JP4771948B2 - Immunogenic composition for Streptococcus agalactiae - Google Patents

Description

  This application claims the benefit of US Provisional Patent Application 60 / 548,789, filed February 26, 2004, which is incorporated by reference in its entirety. Claiming priority from International Patent Application No. PCT / US03 / 29167 filed on May 15th and Attorney Reference No. PP19766.002, the entirety of this PCT / US03 / 29167 is incorporated herein by reference. The

(Field of Invention)
The present invention relates to immunogenic antigens derived from Streptococcus agalactiae (“GBS”) and uses that immunogenic antigen in combination with other GBS antigens to provide wider coverage among different GBS strains. Related to that. In particular, the present invention relates to a composition comprising a combination of two or more GBS antigens, wherein the combination comprises GBS80 or a fragment thereof. The combination can include GBS 80 and at least one other GBS antigen. For example, the combination can include GBS 80 and up to 13 GBS antigens. In preferred embodiments, the combination may include GBS 80 and up to 10 GBS antigens. In a more preferred embodiment, the combination may comprise GBS 80 and up to 5 GBS antigens. In one embodiment, the combination is 2-13 selected from the group of antigens consisting of GBS80, GBS91, GBS104, GBS184, GBS276, GBS305, GBS322, GBS330, GBS338, GBS361, GBS404, GBS690 and GBS691. It can consist of GBS antigen. Preferably, the combination includes GBS 80 in combination with one or more of GBS 104 and GBS 322.

(Background of the Invention)
GBS has been the leading cause of neonatal sepsis and meningitis affecting 0.5-3 per 1000 live babies in the last 20 years, and older age affecting 5-8 per 100,000 population It has emerged as an important cause of morbidity among the population. Current disease management strategies rely on parturition antibiotics and neonatal monitoring, which reduces neonatal case mortality from more than 50% in the 1970s to less than 10% in the 1990s. Nevertheless, there is still considerable morbidity and mortality, and its management is expensive. 15-35% of pregnant women are asymptomatic carriers and are at high risk of transmitting the disease to their infants. The risk of neonatal infection is associated with low serotype-specific maternal antibodies, with high titers being protective. In addition, invasive GBS disease is increasingly recognized in older people with an underlying disease (eg, diabetes and cancer).

  “B” in “GBS” refers to the Lancefield classification, which is based on the antigenicity of carbohydrates that are soluble in dilute acid and called C carbohydrates. Lancefield has identified 13 C carbohydrates and named them A through O, which can be serologically distinguished. The organisms that most commonly infect humans are found in groups A, B, D and G. Within group B, the strains are divided into at least 9 serotypes (Ia, Ib, Ia / c, II, III, IV, V, VI, VII and VIII) based on their polysaccharide capsule structure. Can be. In the past, serotypes Ia, Ib, II and III were uniformly prevalent in normal vaginal migration and early onset sepsis in newborns. Type V GBS has emerged as an important cause of US GBS infections, however, type VI and type VIII strains are prevalent among Japanese women.

  The genomic sequence of serotype V strain 2603 V / R has been published (Ref. 1), and various polypeptides have been identified for use as vaccine antigens (Ref. 2). However, currently in clinical trials, vaccines are based on polysaccharide antigens. Since they suffer from serotype specificity and poor immunogenicity, There is a need for an effective vaccine against agalactiae infection.

  It is an object of the present invention to provide further improved compositions for providing immunity against GBS disease and / or GBS infection. The composition is based on a combination of two or more (eg, three or more) GBS antigens.

(Summary of the Invention)
Applicants have found that immunogenic GBS antigens, in particular GBS 80, are particularly suitable for immunization purposes, especially when used in combination with other GBS antigens. The combination may include GBS 80 and at least one other GBS antigen or up to 13 other GBS antigens. In preferred embodiments, the combination may include GBS 80 and up to 10 GBS antigens. In a more preferred embodiment, the combination comprises GBS 80 and up to 5 GBS antigens. In particular, the present invention relates to a composition comprising a combination of two or more GBS antigens, wherein the combination comprises GBS80 or a fragment thereof. In one embodiment, the combination comprises 2 to 13 GBS antigens selected from the group consisting of GBS80, GBS91, GBS104, GBS184, GBS276, GBS305, GBS322, GBS330, GBS338, GBS361, GBS404, GBS690 and GBS691. It can consist of Preferably, the combination consists of GBS 80, GBS 104 and GBS 322.

  Instead of a full-length antigen, the combination can include an immunogenic fragment of the selected GBS antigen and / or a polypeptide sequence having sequence identity to the selected antigen.

  Preferably, the combination of GBS antigens consists of 3, 4, 5, 6, 7, 8, 9 or 10 GBS antigens. Even more preferably, the combination of GBS antigens consists of 3, 4 or 5 GBS antigens.

(Detailed description of the invention)
The practice of the present invention uses conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology that are within the skill of the art unless otherwise stated. Such techniques are explained fully in the literature. For example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. 19th edition (1995); Methods In Enzymology (S. Colowick and N. Kaplan, ed., Academic Press, Inc.); and Handbook of Experimental Immunology, Volumes I to IV e. Blackwell, ed., 1986, Blackwell Scientific Publications); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Handbook of Surface, CR. Short Protocols in Molec lar Biology, 4th edition (Ausubel et al., Ed., 1999, John Wiley &Sons); Molecular Biology Technologies: An Intensive Laboratory Course, (Ream et al., Ed., 1998, Acre; 2nd edition (Newton & Graham ed., 1997, Springer Verlag); Peters and Dalymplle, Fields Virology (2nd edition), Fields et al. N. Raven Press, New York, NY. checking ...

  All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

(GBS antigen)
As discussed above, the present invention provides an immunogenic composition comprising a combination of two or more GBS antigens, wherein the combination comprises GBS80 or a fragment thereof.

  A combination of GBS antigens can comprise a polypeptide fragment of the identified GBS antigen. The length of the fragment can vary depending on the amino acid sequence of the particular GBS antigen, but the fragment is preferably at least 7 contiguous amino acids (eg, 8, 10, 12, 14). 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more) is there. Preferably, the fragment comprises one or more epitopes derived from the above sequences. Other preferred fragments are: (1) the N-terminal signal peptide of each identified GBS antigen, (2) the identified GBS antigen without the N-terminal signal peptide, and (3) each identified GBS antigen (Up to 10 amino acid residues (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or And the like) are deleted from the N-terminus and / or C-terminus (eg, the N-terminal amino acid residue may be deleted)). Other fragments have removed one or more domains of the protein (eg, removal of a signal peptide, cytoplasmic domain, transmembrane domain, or extracellular domain).

  A combination of GBS antigens can comprise a polypeptide sequence having sequence identity to the identified GBS antigen. The degree of sequence identity may vary depending on the amino acid sequence in question (a), but is preferably greater than 50% (eg, 60%, 65%, 70%, 75%, 80% 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. 99.5% or more). Polypeptides having sequence identity include homologs, orthologs, allelic variants and functional variants of the GBS antigen identified above. Typically, greater than 50% identity between two proteins is considered an indicator of functional equivalence. The identity between proteins is preferably Smith-Waterman as performed in the MPSRCH program (Oxford Molecular) using a gap search of similarity with parameters such as gap opening penalty = 12 and gap extension penalty = 1. Determined by homology search algorithm.

  The polypeptide is of course in various forms (eg, native, fusion, glycosylated, non-glycosylated, etc.) by various means (eg, recombinant expression, purification from GBS, chemical synthesis, etc.). Can be prepared. They are preferably prepared in a substantially pure form (ie substantially free of other streptococcal proteins or host cell proteins) or in a substantially isolated form.

(GBS80)
As discussed above, the present invention relates to the use of GBS 80 in a synergistic combination with other GBS antigens. GBS80 refers to a putative cell wall surface anchor family protein. The nucleotide and amino acid sequences of GBS80 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 8779 and SEQ ID NO: 8780. These sequences are also referred to as SEQ ID NO: 1 and SEQ ID NO: 2.

として上に示す。

As shown above.

  As mentioned above, the combination of the present invention may comprise a fragment of GBS antigen. In some cases, removal of one or more domains (eg, leader sequence region or signal sequence region, transmembrane region, cytoplasmic region or cell wall anchor motif) may result in antigen and / or recombinant expression of the GBS protein described above. Cloning of the encoding gene can be facilitated. In addition, fragments comprising immunogenic epitopes of the above listed GBS antigens can be used in the compositions of the invention.

  GBS 80 contains an N-terminal leader sequence region or an N-terminal signal sequence region indicated by the underlined sequence at the beginning of SEQ ID NO: 2 above. In one embodiment, one or more amino acids from the leader sequence region or signal sequence region of GBS80 are removed. An example of such a GBS80 fragment is SEQ ID NO: 3

として上に示す。

As shown above.

  GBS 80 contains a C-terminal transmembrane region indicated by the underlined sequence near the end of SEQ ID NO: 2 above. In one embodiment, one or more amino acids from the transmembrane region and / or cytoplasmic region are removed. An example of such a GBS80 fragment is SEQ ID NO: 4

として上に示す。

As shown above.

  GBS80 contains an amino acid motif indicating a cell wall anchor: SEQ ID NO: 5 I.PNTG (shown in italics in SEQ ID NO: 2 above). In some recombinant host cell systems, it may be preferred to remove this motif to facilitate secretion of recombinant GBS80 protein from that host cell. Thus, in one preferred fragment of GBS80 for use in the present invention, the transmembrane and / or cytoplasmic region and the cell wall anchor motif are removed from GBS80. An example of such a GBS80 fragment is SEQ ID NO: 6

として上に示す。

As shown above.

  Alternatively, in some recombinant host cell systems, it may be preferred to use the cell wall anchor motif described above to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein can be cleaved during purification, or the recombinant protein can remain attached to either the inactivated host cell or the cell membrane in the final composition. possible.

  In one embodiment, the leader or signal sequence region, the transmembrane and cytoplasmic regions, and the cell wall anchor motif are removed from the GBS80 sequence. An example of such a GBS80 fragment is SEQ ID NO: 7

として上に示す。

As shown above.

  Applicants have identified fragments that are particularly immunogenic of the GBS80 protein described above. This immunogenic fragment is located towards the N-terminus of the protein, which is underlined in the sequence of SEQ ID NO: 2 of GBS80 above as follows. The underlined fragment is SEQ ID NO: 8

として上に示す。

As shown above.

