Patent Grant
9878051
The present invention relates to an immunogenic and non-toxic glycoconjugate comprising Bordetella pertussis LOS-derived oligosaccharide and a pertussis toxin, a method of preparing such glycoconjugate, the pharmaceutical composition, a vaccine composition containing such glycoconjugate, and an application of the glycoconjugate. The glycoconjugate is prepared as a vaccine component for protection against infections caused by Bordetella pertussis.
The present invention belongs to the field of antibacterial glycoconjugate vaccines design.
Bordetella pertussis causes whooping cough, a highly contagious disease involving the respiratory tract, which is especially serious for infants and young children. Despite widespread immunization, in recent years the number of pertussis incidences has increased [19, 20]. The main reason for the resurgence of this vaccine-preventable disease is a waning of vaccine-induced immunity and genetic changes in B. pertussis strains [11]. High reactogenicity of the whole-cell pertussis vaccines and better understanding of the molecular function mechanisms of B. pertussis virulence factors that are also its major protective antigens have led to the introduction of acellular vaccines which are in continuous development [21, 31, 41, 63, 68]. Commonly used acellular pertussis vaccines contain inactivated pertussis toxin since the toxin is the most immunogenic component of B. pertussis [50, 55]. Pertussis toxoid (PTd), diphtheria (DTd) and tetanus toxoids (TTd) were combined into a 3-component vaccine (DTaP). The efficacy of this vaccine against pertussis is approximately 71% and it is lower than for tetanus and diphtheria [69]. All symptoms of tetanus and diphtheria diseases are caused exclusively by toxins, whereas pertussis pathogenesis involves multiple virulence factors. Besides the secretory proteins of B. pertussis (e.g. pertussis toxin), surface molecules such as adhesins and endotoxin are involved in pathogenesis of pertussis [18, 37]. Therefore not only toxin-neutralizing activity is required in the immune defense against pertussis, but also bactericidal activity against surface components of B. pertussis which ensures the bacterial killing. Therefore besides toxin-neutralizing activity, the bactericidal activity against surface components of B. pertussis, ensuring the bacterial killing is required in the immune defense against pertussis.
The bactericidal attack against B. pertussis could targeted the highly exposed lipooligosaccharide (LOS) [73]. Actually, antibodies to LOS are found in the sera of patients with bacterial infections [5, 70]. B. pertussis endotoxin is lacking a typical O-antigen and thus it constitutes a lipooligosaccharide. B. pertussis LOS is composed of a lipid A, a core oligosaccharide and a distal trisaccharide which is a single oligosaccharide unit [10]. Among B. pertussis strains there are also strains having LOS devoid of the terminal trisaccharide, which exhibit lower virulence [9, 14]. Similarly to lipopolysaccharides of other Gram-negative bacteria, LOS is also an important factor showing the endotoxic activity [1]. The LOS plays a role in the pathogenesis of pertussis acting in synergy with exotoxins. Thus, LOS together with the tracheal cytotoxin (TCT) and pertussis toxin (PT) causes a destruction of the ciliated cells of the respiratory tract by activation of cytokine-inducible nitric oxide synthase (iNOS) [15, 37]. LOS is lethal in mice sensitized to histamine. It is pyrogenic and mitogenic in spleen cell culture. It activates macrophages and induces the production of TNF-α. Endotoxic activity of lipid A excludes an application of the LOS as a component in pertussis vaccines. However, the removal of the LOS from vaccines reduces their effectiveness, because this component provides adjuvant properties through induction of interleukin-12 (IL-12) and IL-1β that promote the Th1 and Th17 responses, respectively [23, 24, 39].
However, none of the sugar fragments of LOS have been considered as vaccine antigens since they are not immunogenic components. To overcome the problem in the design of vaccines using the LOS, the non-toxic hapten oligosaccharide was conjugated to a carrier protein [27, 28]. Oligosaccharide from B. pertussis LOS in its complete form (OS, core oligosaccharide substituted by a distal trisaccharide) is a branched dodecasaccharide with a unique structure. It is an evolutionarily stable component which has been found in its unchanged form in clinical isolates therefore it makes for a suitable vaccine candidate.
Immunogenic conjugates of B. pertussis OS with filamentous hemagglutinin (FHA) and B. pertussis OS with bovine serum albumin (BSA) have been previously described [32, 33, 34, 35, 81, 84]. Oligosaccharide fragment of B. pertussis LOS, a pentasaccharide component conjugated to tetanus toxoid (OS-TTd) was also immunogenic [47]. All the described conjugates of B. pertussis oligosaccharide with a carrier protein have induced a strong immune response specific for the oligosaccharide. Moreover, the produced antibodies showed the bactericidal activity specific to LOS presented on B. pertussis surface, leading to complement-mediated destruction of the cell [34, 47]. In vivo, the anti-OS bactericidal antibodies eliminate the bacteria from infected individuals [45]. However, the protein carriers employed, that is BSA and TTd, serve as immunogens, but they do not contribute to the pool of antibodies directed against B. pertussis. FHA is an adhesin of B. pertussis and similarly to LOS it is a surface component. Thus, the abovementioned pertussis vaccines do not combine components that could yield the complete immune response providing the clearance of the pathogen from the organism as well as neutralizing its toxic effects.
In the case of pertussis vaccines, a composition inducing an immune response comprising toxin neutralization and bactericidal activities against B. pertussis, has not been devised to date. Pertussis toxin (PT) which is the strongest immunogen of B. pertussis in its inactivated form is an essential component of pertussis vaccine [53, 38, 77]. PT belongs to the family of AB-type bacterial toxins and it consists of protomer A (S1 subunit) providing an enzymatic activity and oligomer B responsible for binding to serum glycoproteins and eukaryotic cells (S2 to S5 subunits) [8, 38, 40, 56, 53, 57, 62, 65, 76]. PT is able to initiate two types of cellular responses: lectin-like effects of oligomer B and ADP-ribosylation, which disrupts a signal transduction involving guanine nucleotide binding proteins (G proteins). The oligomer B is required for binding of the holotoxin to a receptor on the surface of a target cell and enables translocation of the protomer A catalytic domain into the cell. Studies on the structure-function relationship of pertussis toxin indicated the amino acid residues involved in the toxic activity. This research have allowed to obtain the toxoid (PTd) which is genetically inactivated by amino acids alterations in the active site of S1 subunit and/or in the oligomer B residues involved in receptor binding [25, 46]. The ongoing clinical trials of a live attenuated vaccine containing the genetically inactivated PT are promising [44]. However, due to multiple amino acid residues involved in activity of the toxin it is necessary to define the sites and subsequently to use the modification to obtain a completely non-toxic PTd. The amino acid residues which were significant for PT interactions include the following residues: His35 and Glu129 of the S1 subunit and the residue no. 105 in S2 and S3 subunits, that is Asn105 and Lys105, respectively [2, 3, 36, 65]. Minute alterations in the PT sequence lead to detoxification of the protein, but its antigenic properties remain unchanged [51, 83]. It can be presumed, that modification or blocking of the PT sites that interact with the target receptors, would inactivate PT. The patent application US005445817 as of Aug. 28, 1995 described the inactivation of the pertussis toxin by conjugation to the Streptococcus pneumoniae capsular polysaccharide (Pn14-PT) [82]. However, the generated immune response was exclusively specific for the Pn14.
