activated. In the methods or in the composition of the invention, said donor carbohydrate may be activated by linkage to an activating nucleotide. Said activating nucleotide may be, e.g. CMP, CDP, CTP, UMP, UDP, TDP, AMP or UTP, preferably UDP.
For example, the activation may be mediated by linkage of an activating nucleotide such as CMP, UDP, TDP or AMP. Preferably, the activating nucleotide is UDP. The activation of a carbohydrate by linkage of a nucleotide may be catalyzed by several activating enzymes which are known in the art. Such activating enzymes may be incubated with the at least one donor carbohydrate, at least one acceptor carbohydrate and the at least one CP during step (a) of the in vitro method provided herein. UDP-ManNAc is preferably synthesized from UDP-GlcNAc using the enzyme UDP-GlcNAc-epimerase. In SEQ ID NO: 32, the nucleotide sequence of UDP-GlcNAc-epimerase cloned from Neisseria meningitidis serogroup A is shown, the corresponding polypeptide sequence of UDP-GlcNAc-epimerase is shown in SEQ ID NO: 33.
One particular embodiment of the present invention relates to the herein provided in vitro methods or the herein provided composition, wherein the capsule polymerase is a truncated version of the capsule polymerase of Neisseria meningitidis serogroup X and wherein at least one donor carbohydrate is UDP-GlcNAc. However, another embodiment of the present invention relates to the herein provided in vitro methods or the herein provided composition, wherein the capsule polymerase is a truncated version of the capsule polymerase of Neisseria meningitidis serogroup X and wherein at least one donor carbohydrate is GlcNAc-1-P.
In a further embodiment of the herein described in vitro methods or compositions the capsule polymerase is the capsule polymerase of Neisseria meningitidis serogroup A and at least one donor carbohydrate is UDP-ManNAc. The sugar building block UDP-ManNAc is commercially not available. Therefore, a further advantage of the present invention is that UDP-ManNAc can be synthesized (during the inventive in vitro method for producing Nm capsular polysaccharides) from cheap UDP-GlcNAc. Thus, another embodiment of the present invention relates to the inventive in vitro methods or the inventive composition, wherein the capsule polymerase is the capsule polymerase of Neisseria meningitidis serogroup A and wherein at least one donor carbohydrate is UDP-GlcNAc.
In context of the present invention, UDP-ManNAc is preferably synthesized from UDP-GlcNAc using the enzyme UDP-GlcNAc-epimerase. In SEQ ID NO: 32, the nucleotide sequence of UDP-GlcNAc-epimerase cloned from Neisseria meningitidis serogroup A is shown, the corresponding polypeptide sequence of UDP-GlcNAc-epimerase is shown in SEQ ID NO: 33. Thus, one aspect of the invention relates to the herein provided in vitro methods, wherein in step (a) the capsule polymerase is further incubated with the UDP-GlcNAc-epimerase. In addition, one embodiment of the invention relates to the herein provided composition, further comprising the UDP-GlcNAc-epimerase. For example, in the in vitro methods or compositions of the present invention, the UDP-GlcNAc-epimerase (e.g. CsaA of NmA) may be used in a concentration of 1-2000 nmol, e.g. 10 nmol.
In another embodiment of the inventive in vitro methods and compositions, the capsular polymerase (CP) may be a truncated version of the CP-X or a functional derivative thereof and at least one donor carbohydrate may be GlcNAc-1-phosphate. Said donor carbohydrate GlcNAc-1-phosphate may be further contacted with at least one nucleotide and/or phosphoenolpyruvate (PEP) and auxiliary enzymes. Said nucleotide can be, e.g., UMP, UDP or UTP. Said donor carbohydrate GlcNAc-1-phosphate may further be activated during incubation with the terminally truncated CP-X. In accordance with the herein presented in vitro method, this activation may yield the activated sugar nucleotide UDP-GlcNAc.
Thus, the CP to be applied in the means and methods described herein may be a truncated version of CP-X or a functional derivative thereof and at least one donor carbohydrate may be UDP-GlcNAc or a derivative thereof. Examples for derivatives of UDP-GlcNAc may be compounds that are alkylated or hydroxylated or that comprise additional functional groups, such as carboxylic acids, azides, amides, acetyl groups or halogen atoms; see also “Carbohydrate chemistry” Volumes 1-34, Cambridge [England], Royal Society of Chemistry, loc. cit.
Generally, in context of the present invention, the saccharides described herein may also be labelled forms of these saccharides. For example, the saccharides may be labelled radioactively, such as [14C] or [3H]. Such labelling may be inter alia useful in diagnostic applications and uses of the saccharides described herein. Such diagnostic applications and uses will be further described herein below.
In accordance with the inventive method, the terminally truncated version of the CP-X is contacted with at least one donor carbohydrate and with at least one acceptor carbohydrate during the incubation step (a) of the in vitro method presented herein. Said acceptor carbohydrate may be oligomeric or polymeric CPS of Neisseria meningitidis serogroup X (CPSX), and/or a carbohydrate structure containing terminal GlcNAc residues such as hyaluronic acid, heparin, heparin sulphate or protein-linked oligosaccharides.
In the herein provided methods for producing Nm capsular polysaccharides which have a defined length, it is preferred that the acceptor carbohydrate has a DP≥4. Or, in other words, acceptor carbohydrates with a length of DP≥4 are particularly preferred in the herein described methods for producing Nm capsular polysaccharides which have a defined length. Normally, the acceptor carbohydrates are purified capsular polysaccharides of the respective serogroup and have different length. However, in the herein provided methods for producing capsular polysaccharides with a defined length, and in the herein provided compositions, it is preferred that at least 80% of the acceptor carbohydrates are polysaccharides with a DP of ≥4. The acceptor carbohydrates may also consist of polysaccharides with a DP of ≥4. For example, if the CP-A is used, then the acceptor (or at least 80% of the acceptor carbohydrates) is preferably DP4, DP5, or DP6; more preferably the acceptor (or at least 80% of the acceptor carbohydrates) is a tetramer or pentamer of ManNAc-1P with a free reducing end. If a terminally truncated version of the CP-X is used, then the acceptor (or at least 80% of the acceptor carbohydrates) is preferably DP4, DP5, or DP6; more preferably, the acceptor (or at least 80% of the acceptor carbohydrates) is a tetramer or pentamer of GlcNAc-1P with a free reducing end as shown in
An example of the present invention relates to the in vitro method for producing Nm capsular polysaccharides of NmX which have a defined length, said method comprising the steps:
A further example of the present invention relates to the in vitro method for producing Nm capsular polysaccharides of NmX which have a defined length, said method comprising the steps:
Another example of the present invention relates to the in vitro method for producing Nm capsular polysaccharides of NmX which have a defined length, said method comprising the steps:
In the above examples also other combinations of activated or non-activated donor carbohydrates and acceptor carbohydrates as described herein can be applied. Such other combinations do not defer from the gist of the present invention.
In one embodiment of the present in vitro method, the CP to be used is CP-A or a functional derivative thereof and at least one donor carbohydrate may be UDP-ManNAc or a derivative thereof. Examples for derivatives of UDP-ManNAc may be compounds that are alkylated or hydroxylated or that comprise additional functional groups such as carboxylic acids, azides, amides, acetyl groups or halogen atoms; see also “Carbohydrate chemistry” Volumes 1-34: monosaccharides, disaccharides, and specific oligosaccharides, Reviews of the literature published during 1967-2000, Cambridge (England), Royal Society of Chemistry.
In another embodiment of the in vitro method described herein, the CP is CP-A or a functional derivative thereof and at least one donor carbohydrate is ManNAc-1-phosphate or a derivative thereof. Examples for derivatives of ManNAc-1-phosphate ManNAc-3-O-Ac or ManNAc-4-O-Ac or ManNAc-3,4-di-O-Ac. In the herein provided in vitro method, said donor carbohydrate ManNAc-1-phosphate (ManNAc-1-P) may be contacted with at least one nucleotide and/or phosphoenolpyruvate (PEP) and auxiliary enzymes during step (a) of the in vitro method. Said nucleotide can be, e.g., UMP, UDP and UTP. Said donor carbohydrate ManNAc-1-phosphate may be activated during incubation with CP-A. In accordance with the herein presented in vitro method, this activation may yield the activated sugar nucleotide UDP-ManNAc, or its derivatives UDP-ManNAc-3-O-Ac or UDP-ManNAc-4-O-Ac or UDP-ManNAc-3,4-di-O-Ac. Thus, one aspect of the invention relates to the herein provided in vitro methods, wherein the capsule polymerase is the capsule polymerase of Neisseria meningitidis serogroup A and wherein at least one donor carbohydrate is ManNAc-1-P.
In the herein provided in vitro method, CP-A or a functional derivative thereof is contacted with at least one donor carbohydrate and with an acceptor carbohydrate during the incubation step (a) of the inventive in vitro method. In accordance with the inventive in vitro method presented herein, the acceptor carbohydrate may be oligomeric or polymeric CPS of Neisseria meningitidis serogroup A (CPSA) and/or a carbohydrate structure containing terminal GlcNAc or ManNAc residues such as hyaluronic acid, heparin, heparin sulphate or protein-linked oligosaccharides. As described above, in the herein provided in vitro methods, the acceptor carbohydrate of CP-A may be the cheap and easily commercially available UDP-GlcNAc, as the UDP-GlcNAc-epimerase can be used to convert UDP-GlcNAc into UDP-ManNAc.
An example of the present invention relates to the in vitro method for producing Nm capsular polysaccharides of NmA which have a defined length, said method comprising the steps:
A further example of the present invention relates to the in vitro method for producing Nm capsular polysaccharides of NmA which have a defined length, said method comprising the steps:
Again, the skilled person readily understands that also other combinations of activated or non-activated donor carbohydrates and acceptor carbohydrates as described herein can be applied. Such other combinations do not defer from the gist of the present invention.
As described herein and illustrated in the appended examples, the acceptor carbohydrate is preferred by the CP (e.g. CP-A) if it is presented in non-acetylated form. Moreover, the acceptor is even more preferred if presented in non-acetylated, de-phosphorylated form. Accordingly, one embodiment of the present invention relates to the herein provided in vitro methods or the herein provided composition, wherein the acceptor carbohydrate is non-acetylated and/or dephosphorylated, preferably non-acetylated and dephosphorylated.
Within a CPS obtainable by the inventive method described hereinabove, one or more carbohydrates of the CPS-subunits may be derivatized and may contain, for example, additional functional groups such as acetyl groups, amino groups, alkyl groups, hydroxyl groups, carboxylic acids, azides, amides, or halogen atoms; see also “Carbohydrate chemistry” Volumes 1-34: monosaccharides, disaccharides, and specific oligosaccharides, Reviews of the literature published during 1967-2000, Cambridge (England), Royal Society of Chemistry. Thus, in one embodiment of the inventive in vitro method or composition, the acceptor carbohydrate carries one or more additional functional groups at its reducing end. Said additional functional groups, may be, e.g., acetyl groups, carboxylic acids, azides, amides, or halogen atoms; see also “Carbohydrate chemistry” Volumes 1-34, Cambridge [England], Royal Society of Chemistry, loc. cit.
One embodiment of the present invention relates to the herein provided in vitro method or the herein provided composition, wherein said acceptor carbohydrate is capsule polysaccharide of Neisseria meningitidis serogroup A or X or a carbohydrate structure containing terminal GlcNAc residues. Said carbohydrate structure containing terminal GlcNAc-residues may be hyaluronic acid, heparin sulphate, heparan sulphate or protein-linked oligosaccharides.
As demonstrated in the appended examples, the inventors of the present invention analyzed the minimal length of the priming acceptor for CP-A. Therefore, synthetic compounds which are shown in
Thus, in context of the present invention, a dimer of ManNAc-1P carrying a phosphodiester at the reducing end was identified as minimal acceptor for CP-A or derivatives of CP-A (such as ΔN69-CP-A).
Accordingly, the inventors of the present invention identified two important features of CP-A: (i) the minimal efficient acceptor is a dimer and (ii) the reducing end phosphate group can be extended with rather large chemical groups (such as decyl-ester). This latter finding bears the perspective that chain elongation can be primed with reagents of very high purity and functional groups that facilitate conjugation of glycans to carrier proteins in the vaccine production chain (Costantino (2011) Expert opinion on drug discovery 6 (10), 1045-1066; Bardotti (2008) Vaccine 26 (18), 2284-2296). Besides decyl-ester, chain elongation may be primed with a number of functional groups that allow conjugation to carrier proteins, e.g., Alkyl-azides, Alkyl-amindes or sulfhydryl-reagents chemical functions as described in Costantino et al., 2011 Expt. Opin. Drug. Discov. 6:1053; Table 2.
Accordingly, one embodiment of the present invention relates to the in vitro method of the invention or the composition of the invention, wherein said acceptor carbohydrate is a dimer of ManNAc carrying a phosphodiester at the reducing end. In this embodiment of the present invention, the capsule polymerase may be the capsule polymerase of Neisseria meningitidis serogroup A. Moreover, the phosphate group at the reducing end may be extended with alkyl-azides, alkyl-amindes or sulfhydryl-groups and chemical functions as described in Costantino (2011) Expt. Opin. Drug. Discov. 6:1053; Table 2. For example, in the herein provided in vitro methods or composition, the acceptor carbohydrate may be a disaccharide carrying a decyl-phosphate-ester at the reducing end. As mentioned, in this embodiment of the present invention, the capsule polymerase may be the capsule polymerase of Neisseria meningitidis serogroup A.
If in the herein described in vitro methods or compositions the acceptor is a dimer of ManNAc carrying a phosphodiester at the reducing end (which may be extended with large chemical groups such as a decyl-ester), then the capsular polymerase may be CP-A or a derivative of CP-A (such as ΔN69-CP-A).
The capsule polymerase (CP) which is incubated with the at least one donor carbohydrate in the presented in vitro methods and compositions may be purified. Said CP (or a functional fragment or derivative thereof) may be isolated from Neisseria meningitidis lysates or recombinantly produced. Recombinant production of a CP (or a functional fragment or derivative thereof) may be performed by transferring the DNA coding for the CP (or the functional fragment or derivative thereof) into a vector and using a host (e.g. eukaryotic host cells such as insect cells or yeast or prokaryotic host cells such as Escherichia coli) to express the CP (or the functional fragment or derivative thereof). After expression, the CP (or the functional fragment or derivative thereof) may be purified from the cell lysate. The purification may be carried out following the instructions of the Qiaexpressionist (http://www.qiagen.com/literature/render.aspx?id=128, Qiagen) for bacterial protein purification or by using BD Bioscience Baculo Gold instruction (Cat. No. 560138) for insect cell protein production and purification. For instance, the CP (or the functional fragment or derivative thereof) may be produced by purifying the recombinant CP (or the functional fragment or derivative thereof) from bacteria via his-tag purification following the instructions of the Qiaexpressionist. A CP (or a functional fragment or derivative thereof) may also be synthetically or chemically produced. Therefore solid phase peptide synthesis (SPPS) may be applied.
The donor carbohydrates which are used in the herein provided means and methods may be obtained from commercial sources or may be chemically synthesized (Wolf, Chemistry (2009) 15:7656-64; Wolf, Eur J Cell Biol. (2010) 89:63-75. The acceptor carbohydrates which are used in the inventive means and methods may be obtained by purifying them from Neisseria meningitidis cultures and can be chemically synthesized as described in the attached manuscript (Black (2010) Synthesis of structures corresponding to the capsular polysaccharide of Neisseria meningitidis group A. 4th Baltic Meeting on Microbial Carbohydrates, Abstracts, p. 56 (abstract 5), Hyytiälä Forestry Field Station, Finland; Black (2011) Towards synthetic glycoconjugates based on the structure of Neisseria meningitidis group A capsular polysaccharide. 16th European Carbohydrate Symposium, Abstracts, p. 61 (OL-02), Sorrento, Italy; Nikolaev (2011) From phosphosaccharide chemistry to potential anti-parasite and anti-bacterial carbohydrate vaccines. Carbohydrate Gordon Research Conference, Abstracts, p. 4, Colby College, Waterville, Me., USA.). One embodiment of the invention relates to the herein provided in vitro methods or composition, wherein said acceptor carbohydrate is purified.
As mentioned, the acceptor carbohydrate which is contacted with the donor carbohydrate and the CP may be purified according to the in vitro method described herein. If said acceptor carbohydrate is oligomeric or polymeric CPS of Neisseria meningitidis, it may be hydrolysed. Thus, one aspect of the invention relates to the herein provided in vitro methods or composition, wherein said acceptor capsule polysaccharide is hydrolysed.
It is known in the art that capsule polysaccharides of NmA are immunogenic only if O-acetylated (Berry (2002) Infection and immunity 70 (7), 3707-3713). Therefore, in the herein described in vitro methods for producing capsular polysaccharides an O-acetyltransferase can be used which is able to perform this modification in a nature identical form. Accordingly, one embodiment of the invention relates to the herein provided in vitro method, further comprising O-acetylation of the produced capsule polysaccharides. Said O-acetylation may be performed by contacting the produced capsule polysaccharides with an O-acetyltransferase. Also provided herein is the composition of the invention, further comprising an O-acetyltransferase.