  The immunogenicity of the protein encoded by SEQ ID NO: 7 was determined by PBS, GBS whole cells, GBS80 (full length) and another fragment of GBS80 located closer to the C-terminus of the GBS80 peptide (SEQ ID NO: 9)

に対して比較した。

Compared against.

  Both an active maternal and child immunoassay (Active Material Immunization Assay) and a passive mother and child immunoassay (Passive Material Immunization Assay) were performed on this protein set.

  As used herein, active maternal immunoassay refers to an in vivo protection assay in which female mice are immunized with the test antigen composition described above. The female mice are then born and challenged with a lethal dose of GBS. Serum titers of female mice during the above immunization schedule as well as pup survival after challenge are measured.

  Specifically, the active maternal immunoassay referred to herein used a group of 4 CD-1 female mice (Charles River Laboratories, Calco Italy). These mice were immunized intraperitoneally with selected proteins in Freund's adjuvant on days 1, 21, and 35 before birth. 6-8 week old mice receive 20 μg protein per dose when immunized with a single antigen and 30-45 μg per dose when immunized with a combination of antigens Protein (15 μg of antigen each) was given. The mother's immune response was monitored by using serum samples obtained on days 0 and 49. The female mice gave birth to pups 2-7 days after the last immunization (approximately t = 36-37) and typically had a gestation period of 21 days. Within 48 hours of birth, a dose approximately equal to the amount sufficient to kill 70-90% of unimmunized pups (as determined by empirical data based on experiments collected from the PBS control group) The pups were challenged intraperitoneally with GBS. The dose of GBS challenge is preferably administered in 50 μl of THB medium. Preferably, the pup is challenged 56-61 days after the first immunization. The challenge inoculum was prepared starting from a frozen culture diluted to the appropriate concentration with THB before use. Offspring survival was monitored for 5 days after challenge.

  As used herein, a passive maternal immunoassay is an in vivo in which pregnant mice are passively immunized by injecting rabbit immune serum (or control serum) approximately 2 days before delivery. Refers to a protection assay in The pups are then challenged with a lethal dose of GBS.

  Specifically, the passive maternal immunoassay referred to herein includes a pregnant immunized passively immunized by intraperitoneal injection of 1 ml rabbit immune serum or control serum 2 days prior to birth. A group of CD1 mice was used. Neonatal mice (24-48 hours after birth) are challenged intraperitoneally with a 70% -90% lethal dose of GBS serotype III COH1. Challenge doses obtained by diluting frozen mid-log cultures were administered in 50 μl of THB medium.

  For both assays, the number of live GBS-infected pups was evaluated every 12 hours for 4 days. Statistical significance was assessed by Fisher's exact test.

  The results of each assay for immunization using whole cells of SEQ ID NO: 7, SEQ ID NO: 8, PBS and GBS are shown in Tables 1 and 2 below.

表１および表２に示すように、上記の配列番号７のＧＢＳ８０フラグメントを用いる免疫処置は、上記のチャレンジした仔について、比較対象である配列番号８のＧＢＳ８０フラグメントより実質的に改良された生存率を提供した。これらの結果は、上記の配列番号７のＧＢＳ８０フラグメントが、ＧＢＳ８０の重要な免疫原性エピトープを含み得ることを示す。

As shown in Tables 1 and 2, immunization with the above-described GBS80 fragment of SEQ ID NO: 7 resulted in a substantially improved survival rate for the challenged pups over the comparable GBS80 fragment of SEQ ID NO: 8. Provided. These results indicate that the GBS80 fragment of SEQ ID NO: 7 above can contain important immunogenic epitopes of GBS80.

(Combination including GBS80)
The invention includes a combination of two or more GBS antigens, wherein the combination includes GBS 80 or a fragment thereof. Applicants have discovered that GBS80 is particularly suitable for immunization in combination with other GBS antigens, and that combinations of these antigens provide broader coverage among different GBS strains. did.

  Preferably, the combination of GBS antigens consists of 3, 4, 5, 6, 7, 8, 9 or 10 GBS antigens. Even more preferably, the GBS antigen combination consists of 3, 4 or 5 GBS antigens.

Preferably, the combinations of the present invention provide an immunogenicity that is improved over the immunogenicity of the antigens described above when administered alone. Improved immunogenicity can be measured, for example, by active maternal immunoassay. As discussed above, this assay can be used to measure the serum titer of female mice during the immunization schedule as well as the survival time of pups after challenge. Preferably, immunization with an immunogenic composition of the invention results in the challenged pups as compared to the percent survival from maternal immunization with a single antigen of the composition when administered alone. Result in an increase of at least 2 percentage points (preferably at least 3 percentage points , 4 percentage points or 5 percentage points ). Preferably, the increase is at least 6 percentage points , 7 percentage points , 8 percentage points , 9 percentage points , 10 percentage points , 11 percentage points , 12 percentage points , 13 percentage points , 14 percentage points , 15 percentage points , 16 percentage points . Points , 17 percentage points , 18 percentage points , 19 percentage points , 20 percentage points , 21 percentage points , 22 percentage points , 23 percentage points , 24 percentage points , 25 percentage points , 26 percentage points , 27 percentage points , 28 percentage points , 29 percentage points or 30 percentage points . Preferably, the combination of GBS of the invention comprising GBS80 exhibits an increase in percent survival as compared to the percent survival from immunization with an antigen that is not GBS80 alone.

  According to one embodiment of the present invention, an antigen or combination of fusion proteins comprising part or several parts of the above antigens is combined with 1 to 10 antigens, preferably 1 to 10 or less antigens GBS 80 or a part thereof. Such other antigens include, as non-limiting examples, GBS67, GBS91, GBS104, GBS184, GBS276, GBS305, GBS322, GBS330, GBS338, GBS361, GBS404, GBS690 and GBS691. Still other antigens have been identified in US Application No. 10 / 415,182, filed April 28, 2003, which is hereby incorporated by reference in its entirety.

  For example, the combinations include GBS80, GBS104, GBS322 and GBS276; GBS80, GBS338, GBS330; GBS80, GBS330, GBS104; GBS80, GBS104, GBS404; GBS80, GBS338, GBS104; GBS80, GBS338, GBS404; GBS338, GBS104, GBS404; GBS80, GBS330, GBS404; GBS80, GBS322, GBS104; GBS80, GBS322, GBS276; GBS80, GBS322, GBS91; GBS80, GBS104, GBS276; GBS80, GSB104, BS91; GBS80, GBS91; GBS32 GBS80, GBS322, GBS276; GBS80, GBS322, GBS91; GBS80, GBS104, GBS276; GBS80, GBS104, GBS91; GBS80, GBS276, GBS91; GBS80, GBS690, GBS691; GBS80, BS30, GB3 GBS80, GBS691, GBS305; GBS80, GBS338, GBS305; GBS80, GBS338, GBS361; GBS80, GBS305, GBS361; GBS80, GBS184, GBS691; GBS80, GBS691, GBS338; GBS80, GBS104, BSBS267G And GBS322 mention may be made. Examples of combinations of the present invention that exhibit improved immunogenicity are shown below. A more detailed description of the above GBS antigen referred to in these experiments is given in the examples below.

Example 1: Active mother-child immunoassay of GBS80 combination against GBS80 alone
In this example, the active maternal immunoassay described above was used to determine the percent survival of offspring challenged with GBS type III serotype (COH1 isolate) at t = 56 days. The mother mice were either GBS80 alone, a combination of GBS antigens (GBS antigen with GBS80 and GBS antigen without GBS80), placebo (PBS) or inactivated GBS isolate as shown in Table 3 below. Were immunized according to the active maternal immunoassay scheme discussed above. In these experiments, the challenge dose for COH1 isolate, a type III GBS sufficient to kill 70-90% of unimmunized pups, is approximately equal to 10 × LD50%, where LD50% Are statistically derived half-lethal doses).

表３に示すように、ＧＢＳ８０を含むＧＢＳ抗原の組み合わせは、上記の抗原単独の使用よりも改良された免疫原性を示した。例えば、ＧＢＳ８０単独での免疫処置によって、上記のチャレンジした仔の間で７０％の生存率を得た。ＧＢＳ３３８、ＧＢＳ３３０、ＧＢＳ１０４、およびＧＢＳ４０４と、ＧＢＳ８０との組み合わせを用いる免疫処置によって、上記のチャレンジした仔の間で９５〜１００％の生存率を得た。これは、２５〜３０パーセントポイントの増加である。

As shown in Table 3, the combination of GBS antigens including GBS80 showed improved immunogenicity over the use of the above antigen alone. For example, immunization with GBS80 alone gave 70% survival among the challenged pups. Immunization with a combination of GBS338, GBS330, GBS104, and GBS404 and GBS80 gave a survival rate of 95-100% among the challenged pups. This is an increase of 25-30 percentage points .

In comparison, these antigen combinations that did not contain GBS80 failed to achieve the% survival of GBS80 alone. For example, immunization with GBS338, GBS104, and GBS404 resulted in 65% survival. Replacing any one of these antigens with GBS80 dramatically increased the percent survival to between 97% and 100%. This is an increase of 32-35 percentage points . (See percent survival rates for GBS80, GBS338, GBS101 (97%); percent survival for GBS80, GBS338, GBS404 (100%) and percent survival for GBS80, GBS104, GBS404 (100%)). Immunization with GBS338, GBS330 and GBS104 resulted in 73% survival. Replacing any one of these antigens with GBS80 increased the percent survival to between 95% and 97%.

  These findings indicate that protection from COH1 isolates is increased by the use of GBS 80 in combination with other GBS antigens.

(Example 2: Active mother-child immunoassay of GBS80, GBS322, GBS276, GBS104 single pair combination)
In this example, the active maternal immunoassay described above was used to determine the percent survival of offspring challenged with a type III serotype GBS (COH1 isolate) at t = 56 days. The mother mice were immunized according to the active maternal immunoassay scheme discussed above using a single GBS antigen, a combination of GBS80 and GBS antigen, and placebo (PBS) as shown in Table 4 below. did.