Detoxification of PT during its toxoid formation may introduce various modifications and conformational changes into the protein structure. The PT inactivation methods include a genetic manipulation by substitution of amino acids (Arg9→Lys and Glu129→Gly) in S1 subunit and chemical modifications of the toxin by formaldehyde, glutaraldehyde, tetranitromethane, hydrogen peroxide or a combination of formaldehyde and glutaraldehyde [25, 51, 53]. The effects of the detoxification reaction on PT have not been precisely defined. Physicochemical, immunochemical, spectroscopic and serological analyses of the toxoids have been performed to determine the sites and the effects of modifications caused by the used methods [59, 61, 71, 72, 75, 77]. PT can be detoxified by modifications at different amino acid positions in its A subunit, B oligomer or both. However, research on pertussis toxoid has indicated that there is a possibility of partial reversion of the toxoid to its active form [26, 52]. This residual pertussis toxin in a vaccine preparation can cause the toxic effects on an immunized organism. Enzymatic activity of PT revealed in the preparation could be responsible for the reactogenicity manifested by hypersensitivity to histamine and allergic reactions.
Residual toxicity of a preparation containing pertussis toxoid is monitored to assess its safety and acceptance as a pertussis vaccine. Residual PT in the PTd preparation may be a result of incomplete inactivation of the toxin or its toxicity reversion [26, 52, 79, 80]. In vivo histamine-sensitization test (HIST) is an official safety test for detecting the residual activity of PT in vaccines. However, the HIST test is a lethal test and difficult to standardize. As an alternative to HIST, an in vitro system based on a selective binding of PT to fetuin and subsequent detection with a polyclonal antibody has been developed for the safety of pertussis vaccine [26]. This analysis differentiates between the ability of PT to preferential binding to fetuin compared to the reduced binding of PTd to fetuin [17]. A system to examine both function of PT based on the carbohydrate binding assay in combination with monitoring of the protomer A activity in an enzyme coupled-HPLC (E-HPLC) assay, has also been developed [80]. Enzymatic activity of PT can also be estimated during its interaction with CHO cells as changes in morphology of these cells [7, 22]. The latest proposed alternative relies on an observation of the translocation and internalization of pertussis toxin and toxoids to the target cell based on the direct immunofluorescence labeling using a confocal microscopy [67].
There is a need for a pertussis vaccine containing immunogenic, but completely inactivated components of B. pertussis.
The limited efficacy of currently used pertussis vaccines results from the fact that their components do not induce a complete immune response. As mentioned before, the pertussis vaccine should provide a functional immunity in the form of the toxin neutralization, blocking of bacterial adherence, opsonization, complement activation and bacterial killing [69]. PTd plays an essential role as an antigen for induction of antibodies neutralizing toxic activity of PT. Polyclonal serum against the holotoxin neutralizes its toxic action [29, 54]. However, it was shown that the acellular pertussis vaccine does not have bactericidal activity, which is necessary for immune clearance of the bacteria from respiratory tract [74]. The PTd used in vaccines is a secretory protein, which is loosely associated with the cell, and thus it does not constitute a target for bactericidal antibodies. To overcome this problem, LOS, the main surface component of bacterial cell, was used as the target for bactericidal antibodies that can recognize the surface structure of bacteria and promote killing of bacteria in the presence of complement. It was shown that IgG antibodies against core oligosaccharides of non-capsulated bacteria were protective in humans and induced the complement-dependent bacterial killing [70, 73, 74]. The above-mentioned, anti-pertussis glycoconjugates include OS-tioaminooxylated-BSA conjugate that induced bactericidal antibodies in mice [34] and pentasaccharide-TTd that was immunogenic in rabbits [47]. The pentasaccharide part of the conjugate is a fragment isolated from the LOS of B. pertussis 186 and it comprises a distal trisaccharide, a heptose and an anhydromannose. It has been shown that pentasaccharide-TTd conjugate induced antibodies which were able to bind to B. pertussis in immunofluorescence assays (FACS). Moreover, using STD-NMR techniques it was confirmed that that epitopes which are involved in antigen-antibody recognition are located in the distal trisaccharide and the heptose.
Anti-PT antibodies produced in response to a vaccine containing only PTd as an antigen of B. pertussis, do not kill the bacteria, but they neutralize PT activity. However, the bactericidal activity of antibodies is required for complete clearance of bacteria from the host.
The prior art shows therefore a need for a vaccine comprising an immunogenic components against B. pertussis in the form of fully inactivated.
Surprisingly, the present invention provides a vaccine component that is immunogenic and non-toxic at the same time, including the way it was received.
The present invention is a glycoconjugate comprising the B. pertussis LOS-derived oligosaccharide (OS) or fragment thereof coupled to pertussis toxin (PT) by a covalent bond.
Preferably, the covalent bond is formed by the reductive amination
Preferably, the OS is isolated from the bacterial cell envelope or it is obtained by chemical synthesis.
Preferably, the OS is a core oligosaccharide (an incomplete glycoform, R), a distal trisaccharide or LOS-derived oligosaccharide fragment isolated by specific degradation, especially with periodate oxidation and deamination.
More preferably, the OS is selected from the oligosaccharides of the formula 1, formula 2, formula 3 or formula 4.
Preferably, the OS is a pentasaccharide isolated from B. pertussis LOS or its synthetic equivalent.
More preferably, the pentasaccharide is isolated from B. pertussis 186 LOS by deamination.
In a preferred embodiment, the OS is the pentasaccharide depicted by the formula 5.
Preferably, a content of oligosaccharide or fragment thereof in glycoconjugate with PT is 30-50%.