The O-acetyltransferase which is used in the inventive methods and compositions may be, e.g. the CsaC of NmA. Accordingly, one aspect of the invention relates to the in vitro methods of the invention or the composition of the invention, wherein, wherein the O-acetyltransferase is the polypeptide of any one of (a) to (f):
The function of this O-acetyltransferase comprises the ability to transfer Acetyl-groups from the donor Acetyl-Coenzyme A onto hydroxyl-groups of UDP-ManNAc or oligo- and polymeric structures consisting of ManNAc-1-phosphate units linked together by phosphodiester linkages. Or, in other words this O-acetlytransferase catalyses the formation of an ester-bond between an Acetyl-group and a hydroxyl-group of ManNAc-1-phosphate containing molecule. Thus, one aspect of the invention relates to the in vitro methods of the invention or the composition of the invention, wherein the O-acetyltransferase is the polypeptide of any one of (a) to (f):
In context of the present invention, production and O-acetylation of capsular polysaccharides (e.g. of capsular polysaccharides of NmA) may be performed by using a two-step protocol or in a one-pot reaction.
For example, the O-acetylation of capsular polysaccharides may be performed by incubating in vitro synthesized capsular polysaccharides (e.g. 1 mg) in the presence of an O-acetyltransferase (e.g. 1.2 nmol CsaC of NmA) in a total volume of 0.5 mL. The reaction may be performed in reaction buffer (e.g. a buffer comprising 25 mM Tris pH 7.5 and 50 mM NaCl), started by the addition of an equimolar amount of Acetyl-CoA (e.g. 14 mM final) and allowed to proceed for 4 h at 37° C. Obviously, this protocol may be up or down scale depending on the needed amount of O-acetylated capsular polysaccharides.
As mentioned above, capsule polysaccharides of NmA are only immunogenic if they are O-acetylated. In addition, natural capsule polysaccharides of NmA are O-acetylated in positions 3 and 4 of ManNAc. Thus, if the herein provided in vitro methods are combined with O-acetylation of the produced capsule polysaccharides, then the used capsule polymerase may be CP-A. Thus, one aspect of the invention relates to the herein provided in vitro methods or composition, wherein the capsule polymerase is the capsule polymerase of Neisseria meningitidis serogroup A. The capsule polysaccharides produced by the inventive in vitro methods may be O-acetylated in positions 3 and 4 of ManNAc.
As described herein, the capsule polysaccharides (e.g. the O-acetylated capsular polysaccharides) which are produced by the herein described methods may be used for glycoconjugate vaccine production. Thus, one embodiment of the present invention relates to a method for producing a vaccine comprising the herein provided in vitro method. Said method for producing a vaccine may further comprise covalently attaching the produced capsular polysaccharides to a carrier molecule. Said carrier molecule may be tetanus toxoid or CRM197 (Cross-Reacting Material 197), an inactive form of diphteria-toxin (Micoli et al., 2013; PNAS).
Thus, the invention relates to a method for the production of a vaccine against a strain genus Neisseria comprising the steps of:
Accordingly, the invention relates to a method for the production of a vaccine against a strain or strains of the genus Neisseria, in particular N. meningitidis by combining (a) Nm capsular polysaccharide(s) of the invention with a biologically acceptable carrier.
Another embodiment of the present invention also relates to a capsular polysaccharide which has been produced by the in vitro method of the present invention. The CPS obtainable by the in vitro methods described hereinabove are useful as pharmaceuticals, e.g., as vaccines. In particular, the herein described CPS are advantageous as vaccines in the prophylaxis and treatment of diseases caused by Neisseria meningitidis, such as neisserial meningitidis. Accordingly, one embodiment of the present invention relates to the capsular polysaccharide of the invention for use as a medicament.
As mentioned, the synthetic CPS obtainable by the inventive in vitro method may be used as vaccines. In a preferred embodiment of the present invention, they are used in vaccination of a human subject. Also disclosed is the use of the CPS obtainable by the inventive in vitro method for the preparation of a vaccine. In a specific embodiment of the present invention, the CPS obtainable by the in vitro methods described herein are used as vaccines against meningococcal meningitidis caused by Neisseria meningitidis serogroup A or X. Thus, the medicaments disclosed herein may be useful in pharmaceutical and vaccination purposes, i.e. for the treatment of Neisseria-induced diseases or the vaccination against these pathogens. Therefore, one embodiment of the present invention relates to a vaccine comprising the capsular polysaccharide of the invention, optionally further comprising a pharmaceutically acceptable carrier. Also provided herein is the capsular polysaccharide of the invention or the vaccine of the invention for use in vaccination of a subject. Accordingly, the present invention provides for a method for treating and/or preventing a meningococcal meningitidis comprising the administration of (a) capsular polysaccharide(s) of the present invention or the vaccine(s) of the present invention to a subject in need of such a treatment. Preferably said subject is a human patient.
A “patient” or “subject” for the purposes of the present invention includes both humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient is human being. As indicated, the herein provided capsular polysaccharide(s) or vaccine(s) may be used for the vaccination against meningococcal meningitidis caused by Neisseria meningitidis serogroup A or X.
The medicaments provided herein are pharmaceutical compositions and may comprise the CPS of the present invention. The pharmacological compositions may further comprise antibodies specifically directed against the CPS of the present invention. Such CPS as well as the antibodies directed against the same may be used, inter alia, in vaccination protocols, either alone or in combination. Therefore, the pharmaceutical compositions of the present invention which comprise the CPS of the invention or antibodies directed against these CPS, may be used for pharmaceutical purposes such as effective therapy of infected humans or animals and/or, preferably for vaccination purposes. Accordingly, the present invention relates to pharmaceutical compositions comprising the CPS as described herein and/or antibodies or antibody fragments against the CPS as described herein and, optionally, a pharmaceutically acceptable carrier. In context with the present invention, the pharmaceutical compositions described herein may be used, inter alia, for the treatment and/or prevention of Neisseria-induced diseases and/or infections. Preferably, the pharmaceutical composition of the invention is used as a vaccine as will be further described herein below.
As mentioned, the present invention also relates to pharmaceutical compositions comprising the capsular polysaccharides described herein. Said capsular polysaccharides may be isolated but it is also envisaged that these capsular polysaccharides are to be used in context with other structures, e.g., toxins, adjuvants and the like. Such toxins may, inter alia, function as carriers for the capsular polysaccharides produced by the herein described methods. Numerous methods have been developed to link oligosaccharides covalently to carriers (Lit: (a) Vince Pozsgay, Oligosaccharide-protein conjugates as vaccine candidates against bacteria, Advances in Carbohydrate Chemistry and Biochemistry, Academic Press, 2000, Volume 56, Pages 153-199, (b) Jennings, H. J., R. K. Sood (1994) Synthetic glycoconjugates as human vaccines; in Lee, Y. C. R. T. Lee (eds): Neoglycoconjugates. Preparation and Applications. San Diego, Academic Press, pp 325-371, (c) Pozsgay, V.; Kubler-Kielb, J., Conjugation Methods toward Synthetic Vaccines, Carbohydrate-Based Vaccines, American Chemical Society, Jul. 2, 2008, 36-70); (D) Carl E. Frasch, Preparation of bacterial polysaccharide-protein conjugates: Analytical and manufacturing challenges, Vaccine, In Press, Corrected Proof, Available online 24 Jun. 2009, ISSN 0264-410X, DOI: 10.1016/j.vaccine.2009.06.013.) One example is the covalent coupling of the synthetic CPS molecules provided herein to protein amino-groups by means of reductive amination. As indicated above, for producing a vaccine, the herein produced CPS may be coupled to tetanus toxoid or other carrier proteins to generate a conjugate vaccine.
Thus, the capsular polysaccharide produced by the herein described methods may preferably used as a vaccine, i.e. the compounds provided herein can be employed for the vaccination of a subject. Such a subject may be a mammal and, in a particular embodiment, a human being. The vaccines provided herein are particularly useful in the vaccination against Neisseria.
The medicament/pharmaceutical composition (e.g. the vaccine) of the present invention comprises the CPS produced by the in vitro methods of the invention and may further comprise a pharmaceutically acceptable carrier, excipient and/or diluent. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The pharmaceutical composition of the present invention, particularly when used for vaccination purposes, may be employed at about 0.01 μg to 1 g CPS per dose, or about 0.5 μg to 500 μg CPS per dose, or about 1 μg to 300 μg CPS per dose. However, doses below or above this exemplary ranges are envisioned, especially considering the aforementioned factors. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. However, in particular in the pharmaceutical intervention of the present invention, Neisseria infections can demand an administration to the side of infection, like the brain. Progress can be monitored by periodic assessment. The compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously. The compositions of the invention may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise further agents such as interleukins and/or interferons depending on the intended use of the pharmaceutical composition. The pharmaceutical composition (e.g. the vaccine) of the invention may also be administered as co-therapy together with at least one other active agent. This at least other active agent may be a medicament which is conventionally used as adjuvant or for preventing and/or treating Nm infections. The other active agent may be, e.g., a vaccine, an antibiotic, an anti-inflammatory agent, an interleukin or an interferon.
In a preferred embodiment of the present invention, the pharmaceutical composition as defined herein is a vaccine.
Vaccines may be prepared, inter alia, from one or more CPS as described herein, or from one or more antibodies as described herein, i.e. antibodies against the CPS as disclosed herein. Accordingly, in context with the present invention, vaccines may comprise one or more CPS as described herein and/or one or more antibodies, fragments of said antibodies or derivatives of the antibodies of the invention, i.e. antibodies against the CPS as disclosed herein.
The CPS or the antibodies, fragments or derivatives of said antibodies of the invention which are used in a pharmaceutical composition may be formulated, e.g., as neutral or salt forms. Pharmaceutically acceptable salts, such as acid addition salts, and others, are known in the art. Vaccines can be, inter alia, used for the treatment and/or the prevention of an infection with pathogens, e.g. Neisseria, and are administered in dosages compatible with the method of formulation, and in such amounts that will be pharmacologically effective for prophylactic or therapeutic treatments.
A vaccination protocol can comprise active or passive immunization, whereby active immunization entails the administration of an antigen or antigens (like the capsule polysaccharides of the present invention or antibodies directed against these CPS) to the subject/patient in an attempt to elicit a protective immune response. Passive immunization entails the transfer of preformed immunoglobulins or derivatives or fragments thereof (e.g., the antibodies, the derivatives or fragments thereof of the present invention, i.e. specific antibodies directed against the CPS obtained by the means and methods provided herein) to a subject/patient. Principles and practice of vaccination and vaccines are known to the skilled artisan, see, for example, in Paul, “Fundamental Immunology” Raven Press, New York (1989) or Morein, “Concepts in Vaccine Development”, ed: S. H. E. Kaufmann, Walter de Gruyter, Berlin, N.Y. (1996), 243-264; Dimitriu S, editor. “Polysaccharides in medicinal application”; New York: Marcel Dekker, pp 575-602. Typically, vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in or suspension in liquid prior to injection may also be prepared. The preparation may be emulsified or the protein may be encapsulated in liposomes. The active immunogenic ingredients are often mixed with pharmacologically acceptable excipients which are compatible with the active ingredient. Suitable excipients include but are not limited to water, saline, dextrose, glycerol, ethanol and the like; combinations of these excipients in various amounts also may be used. The vaccine may also contain small amounts of auxiliary substances such as wetting or emulsifying reagents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. For example, such adjuvants can include aluminum compositions, like aluminumhydroxide, aluminumphosphate or aluminumphosphohydroxide (as used in “Gen H-B-Vax®” or “DPT-Impfstoff Behring”), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′2′-dipalmitoyl-sn-glycero-3-hydroxyphaosphoryloxy)-ethylamine (CGP 19835A, also referred to as MTP-PE), MF59 and RIBI (MPL+TDM+CWS) in a 2% squalene/Tween-80® emulsion.
The vaccines are usually administered by intravenous or intramuscular injection. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include but are not limited to polyalkylene glycols or triglycerides. Oral formulation include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions may take the form of solutions, suspensions, tables, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredient, preferably about 25% to about 70%.
Vaccines are administered in a way compatible with the dosage formulation, and in such amounts as will be prophylactically and/or therapeutically effective. The quantity to be administered generally is in the range of about 0.01 μg to 1 g antigen per dose, or about 0.5 μg to 500 μg antigen per dose, or about 1 μg to 300 μg antigen per dose (in the present case CPS being the antigen), and depends upon the subject to be dosed, the capacity of the subject's immune system to synthesize antibodies, and the degree of protection sought. Precise amounts of active ingredient required to be administered also may depend upon the judgment of the practitioner and may be unique to each subject. The vaccine may be given in a single or multiple dose schedule. A multiple dose is one in which a primary course of vaccination may be with one to ten separate doses, followed by other doses given at subsequent time intervals required to maintain and/or to reinforce the immune response, for example, at one to four months for a second dose, and if required by the individual, a subsequent dose(s) after several months. The dosage regimen also will be determined, at least in part, by the need of the individual, and be dependent upon the practitioner's judgment. It is contemplated that the vaccine containing the immunogenic compounds of the invention may be administered in conjunction with other immunoregulatory agents, for example, with immunoglobulins, with cytokines or with molecules which optimize antigen processing, like listeriolysin.
For diagnosis and quantification of pathogens like Neisseria, pathogenic fragments, their derivatives, their (poly)peptides (proteins), their polynucleotides, etc. in clinical and/or scientific specimens, a variety of immunological methods, as well as molecular biological methods, like nucleic acid hybridization assays, PCR assays or DNA Enzyme Immuno Assays (DEIA; Mantero et al., Clinical Chemistry 37 (1991), 422-429) have been developed and are well known in the art. In this context, it should be noted that the nucleic acid molecules of the invention may also comprise PNAs, modified DNA analogs containing amide backbone linkages. Such PNAs are useful, inter alia, as probes for DNA/RNA hybridization. The proteins of the invention may be, inter alia, useful for the detection of anti-pathogenic (like, e.g., anti-bacterial or anti-viral) antibodies in biological test samples of infected individuals. It is also contemplated that antibodies of the invention and compositions comprising such antibodies of the invention may be useful in discriminating acute from non-acute infections. The CPS as provided herein can also be used in diagnostic settings, for example as “standards”, in, e.g., chromatographic approaches. Therefore, the present CPS can be used in comparative analysis and can be used either alone or in combination to diagnostic methods known in the art.
The diagnostic compositions of the invention optionally comprise suitable means for detection. The CPS as disclosed and described herein as well as specific antibodies or fragments or derivatives thereof directed or raised specifically against these CPS are, for example, suitable for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. Solid phase carriers are known to those in the art and may comprise polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, animal red blood cells, or red blood cell ghosts, duracytes and the walls of wells of a reaction tray, plastic tubes or other test tubes. Suitable methods of immobilizing nucleic acids, (poly)peptides, proteins, antibodies, microorganisms etc. on solid phases include but are not limited to ionic, hydrophobic, covalent interactions and the like. Examples of immunoassays which can utilize said proteins, antigenic fragments, fusion proteins, antibodies or fragments or derivatives of said antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Commonly used detection assays can comprise radioisotopic or non-radioisotopic methods. Examples of such immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric assay) and the Western blot assay. Furthermore, these detection methods comprise, inter alia, IRMA (Immune Radioimmunometric Assay), EIA (Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno Assay), FIA (Fluorescent Immuno Assay), and CLIA (Chemioluminescent Immune Assay). Other detection methods that are used in the art are those that do not utilize tracer molecules. One prototype of these methods is the agglutination assay, based on the property of a given molecule to bridge at least two particles.
The CPS of the invention can be bound to many different carriers. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes of the invention.
A variety of techniques are available for labeling biomolecules. Theses techniques are well known to the person skilled in the art and comprise, inter alia, covalent coupling of enzymes or biotinyl groups, iodinations, phosphorylations, biotinylations, random priming, nick-translations, tailing (using terminal transferases) or labeling of carbohydrates. Such techniques are, e.g., described in Tijssen, “Practice and theory of enzyme immuno assays”, Burden, R H and von Knippenburg (Eds), Volume 15 (1985), “Basic methods in molecular biology”; Davis L G, Dibmer M D; Battey Elsevier (1990), Mayer et al., (Eds) “Immunochemical methods in cell and molecular biology” Academic Press, London (1987), or in the series “Methods in Enzymology”, Academic Press, Inc., or in Fotini N. Lamari, Reinhard Kuhn, Nikos K. Karamanos, “Derivatization of carbohydrates for chromatographic, electrophoretic and mass spectrometric structure analysis”, Journal of Chromatography B, Volume 793, Issue 1, Derivatization of Large Biomolecules, (2003), Pages 15-36.
Detection methods comprise, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, etc.
The CPS described herein may be detected by methods known in the art as well as described and exemplified herein. For example, an ELISA (Enzyme-linked immunosorbent assay) based method described herein may be used for the detection and quantification of the chimeric CPS described herein. In this context, the chimeric CPS described herein may be immobilized by an antibody or other binding molecule, such as a lectine or similar, contacting one part or building block of the chimeric CPS. Detection of a second part or building block of the chimeric CPS described herein can be achieved by, e.g., contacting with an antibody or other binding molecule as described herein which is labeled for further detection or a secondary antibody or other binding molecule as described which is labeled for further detection.
As indicated above, the present invention further relates to antibodies specifically binding to the synthetic CPS obtainable by the in vitro methods described herein. The term “antibody” herein is used in the broadest sense and specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. Also human, humanized, camelized or CDR-grafted antibodies are comprised.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be constructed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler, G. et al., Nature 256 (1975) 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). “Antibody fragments” comprise a portion of an intact antibody. In context of this invention, antibodies specifically recognize CPS obtainable by the in vitro method described herein. Antibodies or fragments thereof as described herein may also be used in pharmaceutical and medical settings such as vaccination/immunization, particularly passive vaccination/immunization.