表４に示すように、ＧＢＳ８０と上記の抗原の組み合わせによって、その抗原単独の使用よりも改良された免疫原性を得た。例えば、ＧＢＳ３２２単独での免疫処置によって、上記のチャレンジした仔の間で１７％の生存率を得た。ＧＢＳ３２２と、ＧＢＳ８０および別のＧＢＳ抗原との組み合わせを用いる免疫処置によって、９２〜１００％の生存率を得た。別の例において、ＧＢＳ１０４単独での免疫処置によって、７４％の生存率を得た。ＧＢＳ１０４と、ＧＢＳ８０および別のＧＢＳ抗原との組み合わせを用いる免疫処置によって、９０〜１００％の生存率を得た。別の例として、ＧＢＳ２７６単独での免疫処置によって、３５％の生存率を得た。ＧＢＳ２７６と、ＧＢＳ８０および別のＧＢＳ抗原との組み合わせを用いる免疫処置によって、８２〜９７％の生存率を得た。

As shown in Table 4, the combination of GBS 80 and the above antigens resulted in improved immunogenicity over the use of that antigen alone. For example, immunization with GBS322 alone gave 17% survival among the challenged pups. 92-100% survival was obtained by immunization using a combination of GBS322 and GBS80 and another GBS antigen. In another example, immunization with GBS104 alone gave 74% survival. 90-100% survival was obtained by immunization with a combination of GBS104 and GBS80 and another GBS antigen. As another example, immunization with GBS276 alone yielded a survival rate of 35%. Immunization with a combination of GBS276 and GBS80 and another GBS antigen resulted in 82-97% survival.

  Given the immunogenicity of the combination, the duration of immune response in the mouse model was further analyzed. The mother mice used in the above active mother-to-child immunoassay were mated again and at t = 91 days, at a dramatically higher dose (300 × LD50%), different GBS serotypes (type V, CJB111 isolates) ) Was used to challenge. The parameters of this second stronger challenge were outside the parameters of the standard active maternal immunoassay described above. This was to examine the limitations of immune memory resulting from the immunization of the original mother in the mouse model described above. The signs of immune memory in this model under these conditions are considered significant. As shown in Table 5, even under these extreme conditions, increased survival rates were generally achieved, especially for the combination comprising GBS80 and GBS322 and GBS104. It was surprising to note that the percent survival for GBS80 and the combination of GBS233 and GBS104 was 100% for both the first and second challenges described above.

（実施例３：ＧＢＳ８０と、ＧＢＳ６９０、ＧＢＳ６９１、ＧＢＳ３３８、ＧＢＳ３０５、ＧＢＳ３６１およびＧＢＳ１８４との組み合わせの能動母子免疫アッセイ）
この実施例において、ＧＢＳ抗原のさらなる組み合わせを、再びＧＢＳＩＩＩ型 ＣＯＨ１単離体のチャレンジを用いて上記の能動母子免疫アッセイにおいて使用した。上記の母マウスを、以下の表６に示すＧＢＳ抗原の組み合わせを用いて、上記の能動母子免疫アッセイ計画に従って免疫した。

(Example 3: Active mother-child immunoassay combining GBS80 with GBS690, GBS691, GBS338, GBS305, GBS361, and GBS184)
In this example, additional combinations of GBS antigens were used in the active maternal immunoassay described above, again using the GBS type III COH1 isolate challenge. The mother mice were immunized using the GBS antigen combinations shown in Table 6 below according to the active mother-infant immunoassay scheme described above.

このモデルにおける母マウスをまた、再び交配させて、ｔ＝８４日にて、劇的に高い用量（１００×ＬＤ５０％）で、同じＧＢＳ単離体を生じた仔を用いてチャレンジした。上記の実施例のように、この２度目のより強いチャレンジのパラメーターは、上記の標準的な能動母子免疫アッセイのパラメーターの外側にあった。これは、上記のマウスモデルにおける元の母親の免疫から生じる免疫記憶の限界を調べるようになっていた。表７に示すように、これらの極端な条件下でさえ、生存率のいくつかは、７０％のままであるか、または７０％以上のままであった。驚いたことに、ＧＢＳ８０と、ＧＢＳ１８４とＧＢＳ６９１との組み合わせについての生存率パーセントは、実際に増加した。

Mother mice in this model were also mated again and challenged with pups that gave rise to the same GBS isolate at a dramatically higher dose (100 × LD50%) at t = 84 days. As in the example above, the parameters of this second stronger challenge were outside the parameters of the standard active maternal immunoassay described above. This was to examine the limitations of immune memory resulting from the immunity of the original mother in the mouse model described above. As shown in Table 7, even under these extreme conditions, some of the survival rates remained at 70% or remained above 70%. Surprisingly, the percent survival for the combination of GBS80, GBS184 and GBS691 actually increased.

（実施例４：ＧＢＳ８０と、ＧＢＳ６９０、ＧＢＳ６９１、ＧＢＳ３３８、ＧＢＳ３０５およびＧＢＳ３６１との組み合わせを使用する能動母子免疫アッセイ）
この実施例において、ＧＢＳ抗原のさらなる組み合わせを、今回は、ＧＢＳＶ型のＣＪＢ１１１単離体のチャレンジを用いて、上記の能動母子免疫アッセイにおいて使用した。これらの実験において、免疫処置していない仔の７０〜９０％を殺すのに十分なＧＢＳ Ｖ型のＣＪＢ１１１単離体についてのチャレンジ用量は、６０×ＬＤ５０％とほぼ等しい（ここで、ＬＤ５０％は、統計的に導かれた半数致死量である）。上記の母マウスを、以下の表８に示すＧＢＳ抗原の組み合わせを用いて、上記の能動母子免疫アッセイ計画に従って免疫した。表８に示すように、この特定のＶ型単離体を用いるこの特定のチャレンジ研究において、上記の組み合わせすべてについての生存率は、少なくとも７０％に達した。

Example 4: Active maternal immunoassay using a combination of GBS80 and GBS690, GBS691, GBS338, GBS305 and GBS361
In this example, a further combination of GBS antigens was used in the active maternal immunoassay described above, this time using a GBSV type CJB111 isolate challenge. In these experiments, the challenge dose for GBS V type CJB111 isolate sufficient to kill 70-90% of unimmunized offspring is approximately equal to 60 × LD50% (where LD50% is A statistically derived half-lethal dose). The mother mice were immunized with the GBS antigen combinations shown in Table 8 below, according to the active maternal immunoassay scheme described above. As shown in Table 8, in this particular challenge study using this particular type V isolate, the survival rate for all of the above combinations reached at least 70%.

このモデルにおける母マウスをまた、２度目に交配させ、生じた仔を、ｔ＝８４日にて、劇的に高い用量（６００×ＬＤ５０％）で、上記の同じＧＢＳ単離体を用いてチャレンジした。上記の実施例のように、この２度目のより強いチャレンジのパラメーターは、上記の標準的な能動母子免疫アッセイのものの外側にあった。これは、上記のマウスモデルにおける元の母親の免疫処置から生じる免疫記憶の限界を調べるようになっていた。表９に示すように、これらの極端な条件下でさえ、生存率のいくつかは、７０％より高いままであった。驚いたことに、上記抗原群のうちの２つ（ＧＢＳ８０、ＧＢＳ６９０およびＧＢＳ３３８）ならびに（ＧＢＳ８０、ＧＢＳ６９１およびＧＢＳ３３８）についての生存パーセントは、実際に増加した。

Mother mice in this model were also bred a second time and the resulting pups challenged with the same GBS isolate described above at t = 84 days at a dramatically higher dose (600 × LD50%). did. As in the example above, the parameters of this second stronger challenge were outside that of the standard active maternal immunoassay described above. This was to examine the limitations of immune memory resulting from the immunization of the original mother in the mouse model described above. As shown in Table 9, some of the survival rates remained higher than 70% even under these extreme conditions. Surprisingly, the percent survival for two of the antigen groups (GBS80, GBS690 and GBS338) and (GBS80, GBS691 and GBS338) actually increased.

（実施例５：ＧＢＳ８０と、ＧＢＳ１０４、ＧＢＳ２７６およびＧＢＳ３２２との組み合わせを使用する能動母子免疫アッセイ）
この実施例において、ＧＢＳ抗原のさらなる組み合わせを、今回は異なるＧＢＳ菌株の単離体のチャレンジを用いて、上記の能動母子免疫アッセイにおいて使用した。これらの実験において、異なるＧＢＳ菌株についてのチャレンジ用量は、免疫処置していない仔の６０〜９０％を殺すのに十分であり、１０×ＬＤ５０％に等しい（ここで、ＬＤ５０％は、統計的に導かれた半数致死量である）。上記の母マウスを、以下の表１０に示すＧＢＳ菌株における、８０抗原とＧＢＳ１０４抗原、ＧＢＳ２７６抗原およびＧＢＳ３２２抗原とＧＢＳの組み合わせを用いて、上記の能動母子免疫アッセイ計画に従って免疫した。生存％を、２個の異なるアジュバント（ミョウバンおよびフロイント）とともに上記のＧＢＳの組み合わせを用いて測定した。表１０および表１１に示すように、この特定のチャレンジ研究において、上記のＧＢＳ菌株の全てにおける組み合わせについての生存率が、９６％にまで達した。

Example 5: Active maternal immunoassay using a combination of GBS80 and GBS104, GBS276 and GBS322
In this example, further combinations of GBS antigens were used in the active maternal immunoassay described above, this time using different GBS strain isolate challenges. In these experiments, the challenge dose for the different GBS strains is sufficient to kill 60-90% of unimmunized offspring and is equal to 10 × LD50% (where LD50% is statistically It is the half-lethal dose that was led). The above mother mice were immunized according to the above active mother-to-child immunoassay scheme using a combination of 80 antigen and GBS104 antigen, GBS276 antigen and GBS322 antigen and GBS in the GBS strains shown in Table 10 below. % Survival was measured using the above GBS combination with two different adjuvants (alum and Freund). As shown in Tables 10 and 11, in this particular challenge study, the survival rate for combinations in all of the above GBS strains reached 96%.

従って、その結果、本発明は、２種以上のＧＢＳ抗原の組み合わせを含む組成物を包含し、ここで、その組み合わせは、ＧＢＳ８０またはそのフラグメントあるいはそれらに対する配列同一性を有するポリペプチド配列を含む。

Thus, as a result, the present invention encompasses a composition comprising a combination of two or more GBS antigens, wherein the combination comprises GBS 80 or a fragment thereof or a polypeptide sequence having sequence identity thereto.