Another aspect of the invention is a pharmaceutical composition comprising a glycoconjugate according to the invention and a pharmaceutically acceptable carrier.
In another embodiment, a vaccine composition comprises the glycoconjugate according to the invention, a pharmaceutically acceptable carrier and optionally an adjuvant.
Preferably, the vaccine composition induces the production of PT-neutralizing antibodies and antibodies that are bactericidal against B. pertussis.
Another objective of the invention is a vaccine composition comprising glycoconjugate according to the invention, a pharmaceutically acceptable carrier and optionally an adjuvant for the prevention and treatment of diseases caused by B. pertussis.
It is another objective of the invention to provide a method for preparing a glycoconjugate comprising B. pertussis LOS-derived oligosaccharide or its fragment coupled to the pertussis toxin, characterized in that the OS is isolated from bacteria or synthesized and then the OS is conjugated to PT by reaction of reductive amination. Subsequently, the obtained OS-PT glycoconjugate is purified.
Preferably, the method comprises the steps of:
Preferably, the B. pertussis is grown on Stainer-Scholte liquid medium with an addition of (2,6-di-O-methyl)-β-cyclodextrin.
Preferably, the OS is activated with sodium periodate.
Preferably, the reductive amination is carried out at pH 9.0.
Preferably, the reductive amination is carried out in 0.2 M borate buffer.
Preferably, the OS-PT glycoconjugate is purified by chromatography.
Preferably, a content of oligosaccharide (OS) or fragment thereof in glycoconjugate with PT is 30-50%.
Another aspect of the invention is a glycoconjugate prepared using the methods defined above.
The present invention provides an immunogenic and non-toxic conjugate of B. pertussis LOS-derived oligosaccharide with pertussis toxin. This conjugate shows no residual toxicity monitored using the fetuin-binding assay and no interaction in an assay using CHO cells. Pertussis toxin was inactivated by covalent coupling with LOS oligosaccharide. Presumably, in the conjugate, the oligosaccharide blocks PT at the binding sites for fetuin and glycoproteins of eukaryotic cells. The detoxified PT in OS-PT of the present invention retains its antigenic and immunogenic properties. The obtained OS-PT conjugate is a candidate for use in the pertussis vaccine composition as it constitutes a non-toxic and immunogenic combination of the two components of B. pertussis and thus generates optimal anti-pertussis response.
The abovementioned conjugate of the oligosaccharide with the pertussis toxin combines surface and secretory components of B. pertussis. Antibodies generated in response to the conjugate of the surface antigen, that is LOS, and the secreted PT are expected to neutralize the PT toxic effect and to be bactericidal. A vaccine containing the OS-PT should enhance immunity against pertussis and decrease the number of incidences of this disease. The OS-PT conjugate in vaccine prevents a disease by toxin neutralization and clearance of the pathogen. The conjugate may be an additional component of a complex acellular pertussis vaccine consisting of a secretory proteins. A vaccine comprising the OS-PT induces the production of bactericidal antibodies. Thus, it prevents B. pertussis infection and reduces the spread of pertussis among susceptible individuals.
In summary, the present invention demonstrates that B. pertussis oligosaccharide coupled covalently to pertussis toxin forms an immunogenic and non-toxic conjugate, and that the conjugation of an oligosaccharide with PT inactivates the toxin. The OS-PT conjugate is devoid of enzymatic activity of the protomer A and binding properties of oligomer B as it has been demonstrated in the invention using in vitro assays. The complementary features of these two components of the conjugate, that is an oligosaccharide and the pertussis toxin which are important for an effective pertussis vaccine are summarized in the table below. The conjugate is a combination of surface component B. pertussis, which is the LOS and secretory component, which is pertussis toxin (a). Pertussis toxin is the most potent B. pertussis immunogen. When conjugated to an oligosaccharide it induces the strong response directed specifically to this oligosaccharide (b). The pertussis toxin comprises a variety of epitopes recognized by T and B cells (c). On the other hand, an oligosaccharide has a high thermal and chemical stability, and when coupled to a carrier protein it is able to reach the sites of immune cells accumulation that generate a long-lasting and specific immune response to the oligosaccharide (c). The oligosaccharide as a component of the evolutionarily conserved lipooligosaccharide belongs to the “patterns” which are recognized by the innate immune system, whose activation is essential for complete immune response (d). The generated anti-OS B. pertussis antibodies show protective properties against strains used in current vaccines as well as against clinical isolates. Conformational variability of the protein carrier allows for better oligosaccharide exposure (d). Conjugation of the oligosaccharide with pertussis toxin forms a non-toxic and immunogenic conjugate, which induces the immune response and produces antibodies with bactericidal and neutralizing properties (e) (Table 1).
The invention presents an immunogenic and non-toxic conjugate of Bordetella pertussis LOS-derived oligosaccharide and pertussis toxin (OS-PT) intended as a vaccine that protects against infection and diseases caused by B. pertussis. The conjugate is capable of eliciting antibodies to both of its components, oligosaccharide and PT. Thus, it induces a production of type-specific and protective antibodies against B. pertussis. Antibodies generated in response to the conjugate neutralize the toxic effect of PT and have bactericidal activity against B. pertussis. These antibodies promote a bacterial killing involving a complement. Therefore, the OS-PT conjugate induces a protective effect by neutralization of the toxin and clearance of B. pertussis from the host.
The present invention refers to an OS-PT conjugate in which the PT component was rendered non-toxic during the coupling reaction. For the conjugation reaction the pertussis toxin and the LOS oligosaccharide are used in quantities which cause the toxin inactivation. Preferably, a content of an oligosaccharide in the PT-glycoconjugate is 30-50%. Preferably, a content of an oligosaccharide or fragment thereof in glycoconjugate with PT is 49%. Inactivation of PT in OS-PT preparation is monitored in in vitro assays, such as ELISA test with fetuin and an assay using CHO cells. The OS-PT conjugates with the substitution of 30 and 49% were not active in the fetuin-binding test and the CHO cells assay.
The invention also provides methods for the preparation of the immunogenic and non-toxic oligosaccharide-pertussis toxin conjugate. This method allows for obtaining these two components of the conjugate, that is a LOS-derived oligosaccharide and a protein carrier, that is a pertussis toxin, from the culture of B. pertussis, simultaneously. The possible isolation of both antigens from one source accelerates the vaccine preparation. The oligosaccharide of the invention can be obtained by chemical synthesis.
The oligosaccharide-pertussis toxin conjugate according to the invention is obtained by reductive amination. The conjugation reaction is carried out in 0.2 M borate buffer at pH 9.0.