The term “antibody” includes antigen-binding portions, i.e., “antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind an antigen (such as CPS produced by the herein described methods), comprising or alternatively consisting of, for example, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward; 1989; Nature 341; 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Antibody fragments or derivatives further comprise F(ab′)2, Fv or scFv fragments or single chain antibodies.
The antibodies of the present invention may be used for vaccinating against, treating and/or diagnosing meningococcal meningitidis caused by Neisseria meningitidis serogroup A or X.
The term “carbohydrate” as used herein comprises building blocks such as saccharides and sugars in any form as well as aldehydes and ketones with several hydroxyl groups added. A carbohydrate may contain one or more of said building blocks linked via covalent bonds such as glycosidic linkages. A carbohydrate may be of any length, i.e. it may be monomeric, dimeric, trimeric or multimeric. A carbohydrate may also contain one or more building blocks as side chains linked to the main chain via covalent bonds. A carbohydrate may also contain one or more activated saccharides such as nucleotide sugars. Examples of nucleotide sugars are UDP-GlcNAc, UDP-ManNAc, UDP-GlcUA, UDP-Xyl, GDP-Man and GDP-Fuc.
Herein, the term “nucleic acid molecule” or “polynucleotide” refers to DNA or RNA or hybrids thereof or any modification thereof that is known in the state of the art (see, e.g., U.S. Pat. Nos. 5,525,711, 4,711,955, 5,792,608 or EP 302175 for examples of modifications). The polynucleotide sequence may be single- or double-stranded, linear or circular, natural or synthetic. For instance, the polynucleotide sequence may be genomic DNA, cDNA, mRNA, antisense RNA, ribozymal or a DNA encoding such RNAs or chimeroplasts (Gamper, Nucleic Acids Research, 2000, 28, 4332-4339). Said polynucleotide sequence may be in the form of a plasmid or of viral DNA or RNA. For example, the present invention relates to a nucleic acid molecule having the nucleotide sequence of any one of SEQ ID NOs: 1-8, 16-23 and 32. The present invention also encompasses nucleic acid molecules comprising the nucleic acid molecule of any one of SEQ ID NO: 1-8, 16-23 and 32 wherein one, two, three or more nucleotides are added, deleted or substituted. Such a nucleic acid molecule may encode a polypeptide being active (i.e. functional). The term “activity”, “functionality” or “being functional” as used herein refers in particular to the ability of having substantially the same function as the non-modified polynucleotide.
The present invention further relates to nucleic acid molecules which are complementary to the nucleic acid molecules described above. Also encompassed are nucleic acid molecules which are able to hybridize to nucleic acid molecules described herein. A nucleic acid molecule of the present invention may also be a fragment of the nucleic acid molecules described herein. Particularly, such a fragment is a functional fragment, which means that the fragment has the same function as the non-modified polynucleotide.
The term “hybridization” or “hybridizes” as used herein in context of nucleic acid molecules/polynucleotieds/DNA sequences may relate to hybridizations under stringent or non-stringent conditions. If not further specified, the conditions are preferably stringent. Said hybridization conditions may be established according to conventional protocols described, for example, in Sambrook, Russell “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory, N.Y. (2001); Ausubel, “Current Protocols in Molecular Biology”, Green Publishing Associates and Wiley Interscience, N.Y. (1989), or Higgins and Hames (Eds.) “Nucleic acid hybridization, a practical approach” IRL Press Oxford, Washington D.C., (1985). The setting of conditions is well within the skill of the artisan and can be determined according to protocols described in the art. Thus, the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as 0.1×SSC, 0.1% SDS at 65° C. Non-stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may be set at 6×SSC, 1% SDS at 65° C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
In accordance to the invention described herein, low stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may, for example, be set at 6×SSC, 1% SDS at 65° C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions.
Hybridizing nucleic acid molecules also comprise fragments of the above described molecules. Furthermore, nucleic acid molecules which hybridize with any of the aforementioned nucleic acid molecules also include complementary fragments, derivatives and allelic variants of these molecules. Additionally, a hybridization complex refers to a complex between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed). The terms complementary or complementarity refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence “A-G-T” binds to the complementary sequence “T-C-A”. Complementarity between two single-stranded molecules may be “partial”, in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between single-stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands.
The term “hybridizing sequences” preferably refers to sequences which display a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% and most preferably at least 99% identity with a nucleic acid sequence as described above.
The polypeptides and nucleic acid molecule provided herein preferably show a homology, determined by sequence identity, of at least 80%, more preferably of at least 85%, even more preferably of at least 90%, even more preferably of at least 95%, even more preferably of at least 96%, even more preferably of at least 97%, even more preferably of at least 98, and most preferably of at least 99% identity to a sequence as shown in any one of SEQ ID NOs: 1-3, 9, 10, 16, 17, 20-25 or 28-33 as defined herein above.
The polypeptides and nucleic acid molecules to be employed in accordance with this invention preferably show a homology, determined by sequence identity, to the sequences indicated under any one of SEQ ID NOs: 1-3, 9, 10, 16, 17, 20-25 or 28-33, preferably over the entire length of the sequences compared. The homologous sequences are preferably fragments having a length of at least 100, more preferably at least 200, more preferably at least 300, more preferably at least 400 and more preferably at least 500, more preferably at least 600, more preferably at least 700, more preferably at least 800 and most preferably at least 900 nucleotides which have an identity of at least 80%, more preferably of at least 85%, even more preferably of at least 90%, even more preferably of at least 95%, even more preferably of at least 96%, even more preferably of at least 97%, even more preferably of at least 98, and most preferably of at least 99% with the sequence shown under any one of SEQ ID NOs: 1-3, 9, 10, 16, 17, 20-25 or 28-33, respectively.
If two sequences which are to be compared with each other differ in length, sequence identity preferably relates to the percentage of the (amino acid or nucleotide) residues of the shorter sequence which are identical with the (amino acid or nucleotide) residues of the longer sequence. Sequence identity can be determined conventionally with the use of computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, Wis. 53711), CLUSTALW computer program (Thompson; 1994; Nucl Acids Res; 2; 4673-4680) or FASTDB (Brutlag; 1990; Comp App Biosci; 6; 237-245). Bestfit utilizes the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489, in order to find the segment having the highest sequence identity between two sequences. When using Bestfit or another sequence alignment program to determine whether a particular sequence has for instance 95% identity with a reference sequence of the present invention, the parameters are preferably so adjusted that the percentage of identity is calculated over the entire length of the reference sequence and that homology gaps of up to 5% of the total number of the nucleotides or amino acids in the reference sequence are permitted. When using Bestfit, the so-called optional parameters are preferably left at their preset (“default”) values. The deviations appearing in the comparison between a given sequence and the above-described sequences of the invention may be caused for instance by addition, deletion, substitution, insertion or recombination. Such a sequence comparison can preferably also be carried out with the program “fasta20u66” (version 2.0u66, September 1998 by William R. Pearson and the University of Virginia; see also W. R. Pearson, Methods in Enzymology 183 (1990), 63-98, appended examples and http://workbench.sdsc.edu/). For this purpose, the “default” parameter settings may be used.
The in vitro methods described herein may also be used for automated high-throughput approaches using robotic liquid dispensing workstations. Generally in such high-throughput approaches a plurality of assay mixtures are run in parallel. These mixtures may have the same or different concentrations of the respective ingredients. Typically, one of these mixtures serves as a negative control, i.e. at zero concentration or below the limits of assay detection.
These and other embodiments are disclosed and obvious to a skilled person and embraced by the description and the Examples of the present invention. Additional literature regarding one of the above-mentioned methods, means and uses, which can be applied within the meaning of the present invention can be obtained from the prior art, for instance in public libraries, e.g. with the use of electronic means. For this purpose, public data bases, such as “Medline”, can be accessed via the internet, for instance under the address http://www.ncbi.nlm.nih.gov/PubMed/medline.html. Additional data bases and addresses are known to a skilled person and can be taken from the internet, for instance under the address http://www.lycos.com. An overview of sources and information regarding patents or patent applications in biotechnology is given in Berks, TIBTECH 121 (1994), 352-364.
A number of documents including patent applications, manufacturer's manuals and scientific publications are cited herein. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
The present invention is further described by reference to the following non-limiting figures and examples.
The Figures show:
By using CP-A (i.e. CsaB), the product distribution as well as the product length can be controlled via the donor:acceptor ratio. By using full length CP-X (i.e. CsxA), the product distribution as well as the product length cannot be controlled via the donor:acceptor ratio. Here two product pools are produced, one having small and the other having large polymers.
A: CPSA distribution obtained after CsaB (in particular ΔN69-CP-A) reactions in the presence of 2 mM UDP-GlcNAc and varying amounts of short CPSA oligos (DP1-DP10). It is mentioned that, in pilot experiments, a similar product length control by varying the donor-acceptor ratio was seen for the full length CP-A. The designation “expected DP” indicates the major species of the produced capsular polysaccharides (i.e. the main component) and also specifies the donor-acceptor ratio (i.e. the donor:acceptor quotient). Thus, the concentrations of the acceptor CPSX range from 40-4 μM. Accordingly, from left to right, the used ratios of donor carbohydrate to acceptor carbohydrate are 50:1, 75:1, 100:1, 200:1 and 500:1.
B: Purification of CsxA truncations.
C: Design of CsaB and CsaX truncations. Active truncations are indicated by and asterisk *.
D: CPSX distribution obtained after CsxA reactions in the presence of 5 mM UDP-GlcNAc and varying amounts of short CPSX oligos (DP1-DP7). The fact that the highest amount of acceptor sugar is not detectable in the gel indicates that all detected molecule are synthesised by CsxA. The marker consists of polymer of the size which is used in vaccine production.
E: CPSX distribution obtained after CsxA reactions in the presence of 5 mM UDP-GlcNAc and varying amounts of long CPSX oligos (DP10-DP70). The marker consists of polymer of the size which is used in vaccine production.
By using a truncated version of CP-X (i.e. dC99-CsxA or dN58dC99-CsxA), the product distribution as well as the product length can be controlled via the donor:acceptor ratio. The designation “expected DP” indicates the major species (i.e. the main component) of the produced capsular polysaccharides and also specifies the donor-acceptor ratio (i.e. the donor:acceptor quotient). For example, an “expected DP” of 20, 33, 40, 50, 64, 75, 80, 100, 160, 200, 400, or 500 means a ratio of donor carbohydrate to acceptor carbohydrate of 20:1, 33:1, 40:1, 50:1, 64:1, 75:1, 80:1, 100:1, 160:1, 200:1, 400:1, or 500:1.
A/B: CPSX distribution obtained after dN58-CsxA reactions in the presence of 5 mM UDP-GlcNAc and varying amounts of short CPSX oligos (DP1-DP7).
C/D: CPSX distribution obtained after dC99-CsxA reactions in the presence of 5 mM UDP-GlcNAc and varying amounts of short CPSX oligos (DP1-DP7).
E: CPSX distribution obtained after dN58dC99-CsxA reactions in the presence of 5 mM UDP-GlcNAc and varying amounts of short CPSX oligos (DP1-DP7).
F: CPSA distribution obtained after dN69-CsaB reactions in the presence of 2 mM UDP-GlcNAc and varying amounts of short CPSA oligos (DP1-DP10).
A: Purification of single CPSX oligos.
B-D: Finding the minimal acceptor of CsxA full-length and CsxA truncations.
By using constant donor-acceptor ratios and truncated versions of the CsxA (i.e. dC99-CsxA, dN58-CsxA or dC99dN58-CsxA) the avDP of the produced products can be controlled via the reaction time. 50 nM of capsule polymerase have been used. The marker indicates length from DP10 to DP60.
A: Analysis of the CPSX distribution obtained at different time-points during a CsxA reaction in the presence of 10 mM UDP-GlcNAc and DP7. The ratio of donor carbohydrate to acceptor carbohydrate is approximately 8000:1.
B: Analysis of the CPSX distribution obtained at different time-points during a dN58-CsxA reaction in the presence of 10 mM UDP-GlcNAc and DP7. The ratio of donor carbohydrate to acceptor carbohydrate is approximately 8000:1.
C: Analysis of the CPSX distribution obtained at different time-points during a dC99-CsxA reaction in the presence of 10 mM UDP-GlcNAc and DP7. The ratio of donor carbohydrate to acceptor carbohydrate is approximately 8000:1.
D: Analysis of the CPSX distribution obtained at different time-points during a dN58dC99-CsxA reaction in the presence of 10 mM UDP-GlcNAc and DP7. The ratio of donor carbohydrate to acceptor carbohydrate is approximately 8000:1.
E: Analysis of the CPSX distribution obtained at different time-points during a dC99-CsxA reaction in the presence of 10 mM UDP-GlcNAc and DP5. The ratio of donor carbohydrate to acceptor carbohydrate is approximately 200:1.
F: D: Analysis of the CPSX distribution obtained at different time-points during a dN58dC99-CsxA reaction in the presence of 10 mM UDP-GlcNAc and DP7. The ratio of donor carbohydrate to acceptor carbohydrate is approximately 200:1. A calibration showed that the product pool in the 20′ fraction has an avDP of 15 and the product pool in the 20′ fraction has an avDP of 20.
In case of CP-X (CsxA) the C-terminal truncation (dC99) is not sufficient to prevent aggregate formation at low salt concentrations, however, the protein migrates with an apparent molecular mass resembling the trimer if 500 mM salt are present. In contrast the N-terminal truncation (dN58) prevents larger aggregate formation. The mutant dN58 similar to dN58dC99 migrates with an apparent molecular mass that suggest dimer formation already at low salt concentrations (25 mM NaCl). The dimers formed seem to be stable also in the presence of 500 mM salt.
Starting from 4-MU-Sia, monovalent forms of SiaDW-135/SiaDY were used in iterative steps to generate labelled acceptors suited to separately assay hexosyl- and sialyltransferase activity in the chimeric capsule polymerases of NmW-135 and NmY. Exemplarily shown are the acceptors with Gal as hexose. For simplicity DP—degree of polymerization—is used to describe the different 4-MU-labelled saccharides.
In consecutive enzymatic reactions 4-MU-Sia (4-MU-DP1) was elongated to 4-MU-DP4. Before the start of a new reaction 0.01% of the sample was loaded onto a CarboPac® PA-100 column and products eluted with a curved NaNO2 gradient as shown. MU fluorescence was detected at 375 nm. Overlays of chromatograms obtained with the monovalent forms of SiaDW-135 (A) and SiaDY (B) are shown. Baseline separation of peaks allowed the determination of precise retention times. Of note, the chromatograms show that the conversion of 4-MU-DP1 to 4-MU-DP2 was incomplete under the conditions used, while all other that reactions proceeded to completion.
(A) SiaDW-135 was primed with 1 mM 4-MU-Sia-Gal-Sia (4-MU-DP3) in the presence of 2 mM UDP-Gal and 2 mM CMP-Sia. At indicated time points, product profiles were recorded by HPLC-FD. (B) Enlarged display of the HPLC-FD profile obtained at 40 min and labelling of individual DPs. (C) Normed and weighted peak areas were calculated for each time point and used to construct progress curves. Progress curves obtained in three independent experiments (R1-R3) are shown and document the high reproducibility of the reaction. Peaks marked red represent 4-MU-DP1 and 4-MU, which represent contaminants in the 4-MU-DP3 pool, whereby 4-MU is already present in the starting solution of the substrate 4-MU-Sia and represents the hydrolysis of the substrate. Both peaks remain unchanged over the entire reaction, confirming a step increase in acceptor quality from DP2 to DP3.
Reaction profiles of the monovalent point mutants NmW-135-(E307A)-His6 and NmW-135-(S972A)-His6 were recorded in the presence of both donor sugars using the appropriate or false fluorescent primer. Reactions were run over 30 min with samples taken after 15 sec and 30 min. A: NmW-135-(E307A)-His6 with 4-MU-DP2. The only reaction product formed is 4-MU-DP3. B: NmW-135-(E307A)-His6 primed with 4-MU-DP3. No product is formed. C: NmW-135-(S972A)-His6 primed with 4-MU-DP2. No product was formed. D: NmW-135-(S972A)-His6 primed with 4-MU-DP3. The only product formed is 4-MU-DP4.
A: Schematic illustration of the full length capsule polymerases NmW-135 and NmY. The GT-B folds predicted to comprise hexosyl- and sialyltransferase domain are highlighted in green and purple, respectively. The linker region connecting the GT-B folded domains is shown in black. Mutants made in the course of this project were named according the introduced deletion. All proteins were expressed with N-terminal His6-tag. B: Western blot analysis of expressed proteins as indicated. Lysates prepared from transformed BL21(DE3) cells were separated into soluble and insoluble fraction and after separation on 10% SDS-PAGE analysed by western blotting using an anti-penta-His antibody.
Reaction profiles of the truncates variants NmW-135-(CΔ639)-His6 and NmW-135-(NΔ398)-His6 were recorded in the presence of both donor sugars using the corresponding acceptor. Reactions were run over 30 min with samples taken after 15 sec and 30 min. A: NmW-135-(CΔ639)-His6 with 4-MU-DP3 catalyze the transfer of UDP-Gal by creating 4-MU-DP4. B: NmW-135-(NΔ398)-His6 primed with 4-MU-DP2 catalyze the transfer of CMP-Sia by producing 4-MU-DP3.