  In one embodiment, the combination can consist of 2 to 13 GBS antigens, including GBS80. By way of example, the combination may include GBS 80 and other GBS antigens selected from the group consisting of GBS 80, GBS 91, GBS 104, GBS 184, GBS 276, GBS 305, GBS 322, GBS 330, GBS 338, GBS 361, GBS 404, GBS 690 and GBS 691. . Preferably, the combination includes GBS 80 in combination with one or more of GBS 104 and GBS 322. For example, the combination may include GBS 80, GBS 104, GBS 322, and GBS 67.

  Instead of a full-length antigen, the combination can include an immunogenic fragment of the selected GBS antigen and / or a polypeptide sequence having sequence identity to the selected antigen.

  Preferably, the combination of GBS antigens consists of 3, 4, 5, 6, 7, 8, 9 or 10 GBS antigens. Even more preferably, the combination of GBS antigens consists of 3, 4 or 5 GBS antigens.

  Details of examples of GBS antigens for use in combination with GBS 80 are given below.

(GBS91)
GBS91 refers to GBS C3-binding polypeptide. The nucleotide and amino acid sequences of GBS91 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 8937 and SEQ ID NO: 8938. These sequences are referred to as SEQ ID NO: 10 and SEQ ID NO: 11.

として上に示す。

As shown above.

  GBS 91 contains an N-terminal leader sequence region or an N-terminal signal sequence region indicated by the underlined sequence at the beginning of SEQ ID NO: 11 above. In one embodiment, one or more amino acids from the leader sequence region or signal sequence region of GBS91 are removed. An example of such a GBS91 fragment is SEQ ID NO: 12

として上に示す。

As shown above.

  GBS 91 contains a C-terminal transmembrane region that can be located in the underlined region near the end of SEQ ID NO: 11 above. In one embodiment, one or more amino acids from the transmembrane and cytoplasmic regions are removed. An example of such a GBS91 fragment is SEQ ID NO: 13

として上に示す。

As shown above.

  GBS91 contains an amino acid motif indicating a cell wall anchor: SEQ ID NO: 14 LTKTG (shown in italics in SEQ ID NO: 11 above). In one embodiment, both the transmembrane region and the cell wall anchor motif are removed from GBS91. An example of such a GBS91 fragment is SEQ ID NO: 15

として上に示す。

As shown above.

  In one embodiment, one or more amino acids from the leader sequence region or signal sequence region and one or more amino acids from the transmembrane region and cytoplasmic region are removed from the sequence of GBS91. An example of such a GBS91 fragment is SEQ ID NO: 16

として上に示す。

As shown above.

  In another embodiment, the leader or signal sequence region, the transmembrane and cytoplasmic regions, and the cell wall anchor motif are all removed from the GBS91 sequence. An example of such a GBS91 fragment is SEQ ID NO: 17

として上に示す。

As shown above.

  Further information on GBS91 can be found in WO01 / 25440 (C3 binding polypeptide), WO01 / 32882 (ID-65), WO02 / 31156 (BVH) and Reinscheid et al., Microbiology (2002) 148: 3245-3254 (bsp gene). Each of which is incorporated herein by reference in its entirety.

(GBS104)
GBS104 refers to a putative cell wall surface anchor family protein. It is called emaA protein. The nucleotide and amino acid sequences of GBS104 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 8777 and SEQ ID NO: 8778. These sequences are referred to as SEQ ID NOs: 18 and 19

として上に示す。

As shown above.

  GBS 104 contains an N-terminal leader sequence region or an N-terminal signal sequence region indicated by the underlined sequence at the beginning of SEQ ID NO: 19 above. In one embodiment, one or more amino acid sequences from the leader sequence region or signal sequence region of GBS 104 are removed. An example of such a GBS104 fragment is SEQ ID NO: 20

として上に示す。

As shown above.

  GBS 104 contains a C-terminal transmembrane region and / or cytoplasmic region indicated by the underlined region near the end of SEQ ID NO: 19 above. In one embodiment, one or more amino acids from the transmembrane or cytoplasmic region are removed. An example of such a GBS104 fragment is SEQ ID NO: 21

として上に示す。

As shown above.

  In one embodiment, one or more amino acids from the leader sequence region or signal sequence region as well as one or more amino acids from the transmembrane region or cytoplasmic region are removed. An example of such a GBS104 fragment is SEQ ID NO: 22

として上に示す。

As shown above.

  In other embodiments, the further fragment of GBS 104 is a 830 amino acid fragment of amino acids 28-858 of GBS 104, a 359 amino acid fragment of amino acids 28-387 of GBS 104, a 581 amino acid fragment of amino acids 28-609 of GBS 104, or amino acid 28 of GBS 104. It is provided to contain ˜768 740 amino acid fragments.

(GBS184)
GBS184 refers to a putative lipoprotein. The nucleotide and amino acid sequences of GBS184 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 1977 and SEQ ID NO: 1978. These sequences are also referred to as SEQ ID NOS: 23 and 24.

として上に示す。

As shown above.

  GBS 184 contains an N-terminal leader sequence region or an N-terminal signal sequence region indicated by the underlined sequence at the beginning of SEQ ID NO: 24 above. In one embodiment, one or more amino acids from the leader sequence or signal sequence described above are removed from GBS 184. An example of such a GBS 184 fragment is SEQ ID NO: 25

として上に示す。

As shown above.

(GBS276)
GBS276 refers to C5a peptidase. The nucleotide and amino acid sequences of GBS276 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 8941 and SEQ ID NO: 8942. These sequences are referred to as SEQ ID NOS: 26 and 27.

として上に示す。

As shown above.

  GBS 276 contains an N-terminal leader sequence region or an N-terminal signal sequence region indicated by the underlined sequence at the beginning of SEQ ID NO: 27 above. In one embodiment, one or more amino acids from the leader sequence region or signal sequence region of GBS 276 are removed. An example of such a GBS276 fragment is given in SEQ ID NO: 28

として上に示す。

As shown above.

  GBS 276 contains a C-terminal transmembrane region and / or a C-terminal cytoplasmic region indicated by the underlined sequence near the end of SEQ ID NO: 27 above. In one embodiment, one or more amino acids from the transmembrane or cytoplasmic region of GBS 276 are removed. An example of such a GBS276 fragment is given in SEQ ID NO: 29

として上に示す。

As shown above.

  In one embodiment, one or more amino acids from the leader sequence region or signal sequence region of GBS 276 and one or more amino acids from its transmembrane region or cytoplasmic region are removed. An example of such a GBS276 fragment is SEQ ID NO: 30

として上に示す。

As shown above.

  GBS276 Further details of, the following references: Qi Chen et al., "Immunization with C5a Peptidase or Peptidase-Type III Polysaccharide conjugate Vaccines Enhances Clearance of Group B Streptococci from Lungs of Infected Mice", Infection and Immunity (2002) 70 (11 ): 6409-6415; Beckmann et al., “Identification of Novel Adhesions from Group B Streptococci by Use of Page Display Reveals That C5a Peptidase Mediates Fibrinctin Binding ”Infection and Immunity (2002) 70 (6): 2869-2876; Cheng et al.,“ The Group B Streptococcal C5a Peptidoc s ” 2408-2413; and Cheng et al., “Antibody Against Surface-Bound C5a Peptidase Is Oponic and Initiates Macrophage Killing of Group B Streptococcus. "Infection and Immunity (2001) 69 (4): can be found in 2302-2308.

(GBS305)
GBS305 refers to UDP-N-acetylmuramoylalanine-D-glutamate ligase, which is also referred to as Mur D. The nucleotide and amino acid sequences of GBS305 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 207 and SEQ ID NO: 208. These sequences are referred to as SEQ ID NO: 31 and SEQ ID NO: 32.

として上に示す。

As shown above.

  GBS 305 contains an N-terminal leader sequence region or an N-terminal signal sequence region indicated by the underlined sequence at the beginning of SEQ ID NO: 32 above. In one embodiment, one or more amino acids from the leader sequence region or signal sequence region are removed from GBS 305. An example of such a GBS305 fragment is given in SEQ ID NO: 33

として上に示す。

As shown above.

  GBS 305 contains a C-terminal transmembrane or cytoplasmic region indicated by the underlined sequence near the end of SEQ ID NO: 32 above. In one embodiment, one or more amino acids from the transmembrane or cytoplasmic region are removed from GBS 305. An example of such a GBS305 fragment is SEQ ID NO: 34.

として上に示す。

As shown above.

  In one embodiment, one or more amino acids from the leader sequence region or signal sequence region and one or more amino acids from the transmembrane region or cytoplasmic region are removed from GBS 305. An example of such a GBS305 fragment is SEQ ID NO: 35

として上に示す。

As shown above.

(GBS322)
GBS322 refers to a surface immunogenic protein and is also referred to as “sip”. The nucleotide and amino acid sequences of GBS322 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 8539 and SEQ ID NO: 8540. These sequences are referred to as SEQ ID NO: 36 and SEQ ID NO: 37.

として上に示す。

As shown above.

  GBS 322 contains an N-terminal leader or signal sequence region indicated by the underlined sequence near the beginning of SEQ ID NO: 37. In one embodiment, one or more amino acids from the leader sequence region or signal sequence region of GBS322 are removed. An example of such a GBS322 fragment is given in SEQ ID NO: 38

として上に示す。

As shown above.

(GBS330)
GBS330 refers to pyruvate kinase and is also called “pyk”. The nucleotide and amino acid sequences of GBS330 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 8791 and SEQ ID NO: 8792. These sequences are referred to as SEQ ID NO: 39 and SEQ ID NO: 40.

として上に示す。

As shown above.

(GBS338)
GBS338 refers to Sat D protein. The nucleotide and amino acid sequences of GBS338 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 8637 and SEQ ID NO: 8638. These sequences are referred to as SEQ ID NOS: 41 and 42.

として上に示す。

As shown above.

  GSB 338 may contain an N-terminal leader sequence region or an N-terminal signal sequence region indicated by the underlined sequence at the beginning of SEQ ID NO: 42 above. In one embodiment, one or more amino acids from the leader sequence region or signal sequence region are removed from GBS 338. An example of such a GBS338 fragment is given in SEQ ID NO: 43

として上に示す。

As shown above.