The present invention relates to a method for the preparation of the conjugate of B. pertussis LOS oligosaccharide with the pertussis toxin comprising the steps (Scheme 1):
Following the OS-PT conjugate preparation procedure, the oligosaccharide content in the obtained PT-glycoconjugate is determined. The inability of OS-PT to bind to fetuin and no interaction with CHO cells are tested in in vitro assays.
In one aspect, the vaccine is a non-toxic and immunogenic conjugate of oligosaccharide fragment from B. pertussis LOS with pertussis toxin. B. pertussis LOS oligosaccharide fragment of the present invention may be any OS fragment causing inactivation of the toxin as a result of covalent linking. Oligosaccharide fragment of B. pertussis LOS used for conjugation with pertussis toxin may be selected from the following:
In one aspect of the invention, the vaccine is a non-toxic and immunogenic conjugate of pertussis toxin with a pentasaccharide, in which the pentasaccharide is isolated from B. pertussis 186 LOS by deamination or it is a synthetic equivalent thereof. The pertussis toxin and the pentasaccharide of LOS are used for the conjugation reaction in amounts causing a substitution of the protein that inactivates the toxins. Surprisingly, a single step detoxification effect is achieved without the need for detoxification by chemical or genetic methods. The glycoconjugate of the pentasaccharide-PT shows no binding capacity in an ELISA with fetuin.
The invention provides a formulation of the OS-PT conjugate to use as a vaccine against B. pertussis. This conjugate may constitute an additional component of a pertussis vaccine, besides B. pertussis protein antigens, that acts only by neutralization of toxins. The antibodies produced against components of the vaccine except for LOS do not promote the complement-dependent killing. However, because many virulence factors are involved in the pertussis pathogenesis, including LOS, a direct destructive bactericidal activity is essential. A vaccine containing the OS-PT induces production of bactericidal antibodies and ensures clearance of bacteria from the host. Thus it prevents B. pertussis infection and hampers the disease spread among susceptible individuals.
The term “oligosaccharide” (OS) of the present invention relates to an oligosaccharide or its fragments isolated from B. pertussis lipooligosacccharide (LOS). The oligosaccharide of the invention may also be prepared by chemical synthesis. The B. pertussis oligosaccharide used for conjugation with PT is a core oligosaccharide substituted by a distal trisaccharide (OS, a complete form, RS). The OS is a branched dodecasaccharide having the following structure (FORMULA 1):
In one aspect of the invention, the vaccine is a conjugate of B. pertussis LOS-derived oligosaccharide fragment and pertussis toxin. “The oligosaccharide fragment” of B. pertussis LOS of the present invention is any fragment of the OS causing inactivation of pertussis toxin by a covalent coupling.
B. pertussis LOS oligosaccharide fragment used for conjugation to pertussis toxin can be any fragment selected from the group enumerated below:
In one aspect of the invention, the vaccine is a conjugate of pertussis toxin with the pentasaccharide isolated by deamination of B. pertussis 186 LOS or a synthetic pentasaccharide. The pentasaccharide includes immunodominant epitopes of LOS, which include the terminal trisaccharide and the terminal heptose. The pentasaccharide in the glycoconjugate composition possesses the following structure (FORMULA 5):
On the basis of literature data [4, 13] it can be assumed that this pentasaccharide is a sugar fragment of B. pertussis LOS, which is generated as a result of endosomal processing of LOS by deaminative depolymerization. The deaminated OS may be a fragment of LOS presented to T cells and thus it may induce the production of specific antibodies. The depolymerized LOS may make for an optimal vaccine component.
The coupling of the pentasaccharide to pertussis toxin by reductive amination forms a non-toxic glycoconjugate. Such pentasaccharide-PT glycoconjugate is not capable of binding to fetuin in ELISA test.
The term B. pertussis “lipooligosaccharide” indicates that endotoxin of this bacterium is devoid of a typical O-antigen, which is the O-specific polysaccharide. LOS is not a complex, polymeric molecule, but similarly to lipopolysaccharides (LPS) of other Gram-negative bacteria, it is an essential virulence factor and a protective antigen [1, 73]. In the case of B. pertussis, an oligosaccharide is exposed at the surface of the cell.
The term “conjugate” in this invention means a covalent coupling of a hapten oligosaccharide with a carrier protein, which is pertussis toxin, in order to render the OS more immunogenic. The conjugation is used to obtain a formulation that modulates antibody response to oligosaccharide.
The term “immunogenic” means “inducing immune response” such as a production of antibodies. An immunogenic oligosaccharide-carrier protein conjugate induces the production of antibodies specific for an oligosaccharide. The OS component of LOS applied to the conjugation in the present invention includes a terminal trisaccharide and terminal heptose both coupled to a glucosamine which are epitopes recognized by sera of mice immunized with B. pertussis [5, 47, 34].
The term “pertussis toxin” (PT) refers to an exotoxin secreted by B. pertussis. PT is a protein of molecular weight about 105 kDa and belongs to the AB-type bacterial toxins [8, 38, 40, 56, 57, 58, 62, 65, 76]. It consists of the A subunit having enzymatic activity and the B oligomer exhibiting binding activity to glycoproteins of serum and eukaryotic cells. These two activities of PT are monitored in the interaction test using the CHO cells (the test of A protomer activity) and the fetuin binding assay (the test of B oligomer activity). PT in this invention can be prepared by various isolation methods known in the art [49, 60, 64, 66].
The invention also provides methods for preparation of the immunogenic and non-toxic conjugate of an oligosaccharide and pertussis toxin. The oligosaccharide is obtained by the water-phenol LOS extraction according to the method of Westphal et al, followed by acid hydrolysis of the LOS and subsequent purification processes. PT is isolated by ion exchange chromatography [49, 66].
The OS-PT conjugate is not toxic in both fetuin binding assay and the test using CHO cells. These safety tests are defined as assays for pertussis vaccine. The term “safety tests for pertussis vaccines” means the methods of monitoring of the PT residual activity in the acellular and whole-cell vaccines [79]. The residual PT activity in the vaccines containing PTd may be due to an incomplete inactivation or the reversion to toxicity. There are two methods of establishing the active PT in vaccine preparations: the histamine sensitization test (HIST) and a test using CHO cells. These methods are used to determine the biological activity of the preparation. The safety tests are used to ensure that the PT levels in vaccines are at acceptable level (LOQ, <Limit-of-quantification> for the PT is 8 ng/ml of the vaccine preparation) [26]. An international PT reference material (JNIH-50) is defined as the standard PT preparation (sPT).