Reaction profiles of the truncates variants NmW-135-(NΔ562)-His6 and NmW-135-(NΔ609)-His6 were recorded in the presence of both donor sugars using the corresponding acceptor. Reactions were run over 30 min with samples taken after 15 sec and 30 min. A: NmW-135-(NΔ562)-His6 with 4-MU-DP2 catalyze the transfer of CMP-Sia by creating 4-MU-DP3. B: NmW-135-(NΔ609)-His6 primed with 4-MU-DP2 catalyze the transfer of CMP-Sia by producing 4-MU-DP3.
The enzymatic activity of mutant forms was determined in an HPLC-based assay using fractions of purified enzymes. The HexTF catalysis is shown under A which were incubated with 4-MU-DP3 and the SiaTF catalysis is indicated under B which were incubated with 4-MU-DP2. Values represent means of three independent experiments.
The sequence alignment was performed using the ClustalW2 alignment and secondary structure motif predictions The symbols below the sequence illustrate β-strands (black arrows) and α-helices (lines). Positions where truncations were introduced are indicated with arrows above the sequences. The selected sequence accession numbers from the GenBank are Y13970 (SiaDW-135) and Y13969 (SiaDY).
The mechanism of elongation and product distribution are determined by the nature and extent of interaction with the growing polysaccharide chain. Schematic HPLC profiles of the product distributions are shown at right (DP, degree of polymerization). (a) An extended acceptor binding site confers an increased affinity for longer chains which occupy more of the binding subsites, resulting in uneven chain elongation and a skewed product distribution. For highly processive elongation, the acceptor is retained through many transfer cycles and no intermediates between short and long chains will be observed (top, right). Strict nonprocessive elongation involves dissociation of the enzyme-acceptor complex after each sugar transfer, therefore, all intermediates can be observed in the product distribution (bottom, right). In practice, the precise mechanism is often unclear because the product distributions of less processive and nonprocessive enzymes are indistinguishable16. (b) In the absence of an extended acceptor binding site, the enzyme interacts only with the growing end of the polysaccharide, elongation is nonprocessive and distributive, resulting in uniform elongation of all acceptors. The product profile has a narrow, Poissonian distribution (right).
Clones were sorted into three categories based on their average product dispersity. For each category, the reaction time course (top left), HPLC product profiles at three reaction time points (bottom), and an extract from Supplementary Table 1 (top right) are displayed for four representative clones. (a) High dispersity clones (mean dispersity >1.2) exhibit an exacerbated kinetic lag phase, maximum reaction rates up to 10 times greater than the initial rate, and product profiles strongly skewed towards longer chains. (b) Medium dispersity clones (mean dispersity of 1.1-1.2) exhibit a mild lag phase and less skewed product distributions. (c) Low dispersity clones (mean dispersity <1.1) have no lag phase (initial rate is the maximum reaction rate) and uniformly elongate chains resulting in narrow product distributions. Results for all 51 neutral drift clones are given in Supplementary Data Set 2 and Supplementary Table 1.
Sequencing of 122 ‘neutral’ variants from the first round of screening revealed 163 mutations across the Δ25polySTNmB sequence. Here the mutations are plotted on an alignment of the four bacterial polySTs from Neisseria meningitidis serogroup B (NmB), Neisseria meningitidis serogroup C (NmC), Escherichia coli K1 (EcK1) and Escherichia coli K92 (EcK92). Similarities of homology are shown in different gray scales. Numbering above the alignment indicates residue number in the polySTNmB sequence. Of note is that the mutations are unevenly distributed across the sequence with targeted positions and short clusters showing high mutability.
The Examples illustrate the invention.
Materials and Methods
Freshly transformed E. coli BL21(DE3) were grown at 15° C. in PowerBroth medium for 18 h. At an optical density of OD600=1.0 protein expression was induced by addition of 0.1 mM IPTG and allowed to proceed for a period of approximately 20 h. Pellets from 125 mL expression culture were pelleted by centrifugation (6,000×g, 10 min, 4° C.). After a washing step with PBS, cells were re-suspended in 7.5 ml binding buffer (50 mM Tris pH 8.0, 300 mM NaCl) complemented with 40 μg/ml Bestatin (Sigma), 1 μg/ml Pepstatin (Applichem), 100 μM PMSF (Stratagene) and sonified (Branson Digital Sonifier, 50% amplitude, 8×30 s, interrupted by cooling on ice). After centrifugation at 27,000×g for 30 min, the soluble fractions were directly loaded onto a HisTrap column (GE Healthcare) to enrich the recombinant proteins by immobilized metal ion affinity chromatography (IMAC). Columns were washed with binding buffer (50 mM Tris pH 8.0, 300 mM NaCl) and proteins eluted in step gradients using 10%, 30%, 50% and 100% elution buffer (binding buffer containing 500 mM imidazol). Fractions containing recombinant protein were pooled and the buffer exchanged to storage buffer (50 mM Tris pH 8.0, 50 mM NaCl for CsaA/CsaB; 50 mM Hepes pH 7.05, 100 mM NaCl, 5 mM MgCl2 and 1 mM EDTA for CsaC) using the HiPrep 26/10 Desalting column (GE Healthcare). Isolated proteins were concentrated using Amicon Ultra centrifugal devices (Millipore 30 MWCO). After separation into aliquots, samples were snap frozen in liquid nitrogen and stored at −80° C.
For activity testing a radioactive assay system, which is an adaptation of a protocol described by Weisgerber (1990; J. Biol. Chem. 265, 1578-1587) was used. Briefly, assays were carried out with 5 μl of the soluble fractions of bacterial lysates expressing either recombinant CsaB and CsaA or CsxA or the respective purified and epitope-tagged proteins in a total volume of 25 μl assay buffer (50 mM Tris pH 8.0 or various pH for determination of the pH optimum). Divalent cations were added from stock solutions. The reaction was primed with 5 ng of the respective priming oligosaccharides (hydrolysates of the respective capsule polysaccharides) and started by the addition of 0.05 μmol UDP-GlcNAc (Calbiochem) containing 0.05 μCi UDP-[14C]-GlcNAc (American Radiolabeled Chemicals). Samples were incubated at 37° C. and 5 μl aliquots spotted onto Whatman 3MM CHR paper after 0, 5, 10 and 30 min. Following descending paper chromatography, the chromatographically immobile 14C-labeled CPSA was quantified by scintillation counting.
Activity Testing of CsaB (Together with CsaA) and CsxA by Use of a Multi-Enzyme Spectrophotometric Assay—
1.2 μM of CsaA and 1 μM CsaB were assayed in the presence of 0.25 mM UDP-GlcNAc (Calbiochem), 20 mM MgCl2 and 50 mM Tris pH 8.0 in a total volume of 100 μL. The consumption of UDP-GlcNAc was coupled to nicotinamide adenine dinucleotide (NADH) consumption using the following enzymes/substrates: 0.25 mM adenosine triphosphate (ATP, Roche), 1 mM phosphoenolpyruvate (PEP, ABCR), 0.3 mM (NADH, Roche), 9-15 units/ml pyruvate kinase, 13.5-21 units/ml lactic dehydrogenase (PK/LDH mix Sigma), 0.05 mg/ml nucleoside monophosphate kinase (Roche). Absorption was measured at 340 nm every 30 min using a Biotek EL 808 96-well plate reader.
Results
The Donor:Acceptor Ratio Influences the Product Distribution of CsaB- and CsxA-Reactions in a Different Manner
Previous studies have suggested that conjugate vaccines made with intermediate chain-length oligosaccharides, as opposed to high-molecular-weight polysaccharides, are more immunogenic and better at eliciting T cell-dependent antibody responses” (Bröker M, Fantoni S. Minerva Med. (2007) 98(5):575-89). Very effective glycoconjugate vaccines are made of polysaccharides of intermediate size like for example the quadrivalent glycoconjugate vaccine Menveo (Novartis).
In two recent studies we demonstrated that the capsule polymerases from NmA (CsaB) and NmX (CsxA) were able to synthesise CPS chains in vitro which makes them a very attractive target for in vitro vaccine development. However, the synthesised chains were very long and thus not ready for immediate coupling to the toxin.
With the aim of producing oligosaccharide chains that can be readily coupled to a carrier protein, we wanted to investigate in this study if the product distribution produced by these enzymes can be manipulated.
In a first experiment, we incubated catalytic amounts of the enzymes (50 nM) in the presence of different ratios of UDP-GlcNAc, the donor sugar, and a mixture of small oligosaccharides ranging in size from DP1-DP10, the acceptor sugars. The reaction was allowed to proceed over night and the resulting products were analysed using high percentage PAGE stained with a combination of Alcian blue/silver. The expected DPs, indicated above the gels (
As a molecular weight marker, we used hydrolysed CPSA and CPSX samples that were used in the manufacturing process of Menveo. This way, we could judge immediately if the produced product pool had the desired size.
Indeed, CsaB was able to produce the desired product pool (see
In contrast, we observed a completely different elongation behaviour from CsxA (
Determination of the Minimal Active Domain of CsaB and CsxA
Both CsxA and CsaB belong to the family of stealth proteins that are involved in protecting pathogens from to the host immune defense system (Sperisen, PLoS Comput Biol. (2005) 1(6):e63). All members of stealth harbour, on amino acid level, four conserved regions separated and/or extended by non-conserved regions of varying length.
Determination of the Minimal Active Domain of CsaB and CsxA
Both CsxA and CsaB belong to the family of stealth proteins (Sperisen et al., 2005). All members of stealth harbour four conserved regions on amino acid level separated and/or extended by non-conserved regions of varying length. Based on these sequence characteristics, a schematic alignment of CsaB and CsxA shows that the two enzymes differ mainly in their terminal extensions (
Analysis of the Oligomerisation State
We recently demonstrated that MBP-CsxA-His6 forms complexes >669 kDa in the presence of low amounts of NaCl (25 mM) and partly dissociates into tri- to tetrameric assemblies in high salt conditions (700 mM NaCl) (Fiebig et al., 2014a). The preparative SEC step included in the purification procedure of the truncated CsxA constructs indicated that the termini might influence the oligomerisation behavior of this enzyme. To further interrogate this finding, analytical size exclusion chromatography was performed. ΔC99-CsxA showed salt-dependent oligomerisation behavior similar to full-length-CsxA, suggesting that the C-terminus is not involved in the formation of the correct oligomeric state (
In contrast to CsxA, no data is available about the oligomerisation state of CsaB. Since full-length-CsaB is almost exclusively expressed as ΔN69-truncation (Fiebig et al., 2014b), we used ΔN69-CsaB for SEC analysis. Like CsxA, this construct also forms high-molecular weight complexes >669 kDa in low-salt buffer (
The Termini of CsxA Influence its Elongation Mechanism
Asking if the truncations ΔN58-CsxA, ΔC99-CsxA and ΔN58ΔC99-CsxA showed a difference in their d/a-dependent product profiles, we repeated the experiment shown in
Full-length-CsaB is mostly expressed as ΔN69 truncation with only residual amounts of the original construct present in the purified sample (Fiebig et al., 2014b). Consequently, so far, only the product profile of ΔN69-CsaB has been analysed (
Determination of the Minimal Acceptor Used by CsaB- and CsxA-Constructs
Protocols in glycoconjugate vaccine manufacturing often require free reducing ends for the conjugation of saccharides to the carrier protein (Costantino et al., 2011). Thus, we wanted to determine the minimal length of CPS acceptors with a free reducing end needed for chain elongation for each of the active constructs. We were especially interested if the d/a-dependent length control of ΔN69-CsaB, ΔC99-CsxA and ΔN58ΔC99-CsxA would be influenced by the DP of the acceptor. Acceptors of a single DP were purified from a mixture of hydrolysed, dephosphorylated CPSX and CPSA using anion exchange chromatography (
Full-length-CsxA and the single truncations ΔN58-CsxA and ΔC99-CsxA were able to elongate acceptors with a DP≥2 while the presence of the monomers GlcNAc-1P and GlcNAc did not lead to product signals beyond de novo background (
For ΔN69-CsaB, a first screening of short CPSA acceptors of a single DP showed elongation for chains ≥DP2 (
Analysis of the Time-Resolved Elongation Behavior of CsaB and CsxA
To provide further insight into the elongation mechanism used by the different constructs, we analysed the time-course of the polymerase reactions. For this analysis the donor and acceptor concentrations were increased to allow detection by HPLC-AEC at 214 nm while maintaining a d/a sufficient for the production of long chains. Samples of each reaction were heat-inactivated after the indicated time-points and analyzed via PAGE followed by Alcian-blue/silver staining or HPLC-AEC, which was calibrated using the purified DPs (
During the first 60 minutes, full-length-CsxA mainly synthesized short chains (
The fact that the product profile of ΔC99-CsxA is perfectly controllable via the d/a suggests distributive elongation (
ΔN58ΔC99-CsxA appears to be fully distributive over time and again, produces much shorter chains and narrower distributions compared to ΔC99-CsxA (compare
Experimental Procedures
General Cloning—The generation of MBP-csxA-His6, csaBco-His6 and d69-csaBco-His6 has been reported elsewhere (Fiebig et al., 2014b; Fiebig et al., 2014a). All truncated constructs described herein were amplified by polymerase chain reaction (PCR) using the primers shown in Table 1 and genomic DNA of Nm strain Z2491, csaBco (Fiebig et al., 2014b) or pHC19 (for CsxA sequences)(Fiebig et al., 2014a) as template. After restriction digest using BamHI/XhoI csaB truncations were cloned into pET22b-Strep (Schwarzer et al., 2009) and csxA truncations were cloned into MBP-csxA-His6 (T7) or MBP-csxA-His6 (tac) (Fiebig et al., 2014a).
Expression and Purification of Recombinant CsaB- and CsxA-Constructs—Expression and purification of all CsaB- and CsxA-based sequences was performed as described in (Fiebig et al., 2014b) and (Fiebig et al., 2014a), respectively. For activity testing from lysates and subsequent analysis using the radioactive incorporation assay, csxA and its truncations were expressed from MBP-csxA-His6 (T7)-based constructs using BL21(DE3) as expression host (Fiebig et al., 2014a). Purification was performed using MBP-csxA-His6 (tac)-based constructs and M15(pREP4) as expression host.
Activity testing from lysates was performed using the radioactive incorporation assay as described before (Fiebig et al., 2014b; Fiebig et al., 2014a). The spectrophotometric assay published for CsaB (Fiebig et al., 2014b) was adapted for CsxA resulting in the alterations as follows; (i) no CsaA (epimerase) was used in the reaction mixture, (ii) the reaction was performed using 54 nM of the respective CsxA construct in the presence of (iii) 37 ng CPSX oligosaccharides of avDP18.6.
SDS-PAGE and Immunoblotting—SDS-PAGE and immunoblotting was performed as described before (Fiebig et al., 2014b; Fiebig et al., 2014a).
Purification of CPSA and CPSX Oligosaccharides—CPSA and CPSX oligosaccharide mixtures (Fiebig et al., 2014b; Fiebig et al., 2014a) were dephosphorylated using either acid phosphatase (Sigma) or alkaline phosphatase (CIP, NEB) according to the manufacturer's guidelines. Anion exchange chromatography (AEC) was performed on an ÄKTAFPLC (GE Healthcare) equipped with a MonoQ HR 5/5 column (Pharmacia Biotech) at a flow-rate of 1 ml/min. H2O and 1 M NaCl were used as mobile phases M1 and M2, respectively. Samples were separated using a combination of linear gradients (0% to 5% over 1 ml, 5% to 20% over 10 ml, 20% to 30% over 20 ml). The amount of oligosaccharide in each fraction was estimated from the peak area obtained at 214 nm under the assumption that each residue contributes equally to the absorbance of the respective oligomer. Fractions were dialysed against water (ZelluTrans, Roth, 1 kDa MWCO), freeze-dried and equal concentrations were adjusted with H2O. The identity and DP of the oligosaccharide fractions was confirmed by HPLC-PAD and 1H NMR using established protocols (Berti et al., 2012; Ravenscroft et al., 1999; Costantino et al., 1992).
Analysis of CsaB and CsxA Reaction Products—For the d/a dependent analysis, 100 nM CsaB and 50 nM CsxA constructs were incubated in 25-100 uL reaction buffer (50 mM Tris pH 8.0, 20 mM MgCl2, (2 mM DTT, only for CsxA)) in the presence of 2 mM (CsaB) or 5 mM (CsxA) UDP-GlcNAc and acceptors in a concentration resulting in the d/a ratios indicated in
For the time-dependent analysis, the reaction volume was upscaled to 700 uL. The UDP-GlcNAc concentration was increased to 10 mM and the CsaB concentration was decreased to 50 nM. Aliquots were snap-frozen after the indicated time-points and heat-inactivated for 3-5 min at 98° C. HPLC-AEC and high percentage PAGE followed by Alcian blue/silver staining was performed as described before (Fiebig et al., 2014b).
Analytical Size-Exclusion Chromatography—Roughly 300 ug of recombinant protein was analysed at 280 nm on an ÄKTAFPLC (GE Healthcare) equipped with a Superdex 10/300 GL column (GE Healthcare) using 50 mM Tris pH 8.0 elution buffer supplemented with NaCl as indicated in
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Abstract
Crucial virulence determinants of disease causing Neisseria meningitidis (Nm) species are their extracellular polysaccharide capsules (CPSs). In the serogroups W-135 and Y these are heteropolymers of the repeating units [→6)-α-D-Gal-(1→4)-α-Neu5Ac-(2→]n in NmW-135 and [→6)-α-D-Glc-(1→4)-α-Neu5Ac-(2→]n in NmY. We recently showed that the capsule polymerases, SiaDW-135 and SiaDY, which synthesise these highly unusual polymers, are composed of two GT-B folded glycosyltransferase domains (an N-terminal hexosyl- and a C-terminal sialyltransferase domain) and a linker region (amino acids 399-762) of unknown function. Here we use a mutational approach and synthetic fluorescent substrates to define the boundaries of the hexosyl- and sialyltransferase domains. Our results reveal that the active sialyltransferase domain encompasses a large portion of the linker region and indeed may define a new family in the CAZy classification.