(GBS361)
GBS361 refers to cylI protein. The nucleotide and amino acid sequences of GBS361 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 8769 and SEQ ID NO: 8770. These sequences are referred to as SEQ ID NOs: 44 and 45.

として上に示す。

As shown above.

  GBS 361 may contain an N-terminal leader sequence region or an N-terminal signal sequence region indicated by the underlined sequence at the beginning of SEQ ID NO: 45 above. In one embodiment, one or more amino acids from the leader sequence region or signal sequence region are removed from GBS 361. An example of such a GBS361 fragment is given in SEQ ID NO: 46

として上に示す。

As shown above.

(GBS404)
The nucleotide and amino acid sequences of GBS404 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 8799 and SEQ ID NO: 8800. These sequences are referred to as SEQ ID NOs: 47 and 48.

として上に示す。

As shown above.

(GBS690)
The nucleotide and amino acid sequences of GBS690 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 9965 and SEQ ID NO: 9966. These sequences are referred to as SEQ ID NOs: 49 and 50.

として上に示す。

As shown above.

  GBS 690 contains an N-terminal leader sequence region or N-terminal signal sequence region indicated by the underlined sequence at the beginning of SEQ ID NO: 50 above. In one embodiment, one or more amino acids from the leader sequence region or signal sequence region of GBS 690 are removed. An example of such a GBS690 fragment is given in SEQ ID NO: 51

として上に示す。

As shown above.

(GBS691)
GBS691 refers to an iron compound ABC transporter or a substrate binding protein. The nucleotide and amino acid sequences of GBS691 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 3691 and SEQ ID NO: 3692. These sequences are referred to as SEQ ID NOs: 52 and 53.

として上に示す。

As shown above.

  GBS 691 contains an N-terminal leader sequence region or an N-terminal signal sequence region indicated by the underlined sequence at the beginning of SEQ ID NO: 53 above. In one embodiment, one or more amino acids are removed from the leader sequence region or signal sequence region of GBS691. An example of such a GBS691 fragment is given in SEQ ID NO: 54

として上に示す。

As shown above.

  GBS 691 contains a C-terminal transmembrane or cytoplasmic region indicated by the underlined sequence at the end of SEQ ID NO: 53 above. In one embodiment, one or more amino acids are removed from the transmembrane or cytoplasmic region of GBS691. An example of such a GBS691 fragment is given in SEQ ID NO: 55

として上に示す。

As shown above.

  In one embodiment, one or more amino acids from the leader sequence region or signal sequence region and one or more amino acids from the transmembrane region or cytoplasmic region are removed from GBS 691. One example of such a GBS691 fragment is SEQ ID NO: 56.

として上に示す。

As shown above.

  Additional examples of GBS antigens that can be used in combination with GBS80 are shown below.

(GBS4)
GBS4 refers to another putative cell wall surface anchor family protein. The nucleotide and amino acid sequences of GBS4 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 1 and SEQ ID NO: 2. These sequences are also referred to as SEQ ID NOs: 57 and 58.

として上に示す。

As shown above.

  GBS4 contains an N-terminal leader sequence or N-terminal signal sequence underlined at the beginning of SEQ ID NO: 58 above. In one embodiment, one or more amino acids from the N-terminal leader domain or signal peptide domain of GBS4 are removed. An example of such a GBS4 fragment is SEQ ID NO: 59

として上に示す。

As shown above.

  An additional N-terminal portion of GBS4 can be removed to facilitate recombinant expression. An example of such a GBS4 fragment is SEQ ID NO: 60

として上に示す。

As shown above.

  GBS4 contains a C-terminal transmembrane region underlined at the end of SEQ ID NO: 58 above. In one embodiment, one or more amino acids from the C-terminal transmembrane region are removed. An example of such a GBS4 fragment is SEQ ID NO: 61

として上に示す。

As shown above.

  In one embodiment, both the N-terminal leader domain or N-terminal signal domain and the C-terminal transmembrane domain are removed from the GBS4 sequence. An example of such a GBS4 fragment is SEQ ID NO: 62

として上に示す。

As shown above.

  In yet another embodiment, the N-terminal leader domain or N-terminal signal domain, as well as the N-terminal region and C-terminal transmembrane domain, are removed from the sequence of GBS4. An example of such a GBS4 fragment is SEQ ID NO: 63

として上に示す。

As shown above.

(GBS22)
GBS22 refers to a putative adhesion lipoprotein. The nucleotide and amino acid sequences of GBS22 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 8583 and SEQ ID NO: 8584. These sequences are referred to as SEQ ID NOs: 64 and 65.

として上に示す。

As shown above.

(GBS85)
GBS85 refers to putative cell division protein (DivIB). The nucleotide and amino acid sequences of GBS85 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 215 and SEQ ID NO: 216. These sequences are referred to as SEQ ID NOs: 66 and 67.

として上に示す。

As shown above.

(GBS147)
GBS147 refers to a putative protease. The nucleotide and amino acid sequences of GBS147 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 8525 and SEQ ID NO: 8526. These sequences are referred to as SEQ ID NOs: 68 and 69.

として上に示す。

As shown above.

  GBS 147 contains an N-terminal leader sequence region or an N-terminal signal sequence region indicated by the underlined sequence at the beginning of SEQ ID NO: 69 above. In one embodiment, one or more amino acids from the leader sequence region or signal sequence region of GBS147 are removed. An example of such a GBS147 fragment is given in SEQ ID NO: 70

として上に示す。

As shown above.

  GBS 147 also contains a C-terminal transmembrane region and / or cytoplasmic region that may be located within the underlined sequence near the end of SEQ ID NO: 69 above. In one embodiment, one or more amino acids from the transmembrane and / or cytoplasmic region are removed. An example of such a GBS147 fragment is given in SEQ ID NO: 71

として上に示す。

As shown above.

  In one embodiment, one or more amino acids from the leader sequence region or signal sequence region, and one or more amino acids from the transmembrane region or cytoplasmic region are removed from the sequence of GBS147. . An example of such a GBS147 fragment is given in SEQ ID NO: 72

として上に示す。

As shown above.

(GBS173)
GBS173 refers to an amidase family protein. The nucleotide and amino acid sequences of GBS173 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 8787 and SEQ ID NO: 8788. These sequences are referred to as SEQ ID NOs: 73 and 74.

として上に示す。

As shown above.

  GBS 173 contains an N-terminal leader sequence region or an N-terminal signal sequence region indicated by the underlined sequence at the beginning of SEQ ID NO: 74 above. In one embodiment, one or more amino acids from the leader or signal sequence of GBS 173 are removed. An example of such a GBS173 fragment is SEQ ID NO: 75.

として上に示す。

As shown above.

  GBS 173 may also contain a C-terminal transmembrane region and / or cytoplasmic region that may be located within the underlined sequence near the end of SEQ ID NO: 74 above. In one embodiment, one or more amino acids from the transmembrane or cytoplasmic region of GBS 173 are removed. An example of such a GBS173 fragment is given in SEQ ID NO: 76

として上に示す。

As shown above.

  In one embodiment, one or more amino acids from the leader sequence region or signal sequence region described above and one or more amino acids from the transmembrane region or cytoplasmic region are removed. An example of such a GBS173 fragment is given in SEQ ID NO: 77

として上に示す。

As shown above.

(GBS313)
The nucleotide and amino acid sequences of GBS313 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 4089 and SEQ ID NO: 4090. These sequences are referred to as SEQ ID NOs: 78 and 79.

として上に示す。

As shown above.

(GBS328)
GBS 328 belongs to the 5'-nucleotidase family. The nucleotide and amino acid sequences of GBS328 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 6015 and SEQ ID NO: 6016. These sequences are referred to as SEQ ID NOs: 80 and 81.

として上に示す。

As shown above.

  GBS 328 may contain an N-terminal leader sequence region or an N-terminal signal sequence region indicated by the underlined sequence at the beginning of SEQ ID NO: 81 above. In one embodiment, one or more amino acids from the leader sequence region or signal sequence region of GBS 328 are removed. An example of such a GBS328 fragment is shown in SEQ ID NO: 82.

として上に示す。

As shown above.

  GBS 328 may also contain a transmembrane region and / or a cytoplasmic region. In one embodiment, one or more amino acids from the transmembrane region and / or cytoplasmic region of GBS 328 are removed. An example of such a GBS328 fragment is given in SEQ ID NO: 83

として上に示す。

As shown above.

  In one embodiment, one or more amino acids from the leader sequence region or signal sequence region described above, and one or more amino acids from the transmembrane or cytoplasmic region of GBS328 are removed. An example of such a GBS328 fragment is given in SEQ ID NO: 84

として上に示す。

As shown above.

(GBS656)
GBS656 refers to a putative DNA entry nuclease. The nucleotide and amino acid sequences of GBS656 sequenced from serotype V isolated strain 2603 V / R are shown in Ref. 2 as SEQ ID NO: 9323 and SEQ ID NO: 9324. These sequences are referred to as SEQ ID NOs: 85 and 85.

として上に示す。

As shown above.

(GBS67)
In the following, examples of preferred GBS67 fragments are presented. The nucleotide and amino acid sequences of GBS67 sequence from serotype V isolated strain 2603 are shown in SEQ ID NOs: 87 and 88, respectively.

として上に示す。

As shown above.

  GBS67 contains a C-terminal transmembrane region indicated by the underlined region near the C-terminus of SEQ ID NO: 88 above. In one embodiment, one or more amino acids from the transmembrane region are removed and / or the amino acids are cleaved before the transmembrane region. An example of such a GBS67 fragment is shown below as SEQ ID NO: 89.

ＧＢＳ６７は、細胞壁アンカーを示すアミノ酸モチーフ（ＬＰＸＴＧモチーフ）：配列番号９０ ＩＰＭＴＧを含む（上記の配列番号８８にイタリック体で示した）。いくつかの組換え宿主細胞系において、宿主細胞由来の組換えＧＢＳ６７タンパク質の分泌を促進するためにこのモチーフを除去することが好まれ得る。従って、本発明の使用のためのＧＢＳ６７の１つの好ましいフラグメントにおいて、上記の膜貫通および細胞壁アンカーモチーフが、ＧＢＳ６７から除去される。そのようなＧＢＳ６７フラグメントの例を、配列番号９１

GBS67 contains an amino acid motif indicating a cell wall anchor (LPXTG motif): SEQ ID NO: 90 IPMTG (shown in italics in SEQ ID NO: 88 above). In some recombinant host cell systems, it may be preferred to remove this motif to facilitate secretion of recombinant GBS67 protein from the host cell. Thus, in one preferred fragment of GBS67 for use in the present invention, the above transmembrane and cell wall anchor motifs are removed from GBS67. An example of such a GBS67 fragment is SEQ ID NO: 91

として上に示す。

As shown above.