The OS-PT conjugate can be obtained using the methods of conjugation known in the art. The term “conjugation methods known in the art” encompasses the methods commonly used in the preparation of conjugates. Direct conjugation reactions, reactions employing a linker, reactions with an initial activation of a protein or an oligosaccharide are the methods commonly used for conjugation [27, 28, 32, 33, 34, 35]. The oligosaccharide-pertussis toxin conjugate described herein is obtained by the reductive amination. The conjugation reaction is carried out in 0.2 M borate buffer at pH 9.0. The term “reductive amination” as used herein means that during the reaction a covalent linkage of oxidized LOS oligosaccharide with toxin is formed, followed by reduction of the created bond. The modified Kdo residue of an oligosaccharide reacts with the free amino group of the protein to form a stable conjugate in the presence of a reducing agent. This conjugation of an oligosaccharide and pertussis toxin results in inactivation of the toxin.
We have demonstrated the inability of the OS-PT glycoconjugate to bind to fetuin and similarly the OS-PT glycoconjugate has no effect in an interaction with the CHO cells. Detoxification of pertussis toxin by attachment of the oligosaccharide is a result of blocking of the protein binding sites. During conjugation, an oligosaccharide can bind to the terminal amino groups of the subunits and the amine group of lysine residues in the B oligomer. The enzymatically active S1 subunit contains no lysine, therefore the oligosaccharide can only be bound to its N-terminus. Coupling of the N-terminal residue of the S1 subunit with an oligosaccharide may impair its active site conformation and consequently abolish its enzymatic activity. However, mass spectrometry analysis of the trypsin-digested OS-PT, has indicated a signal corresponding to the peptide possessing an unmodified N-terminal residue. This suggests that the N-terminus of the S1 subunit is not available for the oligosaccharide and no coupling of the oligomer A with OS has occurred (
The conjugation is carried out in borate buffer at pH 9.0. The reaction conditions are essential for the conjugation of OS with PT because as it was demonstrated that PT is unstable in the pH range 4-8 [82].
The OS-PT conjugate induces the production of antibodies specific for an oligosaccharide. The OS-PT conjugate also induces the production of anti-PT antibodies neutralizing the toxic effects of B. pertussis. The term “neutralizing antibodies” means that the pertussis toxin in the glycoconjugate adds the anti-toxin antibodies to a pool of protective anti-OS antibodies. The neutralizing properties of serum induced by the OS-PT enhance the protective anti-pertussis response.
This conjugate combines a surface component, which is the exposed LOS fragment of B. pertussis, namely OS and a secretory protein of B. pertussis such as PT. The antibodies generated in response to the conjugate of the surface OS and the secretory PT neutralize the toxic effect of PT and are bactericidal. The activity of antibodies has been demonstrated using a CHO cells assay and a bactericidal assay. The anti-OS-PT antibodies inhibited the action of PT on the CHO cells and showed the bactericidal activity in the presence of the rabbit complement. The antibodies were able to recognize the LOS on B. pertussis cells and to promote complement-dependent bacterial killing. The vaccine consisting of the OS-PT should provide immune response that prevents from the disease by toxins neutralization and by clearance of the bacteria from the respiratory tract. Thus, it prevents infection caused by B. pertussis and limits the spread of the disease among susceptible individuals. The conjugate may constitute an additional component of an acellular pertussis vaccine consisting only of secretory proteins, such as PTd and FHA.
The invention also provides a pharmaceutical formulation characterized in that it comprises an immunogenic and non-toxic conjugate of the OS-PT. In one embodiment, the composition may contain adjuvants, stabilizers and solvents which are acceptable in the vaccine formulations.
The term “adjuvant” refers to a substance which enhances the post-vaccinal immune response to the administered antigen. The mechanism of action relies on slowing down the release of an antigen and providing a “danger signal” to stimulate the immune system. An adjuvant suitable for the vaccine formulation belongs to the group consisting of: inorganic salts (aluminum phosphate, calcium phosphate), aluminum hydroxide, ISCOM, liposomes, monophosphoryl lipid A (MPL), muramyl dipeptide.
The invention provides a preparation of an immunogenic and the non-toxic oligosaccharide-PT conjugate for use as a vaccine for protection against infections caused by B. pertussis.
In the present invention, an immunogenic and a non-toxic conjugate of a LOS-derived oligosaccharide from Bordetella pertussis 186 with pertussis toxin was prepared as a vaccine for prevention and treatment of diseases caused by B. pertussis. As it is a potential vaccine antigen the immunochemical, serological and immunological properties thereof have been investigated.
This assignment of PT amino acid residues includes the signal sequences and therefore it differs from the numbering of PT sequence presented in the description of the invention.
The invention was illustrated by the following embodiment.
A. The Culture of B. Pertussis
B. pertussis 186 was grown in Stainer-Scholte liquid medium with an addition of (2,6-di-O-methyl)-β-cyclodextrin. The growth was controlled by biotyping using MALDI-TOF MS and MALDI Biotyper method (MBT). Spectral analysis was performed in positive-ion mode giving the protein spectra and in negative-ion mode to obtain profiles of endotoxin (
B. Isolation of a Pertussis Toxin
The pertussis toxin, used as a component of the glycoconjugate in this invention was prepared by isolation from the culture media of B. pertussis 186 using an ion-exchange chromatography method (Method 1) [49, 66]. The method was modified in its final step, by using a buffer with 50% glycerol content during dialysis. The addition of glycerol proved to be necessary to isolate the PT. Briefly, the method employs the CM-Sepharose CL-6B used as a cation-exchange resin. The separation is achieved through the binding of two proteins from B. pertussis 186 culture, PT and FHA to the resin and their subsequent elution as separate fractions by changing the pH and ionic strength. The isolation of PT was monitored by UV-absorbance measurement (
C. Isolation of B. Pertussis 186 Oligosaccharide
LOS was isolated from B. pertussis 186 by water-phenol extraction according to a method of Westphal et al. Then, an oligosaccharide (OS) was isolated from the LOS by acid hydrolysis (1.5% acetic acid). The OS was purified by removing a lipid A during ultracentrifugation (105000×g, 2 h, 4° C.). MALDI-TOF mass spectrometry was used to confirm that the structures of the isolated LOS (
D. Analysis of B. Pertussis 186 LOS and OS by MALDI-TOF Mass Spectrometry
The obtained preparations of B. pertussis 186 bacteria and the isolated LOS and OS were analyzed by MALDI-TOF MS (
The signals in the mass range of m/z 2200-2600 correspond to the core oligosaccharide of the B. pertussis LPS (
MALDI-TOF MS spectrum of B. pertussis oligosaccharide isolated and purified on Dowex H+ resin was recorded in positive-ion mode. A molecular ion at m/z 2312.4 corresponded to the dodecasaccharide (
In the spectrum of the oxidized OS, a mass shift of 30 Da towards the lower mass range was observed in comparison with the signal of the OS (
In addition, in MALDI-TOF MS spectrum of the oxidized OS, the ion at m/z 1337.79 was observed (
Periodate oxidation of B. pertussis 186 OS leads to a heterogeneous mixture of oligosaccharides differing in the number of sugar residues.