Experimental Procedures
Generation of truncation mutants and purification of recombinant proteins. To separate hexosyl- and sialyltransferase the clones pHC4 (SiaDW-135) and pHC5 (SiaDY) (Claus (1997) Mol. Gen. Genet. 257, 28-34) were used as templates and truncation mutants were generated by PCR as shown in
E. coli strains
Synthesis and purification of fluorescently labelled acceptors to prime SiaDW-135 and SiaDY Reactions. Because activity testing in the classical radioactive incorporation assay depends on polymer formation (short oligosaccharide primers are washed out in the paper chromatography step (Weisgerber and Troy (1990) J. Biol. Chem. 265, 1578-1587), the reliable testing of monovalent mutants needed a test system in which single sugar transfers can be identified. We exploited the capability of SiaDW-135 and SiaDY to extend sialic acid derivatives carrying the fluorescent label 2-(4-methylumbelliferyl) (4-MU) at the reducing end. The recombinant C-terminally His6-tagged monovalent full length enzymes NmW-135-(S972A)-His6 and NmY-(S972A)-His6 were used as hexosyltransferases and NmW-135-(E307A)-His6 and NmY-(E307A)-His6 as sialyltransferases. 4-MU-Sia-Gal/Glc were obtained by mixing 4 mM 4-MU-Sia*Na (Sigma or Iris Biotech GmbH) with 4 mM donor sugar UDP-Gal/UDP-Glc (Sigma) and 20 μg ml−1 of the respective hexosyltransferase (NmW-135-(S972A)-His6 or NmY-(S972A)-His6) in reaction buffer (20 mM Tris/HCl pH 8.0, 20 mM MgCl2, and 2 mM DTT). After 24 h at 25° C. enzymes were removed by ultra-filtration (Amicon®Ultra 10 Molecular Weight Cut-Off (MWCO) Millipore) and filtrates containing the reaction products (4-MU-Sia-Gal or 4-MU-Sia-Glc) were lyophilised (Christ; Alpha 1-2 LD plus). After dissolution in water, samples were desalted on P2-gel filtration columns (Bio-Rad). Reaction products were identified by high-performance liquid chromatography using an UFLC-RX system (Shimadzu) coupled to a fluorescence detector (RF-10A XL). Samples were excited at 315 nm and monitored at 375 nm. Although, under the conditions used, this reaction did not give product yields >90% (see
Enzyme testing. Radioactive incorporation assays were performed as described (Romanow, J Biol Chem. (2013) 26; 288(17):11718-30). For activity testing with fluorescent compounds, reactions were carried out in a total volume of 25 μl. Mixtures contained 50 mM Tris/HCl pH 8.0, 20 mM MgCl2, 2 mM DTT, 1 mM acceptor (4-MU-Sia-Gal/Glc, 4-MU-Sia-Gal/Glc-Sia, or 4-MU-Sia-Gal/Glc-Sia-Gal/Glc) plus 2 mM of the nucleotide sugar (UDP-Gal/Glc; Sigma and/or 2 mM CMP-Neu5Ac; Nacalai tesque) depending on the tested enzyme. Reactions were started by addition of 20 μg ml−1 purified enzyme or, if the soluble fraction of bacterial lysates was used as enzyme source, with 72-100 μg ml−1 total protein. After appropriate incubation times, reactions were stopped by shock freezing in liquid nitrogen.
Synthesised polymers were analysed and quantified via the fluorescent tag. Separation of 4-MU-labelled oligo- and polysaccharides was achieved by anion exchange chromatography (CarboPac® PA-100 column, Dionex) using an ultrafast HPLC system (UFLC-RX, Shimadzu) with coupled fluorescence detection (FD; detector RF-10A XL). Before loading onto the CarboPac® PA-100 column, samples were 500-fold diluted in water. For the elution the buffer components A (20 mM NaNO3) and B (1 M NaNO3) were used to establish a curved gradient, reaching 21.65% buffer B over 35 min. The flow rate was set to 0.6 ml min−1 and the column temperature to 50° C. The curved portion of the gradient is described by the following formula in with the index −1.425 describes the slope (LS Solution; Shimadzu; Keys, Glycobiology (2013); 23(5):613-8).
Elution profiles were monitored via fluorescence emission at 375 nm with 315 nm as extinction wave length. Under these conditions the separation of polysaccharides up to a degree of polymerisation (DP)>18 was easily achieved (see results).
SDS-PAGE and immunoblotting. SDS-PAGE was performed under reducing conditions using 2.5% (v/v) β-mercaptoethanol. Western blot analysis was done on a PVDF membrane (Millipore). For detection of the hexahistidine tag, penta-His antibody (Qiagen) was used as first antibody at a concentration of 1 μg ml−1 and detected with 0.05 μg ml−1 IgG1 anti mouse IR Dye800 antibody (Odyssey Infrared Imaging®). Protein bands were visualised and quantified with the infrared fluorescence detection system (LI-COR®) according to the manufacturer's instructions.
Results
Synthesis of Fluorescently Labelled Oligosaccharides. With the intention to physically separate hexosyl (Hex)- and sialyl (Sia)-transferase and functionally express the individual enzyme domains, it was prerequisite to have specific acceptors available that would allow the unequivocal detection of single sugar transfers. As detailed in Experimental Procedures the monovalent mutants NmW-135-(S972A)-His6 and NmY-(S972A)-His6, bearing only HexTF activity were used to generate 4-MU-Sia-Hex (4-MU-DP2), which then was substrate for the monovalent enzymes bearing only SiaTF activity (NmW-135-(E307A)-His6 and NmY-(E307A)-His6). 4-MU-Sia-Hex-Sia (4-MU-DP3) resulting from this reaction, was an efficient acceptor for the HexTFs. The structures of the generated acceptors (exemplarily shown for oligosaccharides with Gal as hexose) are given in
Based on previous experiences with the separation and purification of Sia-containing oligo- and polymers (Glycobiology (2013) 23(5):613-8) a CarboPac® PA-100 column and a curved NaNO3 gradient were used to determine the elution positions of 4-MU-Sia and of the elongated products 4-MU-DP2-4-MU-DP4. Shown in
It is important at this point to mention that trials to start the reaction with 4-MU-Gal and 4-MU-Glc as potential substrates for the SiaTF domains were unsuccessful with both, the wild-type enzymes and the monovalent point mutants NmW-135-(E307A)-His6 and NmY-(E307A)-His6. This finding strongly suggested that the SiaTF domain needs at least a disaccharide to be active or, alternatively, recognition of the acceptor may need the presence of at least one sialic acid residue.
The new assay system was then used to record the wild-type SiaDW-135/Y reactions over time. To visualize the initial reaction steps, the enzymes were primed with 1 mM 4-MU-DP3 in the presence of limited substrate concentrations (CMP-Sia and UDP-Gal/Glc, 2 mM each) and reactions run up to 40 min. Samples were taken between 0.25 min and 40 min as indicated (
To quantify product formation over time, the relative abundance of each synthesized DP at a given time point was calculated and with the help of the LC Solution software (Shimadzu) normed and weighted to the total curve area giving the normed and weighted values according to the formula:
with A being the area underneath a single peak and n the number of transfers needed to form this product. Values were plotted against the reaction time. Data points as shown in (
The illustrated table represents the retention time of appropriate produced capsule polysaccharides of NmW-135 and NmY during the elution with 1 M NaNO3 using an adapted curve gradient on the CarboPac PA-100 column. The elution is based on the charge of oligosaccharides and therefore all polysaccharides with sialic acid on the reducing end are represented by odd numbers and such with hexose on the reducing end are represented by even numbers.
Of note, while carbohydrate polymerases normally show a kinetic lag phase (Breyer, Protein Science 10, 1699-1711 (2001)), the progress curves obtained with SiaDW-135/Y represent typical enzyme reaction kinetic with an initial maximum velocity, indicating that products of progressively longer DP are not better acceptors than the starter compound 4-MU-DP3.
As separation of the two active glycosyltranferase domains was the goal of this study, it was important to reconfirm monovalence (Romanow, J Biol Chem. (2013) 26; 288(17):11718-30) of the single point mutant capsule polymerases NmW-135-(S972A)-His6 and NmW-135-(E307A)-His6, used as positive controls in subsequent experiments. The reaction profiles monitored in the presence of both donor sugars are shown in
Compared to the sialyltransferase reaction, transfer of Gal onto 4-MU-DP3 was slower, with only about 10% of the primer converted to 4-MU-DP4 after 15 sec reaction time. However, in contrast to the sialyl-transferase reaction this hexosyltransferase reaction proceeded to completeness within 30 min (
Truncation Mutagenesis to Separate Hexosyl- and Sialyltransferase in SiaDW-135 and SiaDY. In
Next, we asked, if the linker (parts of the linker) may be part of the active sialyltransferase. To test this assumption, mutant NΔ398-His6, which comprises the entire linker sequence (see
To more precisely define the active sialyltransferase domain additional truncations as shown in
Together the mutational studies demonstrated that hexosyl- and sialyltransferase can be separated in SiaDW-135 and SiaDY and can be expressed as functionally active proteins. Different to the N-terminal GT-B folds, which encode classical hexosyltransferases belonging to CAZy family GT-4), the C-terminal GT-B folds comprise only parts of the functionally active sialyltransferases. The active sialyltransferases start with a predicted helical segment starting at F609 (see also
The sialyltransferase domains in SiaDW-135/Y define a new sialyltransferase family. As discussed previously (Romanow, J Biol Chem. (2013) 26; 288(17):11718-30) Blast search analyses carried out with the C-terminal GT-B folds of SiaDW-135 and SiaDY were unable to assign the sequences to one of the sialyltransferase families existing in the CAZy classification system (Coutinho (2003) J. Mol. Biol. 328, 307-317). Nevertheless, these searchers revealed weak homology to open reading frames in some bacterial proteins of unknown function. With the current knowledge that the functional sialyltransferase domains in SiaDW-135/Y encompass the C-terminal part of the linker region (at least F609-I778), the bioinformatics analyses was repeated and in parallel carried out with the programs BLAST and HMMER. Importantly, HMMER identified more than 30 sequences all comprising bacterial proteins of unknown function. After elimination of identical sequences further analyses by multi-sequence alignments clearly showed high conserved motifs (
The polysialyltransferase (polyST) from Neisseria meningitidis serogroup B (NmB) synthesises a homopolymer of α2,8-linked sialic acid residues known as polysialic acid. The chemical and immunological identity of the bacterial polymer with polysialic acid expressed in the human brain constitutes a highly effective molecular mimicry which contributes to virulence of NmB (Muhlenhoff, Curr. Opin. Struct. Biol. 8, 558-564 (1998); Roberts, Annu. Rev. Microbiol. 50, 285-315 (1996)). Recent studies have highlighted the potential of the NmB-polyST for polysialylation of therapeutic proteins (Lindhout, Proceedings of the National Academy of Sciences (2011), doi:10.1073/pnas.1019266108), and for direct therapeutic application of polysialic acid to tissues in vivo (Maarouf, J. Biol. Chem. 287, 32770-32779 (2012)). Despite the importance of this enzyme, little is known of its structure and function.
We have recently developed two new assay systems which enable high throughput screening and detailed characterisation of polyST activity (Keys, Analytical Biochemistry 427, 107-115 (2012); Keys, Analytical Biochemistry 427, 60-68 (2012)). In this study we have used these methods to conduct a neutral drift experiment—subjecting the NmB-polyST to high mutation rates followed by purifying selection to remove inactive variants—to explore functional regions of the polyST sequence space. Detailed analysis of over 50 drifted variants (with 7.3±3.0 amino acid exchanges per sequence) revealed that some sequences had acquired new modes of chain elongation which correspond to either a processive or distributive mechanism of polymerisation. The study identifies sequence elements which control the mechanism of elongation and the dispersity of polymeric products.
The synthesis of uniform glycan structures is a necessary precursor to their evaluation as therapeutic reagents. We have explored the functional sequence space of a bacterial polysialyltransferase and identified sequence elements controlling the processive or distributive mechanism of chain elongation. The results demonstrate that these properties are independent of enzymatic activity and illuminate a pathway for the synthesis of uniform oligo- and polysaccharide structures for research and therapeutic applications.
The extracellular polysaccharides of diverse bacterial species, which have emerged from symbiotic or pathogenic coevolution with their human hosts, are a rich source of structures with important biological functions and applications as therapeutic reagents (DeAngelis, J. Glycobiology. 23, 764-777 (2013); Pollard Nat. Rev. Immunol. 9, 213-220 (2009) and Boltje, Nat. Chem. 1, 611-622 (2009)) The application of oligo- and polysaccharides will be greatly advanced by strategies to fine tune the functional properties of the polymerizing glycosyltransferases which synthesize these polysaccharides (Boltje, Nat. Chem. 1, 611-622 (2009) and Schmaltz, Chem. Rev. 111, 4259-4307 (2011)). Here we demonstrate that these enzymes can readily be engineered to produce uniform Poissonian product distributions.
Our study focused on the polysialyltransferase from Neisseria meningitidis serogroup B (polySTNmB), which synthesizes α2,8-linked polysialic acid (polySia), a low abundance polymer with unique biological functions and importance as a reagent for research and several emerging therapeutic applications. In mammals, polySia is a dynamically regulated posttranslational modification predominantly found on the neural cell adhesion molecule, NCAM (Mühlenhoff, Neurochem. Res. 38:1134-1143 (2013)). Due to its polyanionic nature, large size, and water binding capacity, polySia acts as a powerful anti-adhesive, globally down regulating cellular interactions and increasing cellular motility (Johnson, J. Biol. Chem. 280, 137-145 (2005)). These unique anti-adhesive properties provide plasticity in the developing and adult nervous system (Rutishauser, Nat. Rev. Neurosci. 9, 26-35 (2008)) and play a role in immune development (Drake, Proc. Natl. Acad. Sci. U.S.A. 106, 11995-12000 (2009)). PolySia is re-expressed at the cell surface by various tumors to promote invasion and metastatic potential (Hildebrandt, Adv. Exp. Med. Biol. 663, 95-109 (2010)). Moreover, some neuroinvasive bacteria produce an extracellular capsule of polySia which aids in evasion of the immune system (Corbett, Adv. Appl. Microbiol. 65, 1-26 (2008)). PolySia is currently being investigated for diverse therapeutic applications, primarily based on the ability to promote plastic processes involved in regeneration in neural tissues (El Maarouf, Adv. Exp. Med. Biol. 663, 137-147 (2010) and El Maarouf, Proc. Natl. Acad. Sci. U.S.A. 103, 16989-16994 (2006)) and its ability to improve the pharmacological profile of therapeutic proteins decorated with polySia (Constantinou, Bioconjugate Chem. 19, 643-650 (2008) and Lindhout Proc. Natl. Acad. Sci. U.S.A. 108, 7397-7402 (2011)). A major step towards these goals is the demonstration that gene therapy and chemical coupling can be avoided in these applications by using the polySTNmB to synthesize polySia directly onto therapeutic proteins (Lindhout, Proc. Natl. Acad. Sci. U.S.A. 108, 7397-7402 (2011)), cultured cells, and neural tissues in vivo (Maarouf, J. Biol. Chem. 287, 32770-32779 (2012)).
A critical feature of enzymes for use in glycoengineering applications is the ability to synthesize uniform products of defined length. The primary feature determining product length and dispersity for polymerases is the extent of their interaction with the growing polysaccharide acceptor (
With very few exceptions (Jing, J. Biol. Chem. 279, 42345-42349 (2004)), the polymerizing glycosyltransferases which have been characterized to date exhibit an extended acceptor binding site and broad product distributions—properties which are not amenable to biotechnological applications. The polySTNmB was recently used for enzymatic polysialylation of therapeutic proteins, however, proteins were modified with a range of chain lengths from 4 to >70 residues at each site (Lindhout, Proc. Natl. Acad. Sci. U.S.A. 108, 7397-7402 (2011)). In this case, the assessment of optimal chain length is impossible, and the assessment of the desired modification is weakened due to averaging over many products. In the ideal case, enzymatic modification would be carried out by perfectly distributive enzymes, resulting in uniform chain elongation and a narrow distribution of product lengths (
Pioneering studies on ribonuclease A by delCardayré and Raines (delCardayre, Biochemistry 33, 6031-6037 (1994)) demonstrated that substrate binding could be modified independently of catalytic activity, enabling the introduction of processivity without altering activity. We proposed that this should hold true for polymerizing glycosyltransferases. As no structural data is available to identify residues interacting with the acceptor substrate, we conducted a small neutral drift to explore the polySTNmB sequence space. In a neutral drift, the target protein is subjected to high mutational loads under selection for the native level of catalytic activity, enabling a broad exploration of functional sequence space and facilitating the emergence of new properties (Gupta, Nat. Methods 5, 939-942 (2008)).