  The compositions of the present invention may also comprise a combination comprising one or more known GBS antigens in combination with GBS80.

  There is an upper limit to the number of GBS antigens present in the composition of the invention. Preferably, the number of GBS antigens in the composition of the present invention is less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 13, Less than, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 6, less than 5, less than 4, or less than 3. Even more preferably, the number of GBS antigens in the composition of the invention is less than 6, less than 5 or less than 4. Even more preferably, the number of GBS antigens in the composition of the present invention is three.

  The GBS antigen used in the present invention is preferably isolated (ie, separate and discrete) from the entire organism, the entire organism having a molecule found in nature, or If the polynucleotide or polypeptide is not found in nature, the polynucleotide or polypeptide is substantially free of other biological macromolecules so that it can be used for the intended purposes described above. Absent.

(Fusion protein)
The GBS antigens used in the present invention may be present in the composition as individual separate polypeptides, but at least two (ie 2, 3, 4, 5, 6, 7, Eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or eighteen antigens are combined into a single polypeptide chain ("hybrid" or "fusion"). "Polypeptides" are preferably expressed. Such fusion polypeptides have two major advantages: First, a polypeptide that can be expressed labile or incompletely can be assisted by adding an appropriate fusion partner that overcomes the problem. Second, commercial production is simplified because only one expression and the necessary purification is used to produce two polypeptides that are useful for both antigenicities. I will provide a.

  The fusion polypeptide may comprise two or more polypeptide sequences from the group consisting of GBS80, GBS91, GBS104, GBS184, GBS276, GBS305, GBS322, GBS330, GBS338, GBS361, GBS404, GBS690 and GBS691. Preferably, the polypeptide sequence is selected from the group consisting of GBS80, GBS104 and GBS322. Most preferably, the fusion peptide comprises a polypeptide sequence derived from GBS80. Accordingly, the present invention includes a fusion peptide comprising a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence and the second amino acid sequence are GBS antigens of the above group of antigens or Selected from the fragments. Preferably, the first amino acid sequence and the second amino acid sequence in the fusion polypeptide contain different epitopes.

(Example 7: Example of fragment for fusion protein derived from GBS104 and GBS322 and GBS80)
Examples of GBS fragments for the fusion protein are provided from GBS322, GBS104 and GBS80. One example of a fragment of GBS322 in the fusion protein is a 407 amino acid fragment from which the signal peptide has been removed. A fragment of GBS 104 can also be incorporated into the fusion protein. Examples of GBS104 fragments include 830 amino acid fragments, 359 amino acid fragments derived from near the N terminus, 581 amino acid fragments derived from near the N terminus, and 740 amino acid fragments derived from near the N terminus. . Examples of GBS80 fragments include a 446 amino acid fragment and a 235 amino acid fragment. Table 13 below summarizes examples of fusion proteins within the range corresponding to full-length GBS proteins and fragments for their positions.

２種、３種、４種、５種、６種、７種、８種、９種または１０種のＧＢＳ抗原由来のアミノ酸配列からなるハイブリッド（または融合物）が、好ましい。特に、２種、３種、４種または５種のＧＢＳ抗原に由来するアミノ酸配列からなるハイブリッドが、好ましい。

Hybrids (or fusions) consisting of amino acid sequences derived from 2, 3, 4, 5, 7, 8, 8, 9 or 10 GBS antigens are preferred. In particular, hybrids consisting of amino acid sequences derived from 2, 3, 4 or 5 GBS antigens are preferred.

  Different hybrid polypeptides can be mixed together in a single formulation. Within such combinations, GBS antigens can exist in more than one hybrid polypeptide and / or as non-hybrid polypeptides. However, it is preferred that the antigen be present as either hybrid or non-hybrid and not as both.

Hybrid polypeptide of the formula _NH 2 -A - can be represented by _{- {X-L-} n -B} -COOH, wherein: X is a GBS antigen or a fragment thereof from the group of antigens as indicated in the L is any linker amino acid sequence; A is any N-terminal amino acid sequence; B is any C-terminal amino acid sequence; and n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

If the -X- moiety has a leader peptide sequence in its wild-type form, it can be included in the hybrid protein or removed. In some embodiments, the leader peptide is removed except for the -X- moiety located at the N-terminus of the hybrid protein (ie, the leader peptide of X ₁ is retained, but X ₂ (... leader of _Xn is removed). This is equivalent to using the leader peptide of X ₁ as possible and partial -A- remove all of the leader peptide.

For each n case of {-XL-}, the linker amino acid sequence -L- may or may not be present. For example, when n = 2, the above hybrids are NH ₂ -X ₁ -L ₁ -X ₂ -L ₂ -COOH, NH ₂ -X ₁ -X ₂ -COOH, NH ₂ -X ₁ -L _{1- X 2 -COOH, NH 2 -X 1} -X 2 -L 2 -COOH, and the like. The linker amino acid sequence -L- is typically short (e.g., 20 amino acids or less, i.e. 19, 18, 17, 16, 15, 13, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include short peptide sequences that facilitate cloning, poly-glycine linkers (ie, Gly _n , where n = 2, 3, 4, 5, 6, 7, 8, 9, 10 or more ), And histidine tags (ie, His _n , where n = 3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable linker amino acid sequences will be apparent to those skilled in the art. A useful linker is GSGGGG, which has a Gly-Ser dipeptide formed from a BamHI restriction enzyme recognition site, thereby assisting in cloning and manipulation, and its (Gly) ₄ tetrapeptide is a representative poly- Glycine linker.

-A- is an optional N-terminal amino acid sequence. This is typically short (e.g. 40 or fewer amino acids, i.e. 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 21, 20, 19, 17, 16, 15, 15, 14, 13 , 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 2, 1). Examples include leader sequences that direct protein transport, or short peptide sequences that facilitate cloning or purification (eg, histidine tag, ie, His _n , where n = 3, 4, 5, 6, 7 , 8, 9, 10 or more). Other suitable N-terminal amino acid sequences will be apparent to those skilled in the art. When X ₁ lacks its own N-terminal methionine, -A- is preferably an oligopeptide providing an N-terminal methionine (eg, 1, 2, 3, 4, 5, 6 , 7 or 8 amino acids).

-B- is an arbitrary C-terminal amino acid sequence. This is typically short (e.g. 40 or fewer amino acids, i.e. 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 21, 20, 19, 17, 16, 15, 15, 14, 13 , 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 2, 1). Examples include sequences that direct protein transport, short peptide sequences that facilitate cloning or purification (eg, including a histidine tag, ie, His _n , where n = 3, 4, 5, 6, 7 , 8, 9, 10 or more), or sequences that enhance protein stability. Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art. Most preferably, n is 2 or 3.

Example 8 Active Maternal Immunoassay Using Fusion Proteins of GBS80, GBS67, and GBS322 Fragments
In this example, GBS antigen fusion protein was used in an active maternal immunoassay using different GBS strain isolate challenges. In these experiments, the immunochallenge dose for the different GBS strains is sufficient to kill about 70-90% of the unimmunized pups, equal to 10 × LD50% (where LD50% is Statistically derived half-lethal dose). Mother mice were immunized according to the active maternal immunoassay scheme described above using a fusion protein of GBS322 and GBS80 antigens in the GBS strain shown in Table 14 below. Percent survival was measured using GBS fusion protein. As shown in Table 14, in this particular challenge study, the survival rate reached up to 79% for the fusion proteins in all of the above GBS strains.

（核酸）
本発明はまた、本発明のＧＢＳ抗原および／またはハイブリッド融合ポリペプチドをコードする核酸を提供する。さらに、本発明は、好ましくは、「高ストリンジェンシー」の条件下（例えば、０．１×ＳＳＣ、０．５％ＳＤＳ溶液中で６５℃）で、これらの核酸にハイブリダイズし得る核酸を提供する。

(Nucleic acid)
The invention also provides a nucleic acid encoding the GBS antigen and / or hybrid fusion polypeptide of the invention. Furthermore, the present invention preferably provides nucleic acids that can hybridize to these nucleic acids under “high stringency” conditions (eg, 65 ° C. in 0.1 × SSC, 0.5% SDS solution). To do.

  The polypeptides of the present invention may be produced in various forms (eg, native forms, fusions, non-glycosylated forms, lipidated forms, etc.) by various means (eg, recombinant expression, purification from cell culture, chemical synthesis, etc.). ) Can be prepared. They are preferably prepared in substantially pure form (ie substantially free of other GAS proteins or host cell proteins).

  Nucleic acids according to the present invention can be prepared in a number of ways (eg, by chemical synthesis, from a genomic library or from a cDNA library, from the organism itself, etc.) and can be prepared in various forms (eg, single stranded, double stranded). Strands, vectors, probes, etc.). They are preferably prepared in substantially pure form (ie substantially free of other GBS nucleic acids or host cell nucleic acids).

  The term “nucleic acid” includes DNA and RNA, and also includes analogs thereof (eg, DNA and RNA containing a modified backbone (eg, phosphorothioate, etc.), and also peptide nucleic acids (PNA) and the like. The present invention includes a nucleic acid comprising a sequence complementary to the above sequence (eg, antisense or probe target).

  The invention also provides a process for producing a polypeptide of the invention, the process comprising culturing a host cell transformed with a nucleic acid of the invention under conditions that induce polypeptide expression. To do.

  The present invention provides a process for producing a polypeptide of the invention, the process comprising synthesizing at least a portion of the polypeptide by chemical means.

  The invention provides a process for producing a nucleic acid of the invention, the process comprising amplifying the nucleic acid using a primer-based amplification method (eg, PCR).

  The invention provides a process for producing a nucleic acid of the invention, the process comprising synthesizing at least a portion of the nucleic acid by chemical means.