The activated OS was used for conjugation with pertussis toxin.
E. Preparation of OS-PT Conjugate
The oxidized oligosaccharide of B. pertussis 186 was used for conjugation with pertussis toxin by reductive amination (Method 2). The oxidized oligosaccharide (20 mg) and PT (1 mg) were used for conjugation. The reaction was performed in a borate buffer at pH 9.0.
The OS-PT conjugate was purified by gel filtration (G3000-SW,
The OS-PT conjugates, with (1) a protein concentration of 0.37 mg/ml (volume 1.2 ml) and 49% content of oligosaccharide and (2) a protein concentration of 1 mg/ml (volume 0.5 ml) and 30% content of oligosaccharide, were prepared. In the chromatogram, the first fraction with retention time of 8.3 minutes represents the conjugate with maximal content of the OS (49%), while the second fraction with retention time of 17 min corresponds to PT substituted to a lesser extent by OS, approximately 30%. Neither of the obtained glycoconjugates, differing in oligosaccharide content, showed enzymatic activity of the pertussis toxin or retained its binding properties in in vitro test. The glycoconjugate fractions were concentrated and stored at 4° C. with the addition of the preservative (0.01% merthiolate). The OS-PT conjugate with 49% content of the OS and the protein concentration of 0.37 mg/ml was used for immunization of rabbits.
F. Analysis of Biological Activities of PT
Fetuin Binding Assay
In the studies of PT, two mechanisms of its biological activity are demonstrated, S1 subunit-dependent activity and the B oligomer-dependent activity. Toxicity of the OS-PT conjugate was examined by reaction with a specific receptor for the toxin that is fetuin (Method 3) [22]. The analysis was performed using the ELISA and an antibody which detects PT associated with fetuin (
G. CHO Cells Assay
The enzymatic activity of PT is observed during an interaction of PT with the CHO cells as a morphological response of the cells [7, 22]. PT induces the CHO cell clustering effect that is inhibited by the presence of anti-PT antibodies used at the neutralizing concentration.
Toxicity analysis of OS-PT relies on the treatment of the CHO cells with the OS-PT preparations (Method 4,
In the present invention, we demonstrate that by combining PT and the OS, we have prepared a conjugate which showed no enzymatic activity of the toxin. In the CHO cell assay, which is the most sensitive test, the OS-PT was at least 106 times less toxic than PT. Toxicity was not observed even at the highest concentrations. The lack of enzymatic activity of the OS-PT also suggests that this preparation does not induce hypersensitivity effect to histamine as a result of reversion of PTd toxicity. It is also important that the enzymatic inactivation of the toxoids correlates with the lack of pathological disorders typical for PT such as leukocytosis, stimulation of sensitivity to histamine, anaphylaxis, hyperinsulinemia [46].
H. Pertussis Toxin Neutralization Test
We have demonstrated that antibodies obtained by immunization of rabbits with B. pertussis 186 LOS-derived oligosaccharide-pertussis toxin conjugate neutralize toxicity of PT in pertussis toxin neutralization test (Method 4,
I. Production of Protective Anti-OS-PT Antibodies
Immunogenicity of the OS-PT conjugate was investigated by immunization of rabbits followed by the determination of antibody levels in the produced immune sera (Method 5). The anti-glycoconjugate antibody titers were determined using ELISA with B. pertussis 186 LOS as a solid phase antigen. The anti-OS-PT antibodies reacted with the LOS (
We have also investigated the level of anti-OS-PT antibodies which are protective against B. pertussis in the bactericidal assay (Method 6). The bactericidal titers of the sera corresponding to the maximum dilution of each serum at which 50% of bacterial colonies were killed, was 800-fold and 400-fold (sera no. 3271 and 3239), respectively.
J. MALDI-TOF Mass Spectrometry Analysis of PT and OS-PT Preparations
To define the OS-PT conjugate, MALDI-TOF mass spectrometry analysis was performed (Method 7). We observed that the signals in the spectra of the OS-PT and PT differed. The identified signals in the MALDI-TOF MS of PT correspond to the molecular weights of the subunits: S4 subunit (m/z 12054.84), S3 (m/z 21864.47), S1 (m/z 26220.90), S5 (m/z 11754.57) (
MALDI-TOF MS spectrum of the OS-PT did not reveal signals corresponding to the PT subunits which were identified in the spectrum of PT. The signal at m/z 33550 may correspond to the S3-S4 dimer (
K. Monitoring of the Oxidation of an Oligosaccharide Using NMR
To define the glycoconjugate of B. pertussis 186 oligosaccharide and pertussis toxin, the process of the OS oxidation in the presence of 0.01 M sodium periodate was analyzed using NMR (
The terminal N-acetylglucosamine has been identified on the basis of the typical chemical shift values for the N-acetyl group in the NMR spectra of an oligosaccharide which was not treated with periodate (δH 1.99 ppm and δc 22.8:
This terminal N-acetylglucosamine is a part of a trisaccharide which is considered as the most immunodominant epitope of B. pertussis LOS [47]. The glycoconjugate containing oxidized oligosaccharide devoid of the terminal GlcNAc may not provide a complete immune response. Another fragment of B. pertussis 186 LOS, namely a pentasaccharide obtained by deamination. As the deamination preserves the immunodominant epitopes of the LOS, the pentasaccharide seems to be an optimal sugar fragment of B. pertussis LOS. The B. pertussis 186 LOS-derived pentasaccharide-pertussis toxin conjugate is one aspect of the present invention.