Our previous experiments have shown that truncation of the N-terminus of the polySTNmB increases soluble expression with little cost to enzymatic activity (Keys, Anal. Biochem. 427, 60-68 (2012)). The Δ25polySTNmB was therefore used as the starting point for the neutral drift. In total, three rounds of diversity generation and screening were conducted. An initial error prone PCR library was screened using an in vivo activity assay (Keys, Anal. Biochem. 427, 60-68 (2012)) and sequencing of 122 clones revealed 163 ‘neutral’ amino acid exchanges (
To examine the functional diversity present in the final pool, the reaction time course and pattern of chain elongation was determined for all 51 enzymes. This was achieved using a recently developed fluorescent acceptor, a 1,2-diamino-4,5-methylenedioxybenzene labeled trimer of sialic acid (DMBDP3), with HPLC separation of reaction products (Keys, Anal. Biochem. 427, 107-115 (2012)) adapted to 96-well format (see Methods). In order to be able to distinguish different modes of elongation and make inferences about substrate binding, it is important that reactions are carried out with a large substrate to enzyme ratio (Levengood, J. Am. Chem. Soc. 133, 12758-12766 (2011)), therefore an approximately 100 fold molar excess of the DMB-DP3 acceptor and a 4000 fold molar excess of the donor sugar, CMP-Neu5Ac, were used. The enzymes exhibited diverse reaction kinetics and product profiles suggesting that sequences had indeed drifted in the direction of both processive and distributive mechanisms of elongation. In order to quantify the tendency of each enzyme to produce uniform or disperse product profiles, the product dispersity (Xw/Xn; where Xw is massaverage DP, and Xn is number-average DP) (Stepto, Polym. Int. 59, 23-24 (2010)) was calculated, and the mean dispersity of products over the reaction course was determined for each of the enzymes. The enzymes exhibited a range of mean dispersity values from 1.06-1.33. As a guide, analysis of the Δ25polySTNmB in the same assay gave a mean dispersity of 1.16, and the dispersity of a uniform polymer sample with Poisson distribution of lengths approaches 1. The sequences were sorted into three categories according to high (>1.2), medium (1.1-1.2), and low (<1.1) dispersity.
The enzymes also exhibited different reaction kinetics which correlated broadly with the measured product dispersity. Enzymes in the high and medium dispersity categories displayed skewed product profiles and kinetic lag phases typical of polymerases with extended substrate binding sites (
Remarkably, the Lys69Gln mutation is exclusively associated with enzymes in the low dispersity category, which display a distributive mechanism of elongation and Poissonian product distributions. The presence of numerous additional mutations in these enzymes indicates that the Lys69Gln exchange plays a dominant role in determining the mechanism of chain elongation. The molecular mechanism by which a single amino acid exchange potently affects chain elongation is of high interest for the rational engineering of related polymerases. We speculate that the mutation either alters interaction with the growing end of the polysaccharide or blocks entry of the polysaccharide to the extended binding site.
A number of single amino acid exchanges were observed to produce an elongation phenotype. The mutations Arg111His, Lys114Met, Lys143Thr and Lys412Ile increase the strength of interaction with chains of DP>10, increasing the observed lag phase and the dispersity of reaction products compared to Δ25polySTNmB (
Previous neutral drift experiments demonstrated that target enzymes were able to maintain native levels of activity under high mutational loads due to the accumulation of “global suppressor mutations”, which stabilized the overall protein structure (Gupta, Nat. Methods 5, 939-942 (2008)). To test if this held true in our neutral drift, despite the considerably smaller screening effort, the most highly accumulated exchanges within the pool were cloned and examined. Impressively, the majority of exchanges which were highly accumulated during the neutral drift were observed to increase the yield of activity when present alone in the Δ25polySTNmB sequence (Table 3), and had little or no effect on product distribution. Notably, the Phe460Ile exchange was the most common mutation, present in 51% of the analyzed clones, and more than tripled the yield of activity when present alone in the Δ25polySTNmB sequence. The results are consistent with previous neutral drift experiments (Gupta, Nat. Methods 5, 939-942 (2008)), and suggest that the accumulated mutations may stabilize the polyST structure.
For the biological function of polymerizing glycosyltransferases it is advantageous to have extended interaction interfaces which bind and increase the rate of synthesis of their polysaccharide products. However, for biotechnological applications, the same interface represents a distinct disadvantage, constituting a barrier to the synthesis of uniform oligo- and polysaccharide structures. The results of this study demonstrate that these interaction interfaces can be readily engineered to yield enzymes with a distributive mechanism and product profiles favorable for research and therapeutic applications.
Discussion
Aiming to identify sequence elements contributing to the extended polysialic acid binding site in the polySTNmB, the in vivo and the in vitro assays were combined into a throughput for exploring the polyST sequence space and identifying relationships with the mode of chain elongation. The sequence space was explored using a neutral drift experiment, where the target sequence is subjected to repeated rounds of mutagenesis and selection for the native level of activity (Bershtein, J. Mol. Biol. 379, 1029-1044 (2008) and Gupta, Nature Methods 5, 939-942 (2008)). Starting from the Δ25polySTNmB, three rounds of mutagenesis and screening were used to generate a pool of 51 polymorphic sequences (7.3±3.0 amino acid exchanges per sequence) with a range of activity between approximately 25% and 300% of the original sequence. Each of these clones was then characterised in detail using the DMB-DP3 acceptor and HPLC assay. The mutated enzymes exhibited diverse modes of chain elongation. At one end of the spectrum were clones with product distributions suggestive of processive elongation. These clones were characterised by an exacerbated kinetic lag phase, a strong preference for elongated acceptor structures, and highly disperse reaction products. At the other end of the spectrum were clones with a highly distributive mechanism of chain elongation. These were characterised by the absence of a lag phase (i.e. initial reaction rate was the maximum reaction rate), no preference for elongated acceptors, and uniform product distributions.
The diverse elongation phenotypes were associated with specific amino acid exchanges. Remarkably, the Lys69Gln mutation was exclusively associated with the twelve clones exhibiting a distributive mechanism of elongation and the most uniform product distributions, indicating a dominant role for this residue in determining the mechanism of chain elongation. The Lys69Arg, His78Leu and Asn100Ile mutations also resulted in a narrowing of the product distribution, albeit less striking than that of Lys69Gln. In contrast, the mutations Arg111His, Lys114Met, Lys412Ile and Lys143Thr were demonstrated to increase activity specifically with acceptors of >10 residues in length, causing broadening of the product distributions.
The fact that most of the exchanges causing an elongation phenotype involve the replacement of basic amino acids suggests that the polyST's extended binding site is lined with basic residues which interact with the negative carboxyl groups of the polysialic acid chain. It is typical of enzymes which act on polymeric substrates to form many weak interactions with the polymer's repeating structural motifs, which prevents the formation of strong specific interactions and is thought to facilitate movement along the polymer (Breyer, Protein Science 10, 1699-1711 (2001)). However, during the initial stages of an elongation reaction, such an extended interaction interface confers an increasing affinity and increasing rate of transfer to chains which occupy more of the binding subsites (Levengood, J. Am. Chem. Soc. 133, 12758-12766 (2011)). The exact strength and extent of this interaction will sensitively affect the observed product distribution by altering the reaction rate in a chain-length dependent manner. This would explain the impressive diversity of product distributions in the neutral drift pool.
In contrast, a distributive mechanism of elongation indicates the absence of extended interactions with the polysaccharide chain and strictly nonprocessive elongation. Thus, distributive enzymes display no chain-length bias, but uniformly elongate the pool of acceptors resulting in narrow product distributions which approach a Poisson distribution (Chang, Journal of Molecular Biology 93, 219-235 (1975)), as observed for polySTs containing the Lys69Gln exchange. These considerations suggest that the Lys69Gln mutation blocks entry of the polysaccharide to the extended binding site. The proposition that residue 69 is involved in acceptor binding is supported by homology models of the polySTNmB structure which place this residue on a surface exposed loop in the vicinity of the proposed acceptor binding site (F. Freiberger and H. Fuchs, unpublished data).
Importantly, the rapid emergence of different mechanisms of elongation, as observed in this study, indicates that polymerising glycosyltransferases can be readily engineered to provide desired product distributions. Most promising in this respect is the introduction of a distributive mechanism with a single amino acid exchange. Distributive enzymes provide uniform (narrow) product distributions and product length is controlled simply by the ratio of donor to acceptor substrates. These are desirable properties for biotechnological applications where control of polysaccharide length is critical for the synthesis of defined structures and for the testing of optimal chain length for specific applications.
Methods
Molecular biology. Plasmids were isolated using NucleoSpin Plasmid™ or Extract™ kits from Machery-Nagel (Steinheim, Germany). Enzymes were purchased from New England Biolabs (Frankfurt, Germany) and all steps carried out according to the manufacturer's instructions. The DNA sequence of the Δ25polySTNmB was optimized for expression in E. coli. according to guidelines set out by Welch and colleagues24 and synthesized by Eurofins MWG Operon (Ebersberg, Germany). The gene was cloned into pET32a-Strep22 via BamHI/NotI restriction sites giving the plasmid p3775.
The first library was created by two rounds of error-prone PCR (epPCR), starting on the p3775 template. Buffer condition 3 (320 μM MnSO4; and an additional 40 μM dGTP) of the Diversify® PCR random mutagenesis kit (Clontec, California, USA) was used with 100 ng of template plasmid in a 50 μl reaction using and the T7/T7-term primer pair (Supplementary Table 2). PCR amplification was carried out with the following program:
Reaction products were separated on an agarose gel, the amplified gene purified with the NucleoSpin Extract™ kit and eluted in 50 μl. Of the eluted product, 3 μl, was used as template in a second epPCR reaction with the same buffer conditions and thermocycler program. The product of the second reaction was cloned via BamHI/NotI restriction sites into the pET32a-Strep vector22.
The protocol for the second library was designed to facilitate a high level of recombination within and between the clusters of high mutability identified from sequencing the first round clones (
First, 122 plasmids from the first-round clones were pooled and used as template for amplification of the coding sequences using the T7/T7-term primer pair. The amplified product was purified via agarose gel electrophoresis and 1.6 μg was digested with 0.5 units of DNaseI (New England Biolabs, Frankfurt am Main, Germany) in 40 μl volume for 1 min 50 sec at 25° C. The reaction was stopped by addition of 30 mM EDTA and incubation at 75° C. for 10 min. DNaseI products were separated by agarose gel electrophoresis and fragments of 50-600 bp were extracted and purified. Four 30 μl assembly reactions contained either 130 ng, 50 ng, 25 ng or 0 ng of the 38 pooled equimolar oligonucleotides (TK229-TK266; Supplementary Table 2), 130 ng of gene fragments, 3% DMSO, 200 μM dNTPs, 0.5 units Phusion® DNA polymerase (New England Biolabs, Frankfurt am Main, Germany) and Phusion® HF buffer. The four assembly reactions were carried out with the following program:
Of each assembly reaction, 5 μl was used as template to amplify full length genes in separate PCRs. Four 50 μl amplification PCRs were carried out with 0.5 units Phusion® DNA polymerase, 6% DMSO, and the TK156/TK157 primer pair (Supplementary Table 2). Reactions were thermocycled according to the following program:
Analysis of 5 μl samples confirmed amplification of similar amounts of the full gene in all reactions. Finally all four reactions were pooled and the products cloned as described for the first library. Sequencing of the library pool ensured incorporation of the desired diversity, and sequencing of 12 random clones indicated a mutation rate of 6±5.9 amino acid exchanges per sequence.
The protocol for the third library was designed to enable fine-grade shuffling of sequences from the second round of screening with no further introduction of mutations. The 22 plasmids from the second-round clones were pooled, coding sequences were amplified and DNaseI digested as above, and gene fragments of 50-200 bp were extracted and purified. A single PCR assembly and amplification reaction was carried out as described above without the addition of synthetic oligonucleotides. The shuffled product was cloned into the expression vector as described above.
In vivo activity screening was carried out as previously described22. Briefly, library DNA was transformed into the screening strain, MB3109. Transformants were plated on Luria-Bertani (LB) agar at ˜2000 colonies per 140 mm plate, and grown for 10-12 h at 37° C. until colonies had reached a diameter of 0.5-1 mm. A nitrocellulose membrane was used to copy colonies, then placed colonyside-up onto inducing plates. After incubation for 3 h at 37° C., colonies were lysed and fixed on the membrane and polySia was detected as previously described22. Positive clones were picked from the master plates and the amount of polySia synthesised by each clone was quantified with the same assay in microtiter plate format22. In each round of screening 10,000-20,000 clones were screened and those producing at least 80% of the wild type level of polySia were considered to be neutrally drifting.
Preparation of recombinant protein. Clones for analysis were transformed into BL21-gold(DE3). Colonies were picked into 150 μl LB media with appropriate antibiotics in 96-well flat bottom plates to allow for observation of optical density (OD) on a plate reader (PowerWave 340, BioTek). Dense overnight cultures were diluted 1:20 into 200 μl PowerBroth (AthenaES, Baltimore, Md., USA) and adjusted to uniform OD600. Cultures were grown at 37° C. until OD600≈1.8, then cooled on ice and supplemented with isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. Protein expression was carried out at 15° C. for 20 h with shaking at 1000 rpm. Cultures typically reached an OD600 of 8.0-9.0. Expression cultures were harvested in two 100 μl aliquots, pellets were washed with PBS, and stored at −80° C. until analysis.
For cell lysis, pellets were resuspended in 100 μl lysis buffer (50 mM Tris pH 8.0, 5% glycerol, 200 μg/ml lysozyme, 10 μg/ml Dnase I, 1 mM PMSF) then subjected to two cycles of freeze-sonication (15 min on ice, snap freezing in liquid nitrogen, and thawing in sonication bath at 23° C.). Unlysed cells and debris were removed by centrifugation at 4000 g for 10 min, and cleared lysates were supplemented with 1 mM EDTA. The final cell free extracts were used directly in activity assays and protein determinations.
Polysialyltransferase assays. The in vitro activity of polySTs was determined as previously described18 except that all steps were carried out in 96-well microtiter plates using a 96-channel pipette for accuracy. The recombinant enzymes were assayed at 25° C. in 100 μl volumes containing 50 mM Tris-HCl pH 8.0, 25 mM KCl, 20 mM MgCl2, 5% glycerol, 200-1000 μM CMP-Neu5Ac, and 5.32 μM DMBDP3. Reactions were started by the addition of 25 μl of cell free extract. Reaction samples were quenched by 10-fold dilution in 100 mM Tris-HCl pH 8.0, 20 mM EDTA, followed by 10 min at 50° C. Stopped samples were centrifuged at 4000 g for 1 h prior to HPLC analysis. Separation of 10 μl samples was carried out on a CarboPac PA-100 column as previously described18. During the neutral drift experiments each plate contained two copies of the Δ25polySTNmB reference sequence and clones with at least 25% of the wild type level of activity were considered to be neutrally drifting.
Protein determinations. Total protein concentrations were determined with the BCA assay (Thermo Scientific, Rockford, Ill., USA) according to the manufacturer's instructions.
Determination of polyST expression level was carried out as previously described22. Briefly, samples of cell free extract were separated by 12% SDS-PAGE. For Western blot analysis, proteins were transferred onto PVDF (polyvinylidene fluoride) membranes (LI-COR Biosciences) and detected with 1 μg/ml mouse anti-penta-his antibody (Qiagen) and anti-mouse IR800 (1:20,000, LI-COR Biosciences) and quantified by comparison with protein standards according to the recommendations of the Odyssey infrared imaging system (LI-COR Biosciences).
Introduction
Neisseria meningitidis serogroup A (NmA) is the major cause of meningococcal disease in the African meningitis belt. Besides of seasonal epidemics that occur with almost annual frequency, NmA has been the cause of severe pandemics in the last century (Stephens (2009) Vaccine, 27 Suppl 2, B71-B77). A major virulence factor of Nm is the negatively charged capsular polysaccharide (CPS). The NmA CPS (CPSA) consists of N-acetyl-mannosamine 1-phosphate units, linked by phosphodiester bonds to give the polymer [→6)-α-D-ManpNAc-(1→OPO3→]n (Liu (1971) JBC 246 (9), 2849-2858). Of note, the six most virulent Nm serogroups (NmA, -B, -C, -W, -Y and -X) wear negative CPSs. Negative charge in CPSA and CPSX is due to the phosphodiester group, while negative charge in CPSB, -C, -W, and -Y results from the incorporation of the sialic acid (Stephens (2009) Vaccine 27 Suppl 2, B71-B77).
Besides of CPSB, which is identical with polysialic acid in the human host, all CPS are immunogenic and cause the production of antibodies that are bacteriotoxic in the presence of complement (Gotschlich (1969) The Journal of experimental medicine 129 (6), 1367-1384; Stephens (2007) Lancet 369 (9580), 2196-2210). In fact, this early observation has made the use of polysaccharide-protein conjugates the gold standard in the development of vaccines against Nm strains. A number of mono- and tetravalent (the latter comprising serogroups A, C, W and Y) conjugate vaccines against Nm have been licensed (Costantino (2011) Expert opinion on drug discovery 6 (10), 1045-1066).