(Purification and recombinant expression)
The GBS antigens of the present invention can be isolated from Streptococcus agalactiae or they can be produced recombinantly (eg, in a heterologous host). Preferably, the GBS antigen is prepared using a heterologous host. The heterologous host can be prokaryotic (eg, a bacterium) or eukaryotic. Preferably, E.I. but other suitable hosts include Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Neisseria lactamica, Neisseria lactium, Neisseria c. It is done.

  Recombinant production of the polypeptide is facilitated by adding a tag protein to the GBS antigen described above and expressed as a fusion protein comprising the tag protein and the GBS antigen. Such a tag protein can facilitate the purification, detection and stabilization of the expressed protein. Suitable tag proteins for use in the present invention include polyarginine tag (Arg-tag), polyhistidine tag (His-tag), FLAG-tag, Strep-tag, c-myc-tag, S-tag, Calmodulin binding peptide, cellulose binding domain, SBP-tag, chitin binding domain, glutathione S-transferase tag (GST), maltose binding protein, transcription termination anti-termination factor (NusA), E. coli. E. coli thioredoxin (TrxA) and protein disulfide isomerase I (DsbA). Preferred tag proteins include His-tag and GST. A full discussion regarding the use of tag proteins can be found in reference 3.

After purification, the tag protein can be removed from the expressed fusion protein as needed (ie, in particular by conditioned enzyme treatment known to those skilled in the art). The general protease used, enterokinase, tobacco etch virus (TEV), thrombin, and factor X _a and the like.

(GBS polysaccharide)
The compositions of the present invention can be further improved by including GBS polysaccharides. Preferably, each of the GBS antigens and saccharides contributes to the recipient's immune response. The combination is particularly advantageous in that the sugars and polypeptides described above provide protection from different GBS serotypes.

  The combined antigens can exist as a single combination in which separate saccharide and polypeptide antigens are administered together, or they can be covalently linked to each other, It can exist as a combined combination.

  Thus, the present invention provides an immunogenic composition comprising (i) one or more GBS polypeptide antigens and (ii) one or more GBS saccharide antigens. Said polypeptide and said saccharide can advantageously be covalently linked together to form a conjugate.

  In between, the combined polypeptide and saccharide antigens described above are preferably two or more GBS serotypes (eg, 2, 3, 4, 5, 6, 7, 8, or More serotypes) (or provide protection from more than one GBS serotype). The polypeptide antigens and saccharide antigen serotypes may or may not overlap. For example, the polypeptide can protect against serotype II or serotype V, while the saccharide can protect against either serotype Ia, serotype Ib or serotype III. . Preferred combinations are the following groups of serotypes: (1) serotype Ia and serotype Ib, (2) serotype Ia and serotype II, (3) serotype Ia and serotype III, (4) serotype Ia And serotype IV, (5) serotype Ia and serotype V, (6) serotype Ia and serotype VI, (7) serotype Ia and serotype VII, (8) serotype Ia and serotype VIII, ( 9) serotype Ib and serotype II, (10) serotype Ib and serotype III, (11) serotype Ib and serotype IV, (12) serotype Ib and serotype V, (13) serotype Ib and Serotype VI, (14) serotype Ib and serotype VII, (15) serotype Ib and serotype VIII, (16) serotype II and serotype III, (17) serotype II and serotype IV, (18 Serotype II And (19) serotype II and serotype VI, (20) serotype II and serotype VII, (21) serotype II and serotype VII, (22) serotype III and serotype IV, ( 23) serotype III and serotype V, (24) serotype III and serotype VI, (25) serotype III and serotype VII, (26) serotype III and serotype VIII, (27) serotype IV and Serotype V, (28) serotype IV and serotype VI, (29) serotype IV and serotype VII, (30) serotype IV and serotype VIII, (31) serotype V and serotype VI, (32 Serotype V and serotype VII, (33) serotype V and serotype VIII, (34) serotype VI and serotype VII, (35) serotype VI and serotype VIII, and ( 6) to protect against serotypes VII and serotype VIII.

  Even more preferably, the above combination comprises the following serotype groups: (1) serotype Ia and serotype II, (2) serotype Ia and serotype V, (3) serotype Ib and serotype II, Protect against (4) serotype Ib and serotype V, (5) serotype III and serotype II, and (6) serotype III and serotype V. Most preferably, the above combination protects against serotype III and serotype V.

  Protection against serotype II and serotype V is preferably provided by polypeptide antigens. The protection against serotype Ia, serotype Ib and / or serotype III can be a polypeptide antigen or can be a saccharide antigen.

  In one embodiment, the immunogenic composition comprises a GBS saccharide antigen and at least two GBS polypeptide antigens or fragments thereof, wherein the GBS saccharide antigen comprises GBS serotype Ia, GBS. A saccharide selected from serotype Ib and GBS serotype III, wherein the GBS polypeptide antigens described above are GBS80, GBS91, GBS104, GBS184, GBS276, GBS305, GBS322, GBS330, GBS338, GBS361, GBS404, GBS690. And a combination of at least two polypeptides or fragments thereof selected from the group of antigens consisting of GBS691. Preferably, the combination includes one or more GBS 80, GBS 104 and GBS 322. Even more preferably, the combination comprises GBS 80 or a fragment thereof.

  In certain embodiments, the combinations of the present invention do not include GBS polysaccharides. In certain embodiments, the combination does not include one or more GBS antigens selected from the group consisting of GBS4, GBS22, GBS85, GBS338, and GBS361.

(Immunogenic composition and medicine)
The composition of the present invention is preferably an immunogenic composition, more preferably a vaccine composition. The pH of the above composition is preferably between 6 and 8, preferably about 7. Its pH can be maintained by the use of a buffer. The above composition can be sterilized and / or free of pyrogens. The composition can be isotonic with respect to humans.

  A vaccine according to the invention can be either a prophylactic agent (ie to prevent infection) or a therapeutic agent (ie to treat infection), but is typically a prophylactic agent. Accordingly, the present invention encompasses a method for therapeutic or prophylactic treatment of Streptococcus agalactiae infection in an animal susceptible to streptococcal infection, the method comprising a therapeutic or prophylactic amount of an immunogen of the present invention. The step of administering the sex composition to the animal as described above.

  The present invention also provides a composition of the present invention for use as a medicament. The medicament is preferably capable of raising an immune response in a mammal (ie it is an immunogenic composition), more preferably a vaccine.

  The invention also provides the use of a composition of the invention in the manufacture of a medicament for increasing an immune response in a mammal. The medicament is preferably a vaccine.

  The present invention also provides a kit comprising a first component comprising a combination of GBS antigens.

  The present invention also provides a delivery device pre-filled with the immunogenic composition of the present invention.

  The invention also provides a method for raising an immune response in a mammal, the method comprising administering an effective amount of the composition of the invention. The immune response is preferably protective and is preferably associated with antibodies and / or cell-mediated immunity. The method can elicit a booster reaction.

  The mammal is preferably a human. Where the vaccine is for prophylactic use, the human is preferably a woman (either maternal age or teenager). Alternatively, the human can be elderly (eg, 50, 55, 60, 65, 70, or 75 years old or older) and have an underlying disease (eg, diabetes or cancer). Where the vaccine is for therapeutic use, the human is preferably a pregnant woman or an elderly person.

  These uses and methods are preferably for the prevention and / or treatment of diseases caused by Streptococcus agalactiae. The above composition may also be effective against other streptococci.

  One method of examining the effectiveness of a therapeutic treatment involves monitoring GBS infection after administration of the composition of the invention. One method of examining the effectiveness of prophylactic treatment involves monitoring the immune response to the GBS antigen in the composition of the invention after administration of the composition.

  The compositions of the invention are generally administered directly to the patient. Direct delivery can be by parenteral injection (eg, subcutaneous, intraperitoneal, intradermal, intravenous, intramuscular, or interstitial space of tissues) or rectal, oral (eg, tablet, spray), vaginal, topical, transdermal. Transdermal {eg see ref. 4} or transcutaneous {see eg refs. 5 and 6}, intranasal {eg see ref. 7}, eye It can be achieved by administration to the inner, ear, lung or other mucosa.

  The present invention can be used to elicit systemic and / or mucosal immunity.

  Dosage treatment can be a single dose schedule or a multiple dose schedule. Multiple doses can be used for an initial immunization schedule and / or a booster immunization schedule. In a multiple dose schedule, the various doses may be given by the same or different routes (eg, initial parenteral and additional mucosa, initial mucosa and additional parenteral, etc.).

  The compositions of the present invention can be prepared in various forms. For example, the composition can be prepared as an injectable, either as a liquid solution or suspension. Solid forms suitable for dissolution or suspension in liquid vehicle prior to injection (eg, lyophilized compositions) can also be prepared. The composition can be prepared for topical administration (eg, as an ointment, cream, or powder). The composition can be prepared for oral administration (eg, as a tablet or capsule, a spray, or a syrup (optionally flavored)). The composition can be prepared for pulmonary administration (eg, as an inhaler with a fine powder or spray). The composition can be prepared as a suppository or pessary. The composition can be prepared for administration to the nose, ear or eye (eg, as a drop). The composition may be in the form of a kit designed such that the combined composition is reformed just prior to administration to a patient. Such a kit may contain one or more antigens and one or more lyophilized antigens in liquid form.

  The immunogenic composition used as a vaccine contains an immunologically effective amount of the antigen, as well as any other components as required. By “immunologically effective amount” is meant that administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prophylaxis. . This amount depends on the health and physical condition of the individual being treated, the age, the taxon of the individual being treated (eg, non-human primate, primate, etc.), the ability of the individual's immune system to synthesize antibodies. Varies depending on the degree of protection desired, the formulation of the vaccine, the medical condition of the treating physician, and other relevant factors. The amount is expected to range over a relatively wide range that can be determined by routine trials.

(Additional composition ingredients)
The compositions of the present invention typically contain one or more “pharmaceutically acceptable carriers” in addition to the compositions described above, which are themselves individuals that receive the composition. Contains any carrier that does not induce the production of antibodies that are detrimental to. Suitable carriers are typically large, slowly metabolized macromolecules (eg, proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and lipid aggregates (eg, oil droplets or liposomes). ). Such carriers are well known to those skilled in the art. The vaccine can also include a diluent (eg, water, saline, glycerol, etc.). In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may be present. A thorough discussion of pharmaceutically acceptable excipients is available in reference 8.