L. MALDI-TOF MS and NMR Spectroscopy Analyses of the Pentasaccharide
The pentasaccharide isolated from B. pertussis 186 LOS was analyzed using NMR and MALDI-TOF MS techniques. The structure of the isolated fragment was determined by two-dimensional NMR spectra (COSY, HSQC, HMBC,
Methods
B. pertussis strain 186 was obtained from the Laboratory of Infection Prevention and Nosocomial Infections of the National Institute of Public Health (Warsaw, Poland). Chinese hamster ovary cells (CHO) were purchased from the German Collection of Microorganisms and Cell Cultures (Germany). The Standard for PT (JNIH-5) was obtained from the National Institute for Biological Standards and Control (NIBSC, UK).
Method 1. Isolation of Pertussis Toxin
Isolation of the pertussis toxin was carried out according to the method using ion-exchange chromatography on CM-Sepharose [49, 66]. To a culture medium of B. pertussis 186 separated from the bacterial cells by centrifugation, solid ammonium sulfate (390 g/l) was added to precipitate the proteins. Following the incubation for 16 h at 4° C. the preparation was centrifuged (29000×g, 30′, 4° C.) the supernatant was discarded and the precipitate was dissolved in 50 mM phosphate buffer at pH 8.0 containing 1 M NaCl. The extract was then centrifuged (17300×g, 30′, 4° C.) and the supernatant was saved. The extraction on the precipitate was repeated three times. The extracts were pooled and dialyzed against 50 mM phosphate buffer containing 2 M urea (buffer A) at pH 8.0. After 16-hours dialysis, the pH of the dialysate was adjusted to pH 6.0 with 1 M H3PO4 and applied to a CM-Sepharose CL-6B (1 cm×16 cm) equilibrated with buffer A at pH 6.0. The isolation process was monitored by measuring the UV absorbance (chromatogram,
Method 2. Synthesis and Purification of the Oligosaccharide-Containing Neoglycoconjugate
The oligosaccharide was dissolved in 0.01 M aqueous sodium periodate solution (NaIO4). The reaction was carried out in the dark at 24° C. for 1 h. In parallel, the control was prepared, which was 0.01 M solution of NaIO4. After one-hour incubation, 10 μl of sample and control was added to 2 ml of water and the extinction at a wavelength of 225 nm was measured. The oxidation reaction was stopped by addition of ethylene glycol (30 μl) and it was left at 24° C. for an additional 1 hour. The oxidized oligosaccharide was purified by HPLC using molecular sieve chromatography (G3000-PW, TSK). The purified oligosaccharide in its oxidized form was freeze-dried. Subsequently, it was dissolved in 1.5 ml of 0.2 M borate buffer at pH 9.0 and added to the carrier protein. After one-hour incubation at 37° C., 15 mg of sodium cyanoborohydride (NaBCNH3) and a drop of chloroform to prevent bacterial growth were added to the solution. The reaction was carried out for 14 days at 37° C. On the 5th and 10th day after the start of the reaction, additional portions of NaBCNH3 (10 mg) were added. The reaction mixture was fractionated on a G3000-SW column equilibrated with phosphate-buffered saline (PBS, pH 7.5). In the collected fractions, the protein concentration was determined by measuring absorbance at a wavelength of 280 nm. The presence of oligosaccharide was determined by dot-blot reaction using the anti-endotoxin serum obtained by immunization with a bacterial mass of B. pertussis 186 and with B. pertussis 186 LOS-derived pentasaccharide-tetanus toxoid conjugate. A content of the oligosaccharide in the conjugate was determined by the phenol-sulfuric acid method. Fractions containing the conjugate were concentrated by ultrafiltration and stored at 4° C. with an addition of merthiolate (0.01%).
Method 3. ELISA Test with a Fetuin as a Solid Phase Antigen
Interactions of PT and the OS-PT with fetuin were investigated according to the method described by Isbrucker et al [26]. 96-well plates (Nunc) were coated with fetuin solution (5 μg/ml, 100 μl) in 0.05 M carbonate buffer at pH 9.6 and incubated overnight. The plates were washed five times with TBS (0.1 M) containing 0.1% Tween-20 and blocked with 3% BSA (100 μl) in TBS, for 1 h at 37° C. Following the washing steps, 100 μl of serial dilutions of the OS-PT and PT were added and incubated for 1 h. After washing, the plates were incubated with 100 μl of anti-pertussis toxin serum (Abcam) 10000-fold diluted. The bound anti-toxin antibodies were detected using goat anti-rabbit IgG conjugated with alkaline phosphatase (BioRad) 10000-fold diluted for 90′ at 37° C. The free antibodies were washed with TBS and subsequently, a color was developed by addition of the p-nitrophenylphosphate (1.5 mg/ml) in carbonate buffer at pH 9.6 with 1 mM MgCl2. The reaction was stopped after 100 minutes by addition of 100 μl of 1M NaOH. Absorbance was measured at λ=405 nm using a Perkin Elmer Mikroplate Reader.
Method 4. Toxicity Assay Using CHO Cells
The clustering of the CHO cells in the presence of PT and the OS-PT was determined according to the method described by Hewlett et al. [18]. In brief, the CHO cells were incubated for 24 h with different concentration of the OS-PT (31 pg/ml to 130 μg/ml range) and PT (0.06 pg/ml to 0.9 μg/ml range). Interaction of the preparations with the CHO cells was observed. The CHO cell were cultured in MEMα medium containing 10% fetal bovine serum. The cells cluster-formation was monitored using Axio Vert. A1 microscope (Zeiss).
Pertussis Toxin Neutralization Test
The level of antibodies neutralizing the pertussis toxin activity was investigated in the PT neutralization assay [16, 46]. Two-fold dilutions of the sera (25 μl) were mixed with 25 μl of toxin (120 pg). Following a 3-hours incubation at 37° C., the CHO cells (1×104) were added and incubated at 37° C. in a 5% CO2 atmosphere. As a positive control, sPT was applied to the CHO culture without serum. The CHO cells alone and the CHO cells in the presence of sera served as a negative controls. The neutralizing titer was defined as the highest serum dilution resulting in a complete inhibition of the clustering effect induced by the native toxin.
Method 5. Immunization of OS-PT Conjugate
Two Termond White rabbits were immunized subcutaneously in the neck with the OS-PT antigen. The vaccine composition comprised the OS-PT (50 μg) with MPL (0.4 mg) as an adjuvant. Immunizations with the neoglycoconjugate were performed according to the following schedule: (i) the first dose—day 0, (ii) the second dose—day 21, (iii) the third dose—day 42. The vaccination was carried out according to the procedures approved by the Local Ethics Commission at IITD PAN in the Directive 54/2009. After completion of the vaccination, the level of anti-neoglycoconjugate antibodies in the sera was determined.