Crucial to the success of vaccination programs in the sub-Saharan meningitis belt is the provision of a safe and high-quality vaccine. MenAfriVac®, a conjugate vaccine with CPSA coupled to tetanus toxoid as carrier protein, has been specifically designed to address these needs (Frasch (2012) Human vaccines & immunotherapeutics 8 (6), 715-724). With under 50 cent per dose (Roberts (2010) Science 330 (6010), 1466-1467), mass vaccination campaigns were possible in Burkina Faso, Mali and Niger and installed herd immunity (Djingarey (2012) Vaccine 30, B40-B45; Caini (2013) Vaccine 31 (12), 1597-1603; LaForce (2011) Eliminating epidemic Group A meningococcal meningitis in Africa through a new vaccine. Health affairs (Project Hope) 30 (6), 1049-1057), protecting not only vaccinated but also non-vaccinated individuals and young children (Kristiansen (2013) Clinical infectious diseases 56 (3), 354-363). Recent progress made with the cloning and functional expression of capsule polymerases (CPs) (Freiberger (2007) Molecular microbiology 65 (5), 1258-1275; Romanow (2013) The Journal of biological chemistry 288 (17), 11718-11730; Peterson (2011) Journal of bacteriology 193 (7), 1576-1582) and the pioneering studies that demonstrate the suitability of the respective recombinant enzymes for the in vitro production of CPSs (McCarthy (2013) Glycoconjugate journal 30 (9), 857-870; Fiebig (2014) Glycobiology 24:150-8), have opened a new perspective for the economic and save production of conjugate vaccines. As NmA is a thread foremost in underdeveloped countries, the development of robust and economic regimes for vaccine production is of utmost relevance. Consequently, the goal of the current study was to isolate the capsular polymerase from NmA and to analyse the capability of the recombinant protein to produce CPSA in vitro. Because the sugar building block UDP-ManNAc is commercially not available, it was clear from the start of this project that a successful production chain depends on the in situ synthesis of UDP-ManNAc from cheap UDP-GlcNAc. Moreover, as CPSA is immunogenic only if O-acetylated (Berry (2002) Infection and immunity 70 (7), 3707-3713), an effective production chain needs, in addition, the O-acetyltransferase, which is able to perform this modification in a nature identical form. Based on these considerations we decided to isolate the three relevant enzymes from NmA and to explore the capacity of the recombinant proteins to work together in vitro to produce bio-identical CPSA.
The chromosomal locus (cps for capsular polysaccharide synthesis) contains the genetic information for CPS synthesis, modification and surface transport. The locus is sub-structured into six regions: A-D, D′ and E (
Here we describe the molecular cloning of the genes csaA, csaB and csaC from NmA, the production of functional recombinant proteins and provide proof for their functional properties. Using a series of synthetic primer compounds the ManNAc-dimer linked together by phosphodiester bonds and carrying a phosphodiester at the reducing end was identified to be the minimal acceptor structure, which was preferred by CsaB if presented in non-acetylated form. Using a two-step production protocol, O-acetylated CPSA was prepared in highly pure form and was recognized by the monoclonal antibody (mAb) 935, suggesting that the in vitro produced polymer represents the bioidentical CPSA.
Results
Cloning and expression of csaA, csaB and csaC; production of recombinant proteins.
Because previous analyses carried out on these genes provided strong evidence that csaA, csaB and csaC encode the UDP-GlcNAc epimerase, the poly-ManNAc-1-phosphoryl transferase (Swartley (1998) Journal of bacteriology 180 (6), 1533-1539) and the O-acetyl transferase (Gudlavalleti (2004) The Journal of biological chemistry 279 (41), 42765-42773), respectively, primers were constructed (see Material and Methods) to amplify these ORFs. The genomic DNA isolated from Nm strain Z2491 was used as a template. Obtained PCR products were cloned into the pET22b-Strep vector (Schwarzer (2009) The Journal of biological chemistry 284 (14), 9465-9474), allowing the expression of recombinant proteins with N-terminal StrepII- and/or C-terminal His6-tag. After transformation into BL21(DE3) and induction of protein expression (see Material and Methods) the distribution of recombinant proteins between the soluble (s) and insoluble (i) fractions of bacterial lysates was analyzed by western blotting against the affinity tags. The recombinant epitope tagged forms of CsaA and CsaC appeared mostly in the soluble fraction (data not shown) and could be purified directly from the bacterial lysates. CsaA was purified by IMAC (immobilized metal ion affinity chromatography) followed by a desalting step and yielded 40 mg protein/L expression culture. Though some additional faint bands were visible in Coosmassie stained SDS-PAGE (
Similarly, the StrepII-CsaB-His6, encoding the putative poly-ManNAc-1-phosphoryl transferase, was well expressed and appeared with >60% in the soluble fraction. However, the major product revealed with the anti-penta-His antibody in western blot migrated with an apparent molecular mass of 50 kDa, strongly deviating from the calculated molecular mass of 67 kDa (
To investigate if translation from the alternative ATG leads to a stable protein, the corresponding truncation Δ69CsaB was cloned with (StrepII-Δ69-CsaB-His6) and without (Δ69-CsaB-His6) the N-terminal StrepII-tag. Test expressions in BL21(DE3) demonstrated the occurrence of proteins of the expected molecular masses, but in repeated experiments the level of expressed protein was significantly lower than for the full length construct. Moreover, to our surprise, the construct cloned with free N-terminus (Δ69-CsaB-His6) was routinely higher expressed than the StrepII-tagged construct (
Consecutively, enzymatic activity within the soluble fractions of the bacterial lysates was determined, with a radioactive incorporation assay previously developed for the poly-GlcNAc-1-phosphoryl transferase from NmX (Fiebig, Glycobiology (2014) 24:150-8). The fractions containing the recombinant CsaB variants were tested in the presence of CsaA and UDP-[14C]GlcNAc. In accordance with the levels of expressed protein (
Optimization of test conditions and determination of CsaB acceptors. As the donor sugar (UDP-ManNAc) used by CsaB must be produced in situ in the epimerase reaction catalyzed by CsaA, the optimization of test conditions needed the presence of both enzymes. As for CPs of other Nm strains, a hydrolysate of CPSA (CPSAhyd) was used to prime the reaction in the presence of the CsaA substrate UDP-GlcNAc. Initial studies carried out to evaluate pH and salt conditions showed best activity values in the presence of 10-20 mM MgCl2 and a pH between 8.0-8.5 (data not shown). Replacement of Mg2+ by Ca2+ or Mn2+ inactivated the enzyme. While these results were similar to what we had seen with CsxA (CP of NmX), the CsaA/CsaB reaction, in contrast to the CsxA reaction, did not show sensitivity against DTT (up to 2 mM were tested).
The natural CPSA is O-acetylated in positions 3 and 4 of ManNAc (Liu (1971) The Journal of biological chemistry 246 (9), 2849-2858; Gudlavalleti et al., Carbohydr Res. 2006), we therefore interrogated if Δ69-CsaBco-His6 recognizes and elongates acetylated and non-acetylated CPSAhyd with the same efficiency. Therefore, the hydrolysis of CPSA was carried out before and after base treatment to obtain O-acetylated (CPSAhyd(OAc)) and de-O-acetylated (CPSAhyd(deOAc)) shorter saccharide chains. Knowing that CPSA hydrolysis results in a large distribution of saccharide chain lengths (ranging in size between degree of polymerization, DP, 1 and 70), anion exchange chromatography was used to separate two fractions, the averaged DP (avDP) 6 (comprising DP1-DP10) and avDP15 (comprising DP10-DP70). Both fractions were used to prime the enzyme reactions as indicated in the subsequent experimental steps.
To quantitatively assess the enzyme reactions, we adapted a spectrophotometric assay previously designed to analyse the CP from NmB (Freiberger (2007) Molecular microbiology 65 (5), 1258-1275). In the multi-enzyme assay shown in
Using this assay the activity of CsaB was determined with CPSAhyd fractions (CPSAhyd(OAc) CPSAhyd(deOAc)) of avDP6 and avDP15 (
Because no information on the minimal length of the priming acceptor for CsaB could be derived from the CPSAhyd fractions, we used well characterized synthetic compounds to interrogate this question. The compounds synthesized are shown in
In vitro synthesis of CPSA-chains. To analyze if long CPSA-chains can be produced with the recombinant enzymes, test reactions were carried out having the enzymes Δ69-CsaBco-His6 and StrepII-CsaA-His6 at equal concentration (50 nM) and the priming oligosaccharide (CPSAhyd(deOAc)-deP of avDP6) in 100-fold molar excess.
Reactions were started by addition of 5 mM UDP-GlcNAc. Control reactions (
Since all relevant 31P NMR signals were identified also in the product formed in control-1, this faint CPSA signal demonstrated that Δ69-CsaBco-His6 is capable to induce product formation de novo in the absence of a priming oligosaccharide. Importantly, this same type of de novo synthesis was shown for the CP of NmX (Fiebig, Glycobiology (2014) 24:150-8). Still, the self-priming capacity came as a surprise in the case of CsaB, were de novo formation of product was not seen if the synthetic compounds 1 or 2 where tested as primers (see
To reduce UDP-ManNAc hydrolysis and simultaneously complete the incorporation of ManNAc-1P into the product, the ratio between CsaA and CsaB was varied as indicated in
With the intention to produce purified, bio-identical CPSA in milligram amounts, the CsaA/CsaB reaction was up-scaled. The long polymers that were obtained in this reaction were purified by anion exchange chromatography (AEC) using a protocol similar to the one described (Fiebig, Glycobiology (2014) 24:150-8). CPSAiv eluted at NaCl clearly separated from all other reaction components (
Finally we explored at analytical scale, if bio-identically O-acetylated CPSA can also be produced in the one pot reaction. Therefore, as described above the reaction mixture was supplemented with recombinant CsaC-His6 and acetyl-CoA. Moreover, control reactions, with single compounds missing (see scheme added to
Discussion
Of all pathogenic Nm serogroups, NmA has caused the most disastrous epidemics in sub-saharan Africa. The prevalence of this pathogen caused an unprecedented attempt in terms of developing a highly effective and economic vaccine, MenAfriVac® (Roberts L. Science (2010) 330:1466-7). With coasts of less than 50 cent per dose, MenAfriVac® enabled mass vaccination campaigns in Burkina Faso, Mali and Niger (LaForce (2011) Eliminating epidemic Group A meningococcal meningitis in Africa through a new vaccine. Health affairs (Project Hope) 30 (6), 1049-1057; Djingarey (2012) Vaccine 30, B40-B45; Caini (2013) From Agadez to Zinder: estimating coverage of the MenAfriVacΓäó conjugate vaccine against meningococcal serogroup A in Niger, September 2010-January 2012. Vaccine 31 (12), 1597-1603), which installed herd immunity, leading to the protection not only for vaccinated but also for non-vaccinated individuals and in particular for young children (Kristiansen (2013) Clinical infectious diseases 56 (3), 354-363).
All NmA vaccines licensed today are glycoconjugate vaccines, with CPSA oligosaccharides coupled to carrier proteins. CPSA oligosaccharides are thereby hydrolysis products of CPSA isolated from large scale NmA cultures (Bardotti (2008) Vaccine 26 (18), 2284-2296). To avoid the significant coast and biohazard in association with large scale NmA cultures and the pyrogen-free production of polysaccharides, the enzyme catalyzed in vitro synthesis of CPSA would provide an attractive alternative. Towards this goal, we describe in this study, the molecular cloning and functional expression of the three enzymes (UDP-GlcNAc 2-epimerase, CsaA; NmA capsule polymerase, CsaB; O-acetyltransferase, CsaC) that are part of the capsular biosynthesis complex in NmA and represent the minimal number of enzymes needed to produce immunologically active CPSAOAc in vitro starting from economic precursors. Using the well characterized BL21(DE3) strain as expression host, C-terminally His6-tagged versions of CsaA and CsaC could be purified in high quality and remarkable quantity (CsaA 40 mg and CsaC 96 mg per liter bacterial culture).
In CsaB a second start codon was identified starting with methionine-69 bp upstream of the first. Use of this second start codon generated a stable C-terminally His6-tagged protein. However, the level of expressed protein could be significantly increased (to 60 mg/L bacterial culture) after the cDNA sequence was optimized for codon usage in BL21(DE3). If the second start codon is actually used in the natural environment cannot be answered based on the current data. However, because a multi-sequence alignment demonstrated significant similarity between the N-termini of CsaB and other capsule polymerases starting with the first methionine (data not shown), it is likely that the first and not the second start codon is used in the natural environment.
Using a two-step protocol (in test reactions even a one pot reaction; see
When the CsaA/CsaB reaction was carried out under suboptimal conditions (meaning that CsaA concentrations were higher than CsaB concentrations) UDP was formed as a side product. Based on control experiments and on earlier literature data the occurrence of UDP indicates that CsaA similar to UDP-N-acetylglucosamine 2-epimerase from E. coli, epimerizes UDP-GlcNAc to UDP-ManNAc by forming UDP and 2-acetamidoglucal as intermediate products (Sala (1996) Journal of the American Chemical Society 118, 3033-3034). If present in access, the epimerase releases these intermediates into solution (Sala Journal of the American Chemical Society 118, 3033-3034). Logically, the side reaction was avoided by adjusting the ratio in enzyme concentrations [CsaA]<[CsaB].
O-acetyltransferases from encapsulated bacterial strains have been cloned and functionally characterized (Bergfeld (2007) The Journal of biological chemistry 282 (30), 22217-22227). In these studies addressing also structure-function-relationships the enzymes could be grouped into two families. While O-acetyltransferases of NmW-135 and NmY group into the superfamily of left-handed β-helix (LβH) proteins the O-acetyltransferases isolated from NmA (CsaA) has sequence homology to members of the α/β-hydrolase fold family, harboring a catalytic serine-tried which is part of ‘nucleophilic elbow’ motif (Bergfeld (2007) The Journal of biological chemistry 282 (30), 22217-22227; Bergfeld (2009) The Journal of biological chemistry 284 (1), 6-16). The α/β-hydrolase fold positions one of the catalytic serine-residues to the tip of a narrow turn. This structural motif may impact substrate recognition by the enzyme.
In the current study, we provide clear evidence that the acceptor quality of CPSAhyd increases significantly after release of O-acetyl-groups. Similarly, the synthetic disaccharide carrying 3-O-Ac-groups on ManNAc (compound 6) was a significantly less efficient primer than the respective compound (5) without O-acetyl-groups. These data argue for that O-acetylation takes place during or after the polymer is formed. This type of size-dependent substrate recognition has already been identified for other O-acetyltransferase that act on sugar polymers. With CPSAhyd a steep increase in activity was seen after dephosphorylation of the non-reducing ends. We previously demonstrated that acidic hydrolysis results (up to 70%) in fragments that are carrying phosphate at the non-reducing end. Since removal of phosphate improved the acceptor quality, it can be concluded that chain elongation proceeds by transfer of monosaccharide-phosphates to the non-reducing end.
The use of the synthetic compounds identified two other important features of CsaB: (i) the minimal efficient acceptor is the dimer and (ii) the reducing end phosphate group can be extended with rather large chemical groups (decyl-ester in the compounds tested in this study). This latter finding bears the perspective that chain elongation can be primed with reagents of very high purity and functional groups that facilitate conjugation of glycans to carrier proteins in the vaccine production chain.
Of note is the fact that CsaB is capable of self-priming. The mechanism underlying the de novo start of chain polymerisation is not understood, but was similarly observed with the CP of NmX (Fiebig, Glycobiology (2014) 24:150-8). In the case of CsaB the interpretation of the phenomenon is however further complicated by the fact that no such reaction was detectable in the presence of the artificial compounds 1-4. A possible explanation may be that these compounds compete against UDP-ManNAc in the active site pocket and thus block the initiation of the chain. However, more experimental work is needed to understand and explain this reaction.
Taken together, our study lays a new basis for the development of efficient and economic protocols with which immunologically active and highly pure CPSAOAc can be synthesized in vitro and used for glycoconjugate vaccine production. We provide initial evidence that CPSAOAc can be produced in a one pot reaction and purified to homogeneity in a single step. The used enzymes are without biohazard and provide the perspective that production chains can be established also in areas that are lacking advanced infrastructural conditions.
Material and Methods
General cloning—The genomic DNA isolated from Nm strain Z2491 was a kind gift from Dr. Heike Claus (Institute for Hygiene and Microbiology, University of Würzburg). The csaB sequence was codon optimized for use in E. coli BL21(DE3) using the Gene Designer software package (DNA 2.0) (Villalobos (2006) BMC bioinformatics 7, 285) and the codon frequency tables published by Welch (2009; PloS ONE 4 (9)). The mean codon frequency for each amino acid was calculated from the codon frequency tables FreqA and FreqB (Welch (2009) PloS ONE 4 (9)) and the resulting codon frequency table used as template for the in silico generation of csaBco. csaBco flanked 5′ by a BamHI site and 3′ by a XhoI site was synthesized from Eurofins MWG Operon. All other csaA-C sequences described herein were amplified by polymerase chain reaction (PCR) using the primers shown in Table 4 and genomic DNA from Nm strain Z2491 or csaBco as template. PCR products were cloned via the restriction sites shown in Table 4 into the corresponding sites of the vector pET22b-Strep (Schwarzer (2009) The Journal of biological chemistry 284 (14), 9465-9474) driving the expression of recombinant proteins under the control of the T7 promoter. PCR products digested with BglII were cloned into the BamHI site of pET22b-Strep.
Expression and purification of recombinant CsaA, CsaB and CsaC—Freshly transformed E. coli BL21(DE3) were grown at 15° C. in PowerBroth medium for 18 h. At an optical density of OD600=1.0 protein expression was induced by addition of 0.1 mM IPTG and allowed to proceed for a period of 20 h. In test expressions, 0.2 ml of culture-volume were pelleted with 16,000×g for 1 min. Cell pellets were lysed with 0.1 ml lysis buffer (50 mM Tris pH 8.0, 2 mM EDTA, 0.1 mg/ml lysozyme). The lysis was intensified by 3 cycles of sonication (Branson sonifier 450, 100% amplitude) interrupted by 3 min of cooling on ice. Soluble and insoluble fractions were separated by centrifugation (16,000×g, 30 min, 4° C.), the supernatant mixed (1:1) with Laemmli-buffer and used for PAGE as described below.
For protein purification, pellets from 125 mL expression culture were pelleted by centrifugation (6,000×g, 10 min, 4° C.). After a washing step with PBS, cells were re-suspended in 7.5 ml binding buffer (50 mM Tris pH 8.0, 300 mM NaCl) complemented with 40 μg/ml Bestatin (Sigma), 1 μg/ml Pepstatin (Applichem), 100 μM PMSF (Stratagene) and sonified (Branson Digital Sonifier, 50% amplitude, 8×30 s, interrupted by cooling on ice). After centrifugation at 27,000×g for 30 min, the soluble fractions were directly loaded onto a HisTrap columns (GE Healthcare) to enrich the recombinant proteins by immobilized metal ion affinity chromatography (IMAC). Columns were washed with binding buffer (50 mM Tris pH 8.0, 300 mM NaCl) and proteins eluted in step gradients using 10%, 30%, 50% and 100% elution buffer (binding buffer containing 500 mM imidazol). Fractions containing recombinant protein were pooled and the buffer exchanged to storage buffer (50 mM Tris pH 8.0, 50 mM NaCl for CsaA/CsaB; 50 mM Hepes pH 7.05, 100 mM NaCl, 5 mM MgCl2 and 1 mM EDTA for CsaC) using the HiPrep 26/10 Desalting column (GE Healthcare). Isolated proteins were concentrated using Amicon Ultra centrifugal devices (Millipore 30 MWCO). After separation into aliquots, samples were snap frozen in liquid nitrogen and stored at −80° C.
SDS-PAGE and immunoblotting—SDS-PAGE was performed under reducing conditions using 2.5% (v/v) 1-mercaptoethanol and 1.5% (w/v) SDS. Proteins were stained using Roti-Blue (Carl Roth GmbH) according to the manufacturer's guidelines. For western blot analysis samples and standard proteins were blotted onto PVDF membranes (Millipore). His-tagged proteins were detected with 0.5 μg/ml anti-penta-His antibody (Qiagen) and goat anti-mouse IR680 or goat anti-mouse IR800 antibody (LI-COR) as second antibody. Second antibodies were used in a 1:20,000 dilution.
Preparation of CPSA oligosaccharides—CPSA oligosaccharide samples with an averaged degree of polymerization (avDP) of 6 and 15, respectively, were generated by acidic hydrolysis of long CPSA chains isolated from bacterial cultures (CPSAn). Therefore, solutions containing 2.5 mg CPSA/mL sodium acetate buffer (50 mM sodium acetate, pH 4.8) were incubated at 73° C. for 6 h and two pool fractions (avDP 6 and 15, respectively) were purified by anionic exchange chromatography (Q-Sepharose column, GE Healthcare) using a sodium acetate gradient. The avDP and the dispersion of saccharide chains was determined by 31P NMR and High Performance Anionic Exchange Chromatography-Pulsed Amperometric Detection (HPAEC-PAD) analysis following an established protocol (Berti (2012) Vaccine 30 (45), 6409-6415). If used in enzymatic reactions, hydrolyzed CPSA (CPSAhyd) was dephosphorylated (CPSAhyd-deP) using acid phosphatase (Sigma) according to the manufacturer's guidelines.
Activity testing of CsaA/CsaB by use of a radioactive assay system—CsxA/CsaB activity was analysed using an adaptation of a radioactive incorporation assay previously described for the N-acetylglucosamine-1-phosphate-transferase from NmX (Fiebig, Glycobiology (2014) 24:150-8). Briefly, assays were carried out with 5 μl of the soluble fractions of bacterial lysates expressing either recombinant CsaB or CsaA (see
Activity testing of CsaA/CsaB by use of a multi-enzyme spectrophotometric assay—1.2 μM of CsaA and 1 μM CsaB were assayed in the presence of 0.25 mM UDP-GlcNAc (Calbiochem), 20 mM MgCl2 and 50 mM Tris pH 8.0 in a total volume of 100 μL. The consumption of UDP-GlcNAc was coupled to nicotinamide adenine dinucleotide (NADH) consumption using the following enzymes/substrates: 0.25 mM adenosine triphosphate (ATP, Roche), 1 mM phosphoenolpyruvate (PEP, ABCR), 0.3 mM (NADH, Roche), 9-15 units/ml pyruvate kinase, 13.5-21 units/ml lactic dehydrogenase (PK/LDH mix Sigma), 0.05 mg/ml nucleoside monophosphate kinase (Roche). Absorption was measured at 340 nm every 30 min using a Biotek EL 808 96-well plate reader.
Physicochemical analysis of CPSAiv. To produce sufficient CPSA (CPSAiv) for PAGE and NMR analyses, 0.84 nmol (1.2 μM final) of StrepII-CsaA-His6 and 37.5 pmol (50 nM final) of Δ69CsaBco-His6 in reaction buffer (50 mM Tris pH 8.0, 20 mM MgCl2) were incubated with 5 mM UDP-GlcNAc and 6.8 μg of CPSAhyd of avDP6 after de-O-acetylation and dephosphorylation (CPSAhyd(deOAc)-deP). The total reaction volume was adjusted to 750 μl. In control 1 also StrepII-CsaA-His6 was used at 50 nM. Reactions as well as control samples were incubated over night at 37° C. 5 μl of each sample were then used for separation on high percentage (25%) PAGE and visualized by a combined Alcian blue/silver staining procedure (Min (1986) Analytical biochemistry 155 (2), 275-285).
The residual sample was freeze-dried, solubilized in 0.75 ml deuterium oxide (D2O, 99.9% atom D; Aldrich) to give a concentration of 0.5-1 mg saccharide and used for product characterization by NMR. All the 1H and 31P NMR experiments were recorded as previously described (Fiebig, Glycobiology. (2014) 24:150-8).
HPLC-AEC was performed on a Prominence UFLC-XR (Shimadzu) equipped with a CarboPac PA-100 column (2×250 mm, Dionex). Samples were separated as described by (Keys (2012) Analytical biochemistry 427 (2), 107-115) with the minor adjustment that H2O and 1M NaCl were used as mobile phases M1 and M2, respectively. 5 μl of the samples were loaded for the detection of nucleotides at 280 nm and 50 μl for the detection of CPSA at 214 nm. Products were separated using an elution gradient consisting of a −2 curved gradient from 0 to 30% M2 over 4 min followed by a linear gradient from 30 to 84% M2 over 33 min. Enzyme concentrations were used as indicated in
Analysis of 2-acetamidoglucal—Assignments of 1H NMR spectrum were in agreement with those reported in literature (29). 1H NMR (D2O, 400 MHz): δ=6.68 (d, 1 H, J1,2 1.0, H-1), 4.25 (dd, H 1, J3,4 6.5 Hz, H-3), 3.99 (dt, 1 H, J4,5 8.4, J5,6a=J5,6b 4.2 Hz, 6.5 Hz, H-5), 3.86 (d, 2 H, H-6), 3.77 (dd, 1 H, H-4), 2.05 (s, 3 H, CH3CO). Significant signals from 13C NMR (D2O, 100 MHz): δ=141.47 (C-1), 78.70 (C-5), 68.70 (C-3), 68.36 (C-4), 59.84 (C-6), 21.84 (2×CH3CO).
In vitro synthesis, purification and immunological analysis of CPSAiv and CPSAiv(OAc)—To generate CPSAiv, 10 nmol CsaA and 16 nmol CsaB were incubated over night at 37° C. in reaction buffer (50 mM Tris pH 8.0, 20 mM MgCl2) complemented with 10 mM UDP-GlcNAc in a total volume of 9 ml. The reaction was primed with 1 μg of CPSAhyd(deOAc)-deP of avDP6. Acetylation of 1 mg CPSAiv was performed for 4 h at 37° C. in the presence of 1.2 nmol CsaC and 14 mM acetyl-CoA (Sigma) in a total volume of 0.5 ml acetylation buffer (25 mM Tris pH 7.5, 50 mM NaCl). Both CPSAiv and CPSAiv(OAc) were purified via anion exchange chromatography (AEC) using a MonoQ HR5/5 column (Pharmacia biotech) at a flow rate of 1 ml/min and a linear sodium chloride gradient. CPS containing fractions eluting at 540 mM NaCl were pooled, dialysed (ZelluTrans, Roth, 1 kDa MWCO) against water and freeze dried for further analysis. For dot blot analyses, small aliquots of the purified CPSAiv and CPSAiv(OAc) was spotted onto nitro-cellulose (Whatman) and incubated with mAb 932 specifically directed against CPSA(OAc) (mAb 935 was generated in the laboratory of Prof. Dr. D. Bitter-Suermann, Hannover Medical School, Institute for Medical Microbiology, and was kindly provided for this study) in a 1:10,000 dilution. Dot blots were developed with goat anti-mouse IR800 antibody (LI-COR) in 1:20,000 dilution.
The materials and methods for this Example are described herein above and also shown in Fiebig, Glycobiology (2014) 24:150-8.
The secondary structure prediction software PHYRE (Kelley et al., 2009, Nat Protoc. 4, 363-371) was used to predict secondary structure elements within the CsxA amino acid sequence. Amplification of csxA using the truncation primers FF52 and FF53, annealing upstream of the conserved region (CR) 1 and downstream of CR2 in unstructured regions where no secondary structure was predicted, resulted in a dN58- or dC99-truncated CsxA, respectively.
The PCR products were cloned into a modified pET43.1 vector enabling the expression of the truncated genes as N-terminally MBP- and C-terminally His6-tagged fusion constructs. After expression in BL21(DE3)Gold followed by lysis of the pelletized cells, soluble fractions were used in a radioactive incorporation assay. Following SDS-PAGE and subsequent immuno-blotting against the His6-tag, the Odyssey-Software could be used to determine the amount of the respective constructs within the soluble and the insoluble fraction. The former was used to normalize the enzymatic activity obtained in the radioactive incorporation assay.
Judging from the western blot (
With the aim of analyzing the influence of the termini towards the solubility of CsxA and furthermore defining the minimal catalytic domain of the enzyme, the truncated constructs shown in
Group II polymerases are supposed to be part of the capsule transport complex (Steenbergen (2008) Mol. Microbiol. 68, 1252-1267). The improved solubility could indicate that hydrophobic aa within the first 58 aa of the CsxA sequence are exposed on the enzyme's surface to undergo protein-protein interactions with other components of the capsule transport complex.
The truncation of the C-terminus led to an increase of activity by 61% (compared to full-length). However, solubility was reduced by 50%. Since the C-terminus might play a role in the formation of the correct oligomerisation state of CsxA, the loss of the correct oligomerisation of ΔC99CsxA might explain the reduced solubility. However, further investigation is needed to understand these structure-function relationships.
The truncation studies showed that the minimal catalytic domain of CsxA can be reduced to a polypeptide of 329 aa, which corresponds to a reduction of 33% of the full-length sequence. Furthermore, the results indicate that the truncated termini do not contain aa of CsxA's active center.
Further Studies
In later studies, the ΔN58ΔC99 double truncation was generated and cloned, as well as all afore mentioned construct, into optimised expression vectors. The optimised expression vector is described in Fiebig (Glycobiology (2014) 24:150-8) as MBP-CsxA-His6 (tac).
In accordance with the results described above, the truncation of the N-terminus led to an increase in purification yield by more than 500%, whereas the purification yield of the C-terminal truncation was comparable to the full-length construct (
Activity of the purified constructs, measured using an adaption of the multi-enzyme assay described by Freiberger et al. (2007, Molecular Microbiology 65, 1258-1275), confirmed the increased activity of both single CsxA truncations (
Analytical size-exclusion chromatography showed that the C-terminus is not involved in oligomerisation/aggregation, since the C-terminal truncation, as well as the full-length enzyme (see Fiebig, Glycobiology. (2014) 24:150-8) forms aggregates in low salt buffer (
The materials and methods for this Example are described herein above and also shown in Fiebig, Glycobiology (2014) 24:150-8.
The C-terminally (e.g. ΔC99-CP-X) as well as the C- and N-terminally (e.g. ΔN58ΔC99-CP-X) truncated version of CP-X show a distributive polymerisation (
Preparation of the Acceptor Substrate and Determination of the Donor:Acceptor Ratio (d/a).
Oligosaccharide fractions were prepared as described in Example 1 and fractions containing DP2-10 were pooled to get a mixture of short oligosaccharides ranging from DP2-10. Using the ΔN58ΔC99 CsxA truncation, the donor to acceptor ratio was experimentally determined using 10 mM UDP-GlcNAc and variable amounts of acceptor substrate in over-night reactions to give a product distribution ranging from DP10-DP60 (
Expression of the Enzyme and Coupling of the Construct to the Column
ΔN58ΔC99 CsxA was expressed in a 50 mL culture volume and purified via His-Trap as described in Fiebig et al 2014a. Briefly, the cells were lysed by sonication in binding buffer (50 mM Tris pH 8.0, 1 mM DTT, 500 mM NaCl) and coupled to a 1 mL His-Trap column. The column was washed with binding buffer containing 37.5 mM imidazole to remove cell debris and non-specifically bound protein before it was equilibrated with a buffer only containing 50 mM Tris pH 8.0 and 1 mM DTT.
Production of CPSX Using ΔN58ΔC99 CsxA Coupled to a Solid Phase
The reaction mix containing 50 mM Tris pH 8.0, 10 mM UDP-GlcNAc, 20 mM MgCl2, 1 mM DTT and the amount of acceptor determined in
Following the consumption of the donor substrate UDP-GlcNAc and the production of the second reaction product UMP at 280 nm revealed that the reaction was finished after 20 min and shows that ΔN58ΔC99 CsxA is active even if it is bound/coupled to a solid phase (His-Trap beads) (
The present invention refers to the following nucleotide and amino acid sequences:
meningitidis serogroup A, coding sequence
The sequences provided herein (e.g. the sequences corresponding to CP-A or fragments thereof) may be linked to (a) tag(s). For example, the sequences may have a StrepII-thrombin-tag at the N-terminus and/or a his-tag at the C-terminus. The constructs may be prepared by using appropriate restriction sites. For example, CP-A-constructs may be cloned by using BamHI/Bg1II and XhoI. It is mentioned that, if the N-terminus is extended (e.g. by tags), then ATG/Met1 can be omitted. Accordingly, a construct comprising the full length CP-A may comprise not 545 but 544 CP-A-amino acids. SEQ ID NOs: 34 and 37 show an example for a StrepII-thrombin-BamHI/Bg1II-CP-A-XhoI-his construct. In context of the invention, all herein disclosed sequences may be used to produce constructs as demonstrated for CP-A in SEQ ID NOs: 34 and 37.
The sequences provided herein (e.g. the sequences corresponding to Δ69-CP-A) may be cloned into the vector by using NdeI/XhoI. SEQ ID NOs: 40 and 43 show an example for a NdeI-ΔN69-CP-A-XhoI-his construct. In context of the invention, all herein disclosed sequences may be used to produce constructs as demonstrated for the codon optimized ΔN69-CP-A in SEQ ID NOs: 40 and 43.
The sequences provided herein (e.g. the sequences corresponding to CP-X or fragments thereof) may be linked to (a) tag(s). For example, the sequences may have an N-terminal MBP-tag followed by the protease resistant linker S3N10, and/or a C-terminal his-tag. The sequences provided herein (e.g. the sequences corresponding to CP-X or fragments thereof) may be cloned into the vector by using the restriction sites BamHI and/or XhoI. In particular in the constructs of CP-X (or fragments thereof), if the N-terminus is not truncated, then the ATG1/Met1 may not be in the construct. SEQ ID NOs: 47 and 49 show an example for a MBP-S3N10-BamHI-CP-X-XhoI-his construct. In context of the invention, all herein disclosed sequences may be used to produce constructs as demonstrated for CP-X in SEQ ID NOs: 47 and 49.
The sequences provided herein (e.g. the sequences corresponding to ΔN65ΔC10-CP-X) may have a free N-terminus. These constructs may be used, inter alia for crystallization approaches. For example, the sequences may be cloned into a vector by using NdeI and/or XhoI. SEQ ID NOs: 51 and 52 show an example for an NdeI-ΔN65ΔC10-CP-X-XhoI-his construct. In context of the invention, all herein disclosed sequences may be used to produce constructs as demonstrated for ΔN65ΔC10-CP-X in SEQ ID NOs: 51 and 52.
This application is a national phase application under 35 U.S.C. § 371 of International Application No. PCT/EP2015/058399, filed Apr. 17, 2015, which claims benefit of U.S. Provisional Application No. 61/980,819, filed Apr. 17, 2014, the entire contents of each of which are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2015/058399 | 4/17/2015 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2015/158898 | 10/22/2015 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20060073168 | Stephens | Apr 2006 | A1 |
| Number | Date | Country |
|---|---|---|
| 1490409 | Dec 2008 | EP |
| WO 2007000322 | Jan 2007 | WO |
| WO 2008011201 | Jan 2008 | WO |
| WO 2008011201 | Jan 2008 | WO |
| WO 2011023764 | Mar 2011 | WO |
| WO 2011023764 | Mar 2011 | WO |
| WO 2013174832 | Nov 2013 | WO |
| Entry |
|---|
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| Number | Date | Country | |
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| 20170037440 A1 | Feb 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61980819 | Apr 2014 | US |