Method 6. Serum Bactericidal Assay
The rabbit sera were inactivated at 56° C. for 30 min and diluted 10-fold and 2-fold in PBS with 0.15 mM CaCl2, 0.5 mM MgCl2 and 0.1% BSA (buffer A, pH 7.4). B. pertussis 186 was diluted to ˜300-500 bacteria in 25 μl of buffer A. 140 μl of buffer A was mixed with 45 μl of serum, 25 μl of bacterial suspension and 15 μl of complement (a rabbit complement, Biomed, Lublin). The mixture of bacteria in buffer A with complement and without serum was used as a control. Following an incubation for 60 min at 37° C., 100 μl of each mixture was plated onto charcoal agar containing a sheep blood and incubated for 4 days. The colonies were then counted. The bactericidal titer of serum was defined as the highest dilution at which the 50% of bacterial colonies were killed [30, 39]. The lowest titer giving the desired bactericidal effect was observed for the dilutions of 800-fold and 400-fold.
Method 7. MALDI-TOF MS Analysis
MALDI-TOF MS analyses were performed on Autoflex III and Ultraflex instruments (Bruker Daltonics). Spectra were acquired in negative and positive ion modes. Preparations of PT, the conjugates and the tryptic digest fragments were mixed with appropriate matrices such as: synapinic acid (SA), dihydroxyacetophenone (DHPA), dihydroxybenzoic acid (DHB), a mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid (sDHB) and α-cyano-4-hydroxycinnamic acid (α-HCCA). The samples were analyzed without desalting or were desalted using C4 resin (Ziptip, Millipore, eluents: ACN/0.1% TFA with increasing content of ACN: 50, 80, 95%) or were dialyzed against 0.1% TFA (Microcon, 10 kDa MWCO). Peptide fragments were separated using a C18 column (Dionex) employing a gradient of H2O/0.1% TFA and ACN/0.1% TFA.
The digested preparations were characterized using a trypsin (Sequencing Grade Modified Trypsin, Porcine, Promega) according to the manufacturer's instruction. Briefly, 20 μg of protein was dissolved in the reaction mixture containing 2 M DTT, 0.1% SDS, and filled up with Tris-HCl buffer (pH 8.0) to the volume of 0.1 ml. The mixture was heated at 95° C. for 15 minutes. After cooling, a trypsin was added to achieve a trypsin:protein ratio of 1:20 (w/w) and the mixture was incubated at 37° C. for 24 hours. The reaction was terminated by freezing the sample. The digestion products were analyzed directly by MALDI-TOF MS and using the reverse-phase chromatography (C18, Dionex) interfaced to a MALDI-TOF MS mass spectrometry (Ultraflex). The preparations were dissolved in an initial solvent A: 0.1% TFA in water. Subsequently, a linear gradient at a flow rate of 300 nl/min was applied starting with 2% of solvent B: 0.1% TFA in ACN to 100% of solvent B. The separated peptides were analyzed by MALDI-TOF MS and were subjected to the laser-induced fragmentation (LID). Identification of the peptide sequences was performed with Biotools software and a Mascot database.
Method 8. Isolation of the Pentasaccharide from B. pertussis 186 LOS
The pentasaccharide was obtained by deamination of B. pertussis 186 lipooligosaccharide (Scheme 3). 50 mg LOS was suspended in a solution of water/5% sodium nitrite/30% acetic acid (1:1:1, v/v/v) and stirred for 4 hours at room temperature. The products of LOS deamination were separated by ultracentrifugation (2 h, 200000×g, 4° C.).
The supernatant was freeze-dried and the product was purified on a column of Bio-Gel P-2 (Bio-Rad) in a pyridine/acetic acid/water buffer, pH 5.6. The yield of the pentasaccharide isolation was ˜4 mg.
The precipitated fraction of the LOS obtained by deamination contains a heterogeneous mixture of core oligosaccharides differing in the number of sugar residues (Scheme 4), which were identified by MALDI-TOF MS (
Method 9. NMR Spectroscopy
NMR spectra of the isolated pentasaccharide were obtained for 10% 2H2O solution with a Bruker Avance III 600 MHz spectrometer (Bruker). Spectra were acquired at 30° C. and the WATERGATE pulse sequence was applied.
| Number | Date | Country | Kind |
|---|---|---|---|
| 404247 | Jun 2013 | PL | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2014/061944 | 6/4/2014 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2014/195881 | 12/11/2014 | WO | A |
| Number | Date | Country |
|---|---|---|
| 0471954 | Feb 1992 | EP |
| 9404195 | Mar 1994 | WO |
| 2011110531 | Sep 2011 | WO |
| 2012106251 | Aug 2012 | WO |
| Entry |
|---|
| Melvin et al. Nat Rev Microbiol. Apr. 2014; 1294:274-288. |
| Kubler-Kielb Joanna et al: “Oligosacchande conjugates of Bordetella pertussis and bronchiseptica induce bactericidal antibodies, an addition to pertussis vaccine.”, Proceedings of the National Academy of Sciences of The United States of America Mar. 8, 2011, vol. 108, No. 10, Mar. 8, 2011(Mar. 8, 2011), pp. 4087-4092. |
| Nieoziela Tomasz et al: “Epitope of the vaccine-type Bordetella pertussis strain 186 lipooligosaccharide and antiendotoxin activity of antibodies directed against the terminal pentasaccharide-tetanus toxoid conjugate.”, Infection and Immunity Nov. 2005, vol. 73, No. 11. Nov. 2005 (200541)1 pages 7381-7389. |
| Caroff M et al: “Structure of the Bordetella pertussis 1414 endotoxin”, FEBS Letters 20000714 NL, vol. 477, No. 1-2, Jul. 14, 2000(Jul. 14, 2000), pp. 8-14. |
| Robbins John B et al: “Toward a new vaccine for pertussis.”, Proceedings of the National Academy of Sciences of The United States of America Mar. 4, 2014, vol. 111, No. 9, Mar. 4, 2014 (Mar. 4, 2014), pp. 3213-3216. |
| Anderson P et al: “Immunogens Consisting of Oligosacchardes From the Capsule of Haemophilus-Influenzae Type B Coupled to Diphtheria Toxoid or the Toxin Protein CRM-197”, Journal of Clinical Investigation, vol. 76, No. 1, 1985, pp. 52-59. |
| Number | Date | Country | |
|---|---|---|---|
| 20160114051 A1 | Apr 2016 | US |