  The vaccine of the present invention can be administered in conjunction with other immunoregulatory factors. In particular, the composition usually contains an adjuvant.

  Preferred further adjuvants include, but are not limited to, one or more of the following:

(A. Inorganic-containing composition)
Suitable mineral-containing compositions for use as the adjuvants of the present invention include inorganic salts such as aluminum and calcium salts. The present invention provides inorganic salts (eg, hydroxide (eg, acid hydroxide), phosphate (eg, hydroxyphosphate, orthophosphate), sulfate, etc. {eg, See chapters 8 and 9}), or a mixture of different inorganic compounds, which take any suitable form (eg gels, crystals, amorphous, etc.) and are adsorbent Preferably there is. The inorganic-containing composition can also be formulated as metal salt particles. See reference 10.

(B. Oil emulsion)
Suitable oil emulsion compositions for use as adjuvants of the invention include squalene-water emulsions (eg, MF59 (5% squalene formulated into submicron particles using a microfluidizer. , 0.5% Tween 80, and 0.5% Span 85)). See WO90 / 14837. Frey et al., “Comparison of the safety, tolerability, and immunogenicity of a MF59-adjuvant influenza, 42 in cc, and in non-adjuvant influenza.

In particular, a preferred adjuvant for use in the above composition is a submicron oil-in-water emulsion. Preferred submicron oil-in-water emulsions for use herein are optionally squalene / water emulsions containing varying amounts of MTP-PE (eg 4-5% w / v squalene). 0.25-1.0% w / v Tween 80 ^™ (polyoxyethylene sorbitan monooleate), and / or 0.25-1.0% Span85 ^™ (sorbitan trioleate), and optionally N -Acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE)) Micron oil-in-water emulsions (eg, “MF59” (International Publication No. WO 90/14837; US Pat. No. 6,299,884 and No. 6,451,325, which are incorporated herein by reference in their entirety; and Vaccine Design: The Subunit and Adjuvant Approach (Edited by Powell, MF and Newman, MJ) Pleum. Press, New York, 1995, pp. 277-296, Ott et al., “MF59-Design and Evaluation of a Safe and Potent Adjuvant for Human Vaccines” is a submicron oil-in-water emulsion F, 59). 4 to 5% w / v squalene (e.g., 4.3%), include 0.25~0.5% w / v Tween80 ^TM, and 0.5% w / v Span85 ^TM, and respond as required And various amounts of MTP-PE formulated into submicron particles using a microfluidizer (eg, Model 110Y microfluidizer (Microfluidics, Newton, Mass.)), Eg, MTP-PE. May be present in an amount of about 0-500 μg per dose, more preferably 0-250 μg per dose, and most preferably 0-100 μg per dose. The term “MF59-0” refers to the submicron oil-in-water emulsion described above lacking MTP-PE, while the term MF59-MTP refers to a formulation containing MTP-PE. , “MF59-100” contains 100 μg MTP-PE per dose, etc. . MF69, another submicron oil-in-water emulsion for use herein, is 4.3% w / v squalene, 0.25% w / v Tween 80 ^™ , and 0.75% w / v Includes Span85 ^™ , and optionally MTP-PE. Yet another submicron oil-in-water emulsion is 10% squalene, 0.4% Tween80 ^™ , 5% pluronic block polymer L121, and thr-MDP (also microfluidized into submicron emulsions). MF75, also known as SAF. MF75-MTP means an MF75 formulation containing MTP (eg, 100-400 μg MTP-PE per dose).

  Submicron oil-in-water emulsions, methods of making the submicron oil-in-water emulsions and immunostimulatory factors (eg, muramyl peptides) for use in the above compositions are described in International Publication No. WO90114837 and US Pat. Nos. 6,299,884 and 6,451,325 are described in detail and are incorporated herein by reference in their entirety.

  Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) can also be used as adjuvants of the invention.

(C. Saponin formulation)
Saponin formulations can also be used as adjuvants of the invention. Saponins are a heterogeneous group of sterol glycosides and triterpenoid glycosides that are found even in the bark, leaves, stems, roots and flowers of a wide range of plant species. Saponins from the bark of Quillia saponaria Molina tree have been extensively studied as adjuvants. Saponins can also be obtained commercially from Smilax orna (sarsaparilla), Gypsophila paniculata (brides veils), and Saponaria officialalis (soap root). Saponin adjuvant formulations include purified formulations (eg, QS21), as well as lipid formulations (eg, ISCOM).

  Saponin compositions have been purified using high performance thin layer chromatography (HP-LC) and reverse phase high performance liquid chromatography (RP-HPLC). Specific fractions purified using these techniques have been identified and include QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A method for generating QS21 is disclosed in US Pat. No. 5,057,540. Saponin formulations may also contain sterols (eg, cholesterol) (see WO96 / 33739).

  The combination of saponin and cholesterol can be used to form unique particles called immune stimulating complexes (ISCOMs). ISCOMs typically also include phospholipids (eg, phosphatidylethanolamine or phosphatidylcholine). Any known saponin can be used for ISCOMs. Preferably, the ISCOM includes one or more Quil A, QHA and QHC. ISCOMs are further described in EP0109942, WO96 / 11711 and WO96 / 33739. If necessary, the ISCOM may lack additional detergent. See reference 11.

  A comprehensive review of the development of saponin based adjuvants can be found in reference 12.

(C. Virosome and virus-like particles (VLP))
Virosomes and virus-like particles (VLPs) can also be used as adjuvants of the invention. These structures generally comprise one or more proteins from the virus that are combined as needed or formulated with phospholipids. They are generally non-pathogenic and non-replicating and generally do not contain any natural viral genome. The viral protein can be produced recombinantly or can be isolated from the entire virus. These viral proteins suitable for use with virosomes or VLPs include influenza virus (eg, HA or NA), hepatitis B virus (eg, core protein or capsid protein), hepatitis E virus, measles virus, Sind Bisvirus, rotavirus, foot-and-mouth disease virus, retrovirus, Norwalk virus, human papilloma virus, HIV, RNA-phage, Qβ-phage (eg coat protein), GA-phage, fr-phage, AP205 phage, and Ty (For example, the protein derived from retrotransposon Ty protein p1) is mentioned. VLPs are further discussed in WO03 / 024480, WO03 / 024481, and reference 13, reference 14, reference 15 and reference 16. Virosomes are further discussed, for example, in reference 17.

(D. Bacterial derivative or microbial derivative)
Adjuvants suitable for use in the present invention include the following bacterial or microbial derivatives.

(1) Non-toxic derivatives of enterobacterial lipopolysaccharide (LPS) Such derivatives include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred “small particle” form of 3 De-O-acylated monophosphoryl lipid A is disclosed in EP 0 694 454. Such “small particles” of 3dMPL are small enough to be filter sterilized through a 0.22 micron membrane (see EP 068454). Other non-toxic LPS derivatives include monophosphoryl lipid A mimics (eg, aminoalkyl glycosaminide phosphate derivatives (eg, RC-529)). See, for example, Reference 18.

(2) Lipid A derivative Examples of the lipid A derivative include derivatives of lipid A derived from Escherichia coli (for example, OM-174). OM-174 is described in Reference Document 19 and Reference Document 20, for example.

(3) Immunostimulatory oligonucleotides Immunostimulatory oligonucleotides suitable for use as adjuvants of the present invention include CpG motifs (followed by guanosine followed by unmethylated cytosine bound by phosphate bonds). Nucleotide sequence comprising). Bacterial double-stranded RNA or oligonucleotides containing palindromic or poly (dG) sequences have also been shown to be immunostimulatory.

  The CpG described above can include nucleotide modifications / analogs (eg, phosphorothioate modifications), which can be double-stranded or single-stranded. If desired, the above guanosine can be replaced with an analog (eg, 2'-deoxy-7-deazaguanosine). See Reference 21, WO 02/26757 and WO 99/62923 for examples of possible analog substitutions. Adjuvant effects of CpG oligonucleotides are further described in ref. 22, ref. 23, WO 98/40100, US Pat. Nos. 6,207,646, 6,239,116 and 6,429,199. Has been discussed.

  The CpG sequence described above can be related to TLR9 (eg, motif GTCGTT or TTCGTT). See reference 24. The CpG sequence can be specific to induce a Th1 immune response (eg, CpG-A ODN), or it can be more specific to induce a B cell response (eg, CpG-B ODN). Can be CpG-A ODN and CpG-B ODN are discussed in refs. 25 and 26 and WO 01/95935. Preferably, the CpG is CpG-A ODN.

  Preferably, the CpG oligonucleotide is constructed so that the 5 'end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences can be joined at their 3 'ends to form an "immunomer". See, for example, Reference 27, Reference 28, Reference 29, and WO 03/035836.

(4) ADP-ribosylating toxins and their detoxified derivatives Bacterial ADP-ribosylating toxins and their detoxified derivatives can be used as adjuvants of the invention. Preferably, the protein is E. coli. from E. coli (ie, E. coli heat labile enterotoxin “LT”), cholera (“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in WO 95/17211 and in WO 98/42375 as a parenteral adjuvant.

  Preferably, the adjuvant is a detoxified LT variant (eg, LT-K63).

(E. Human immunomodulator)
Suitable human immunomodulators for use as adjuvants of the invention include cytokines (eg, interleukins (eg, IL-1, IL-2, IL-4, IL-5, IL-6, IL- 7, IL-12, etc.), interferon (eg, interferon-γ)), macrophage colony stimulating factor, and tumor necrosis factor.

(F. Bioadhesive and mucoadhesive)
Bioadhesives and mucoadhesives can also be used as adjuvants of the invention. Suitable bioadhesives include esterified hyaluronic acid microspheres (ref. 30) or mucoadhesives (eg, poly (acrylic acid), polyvinyl alcohol, polyvinyl pyrrolidone, polysaccharides and crosslinked derivatives of carboxymethyl cellulose). . Chitosan and its derivatives can also be used as adjuvants of the invention. For example, reference 31.

(G. Fine particles)
Microparticles can also be used as adjuvants of the invention. Microparticles (ie, particles having a diameter of about 100 nm to about 150 μm, more preferably a diameter of about 200 nm to about 30 μm, and most preferably a diameter of about 500 nm to about 10 μm), biodegradable and non-toxic materials (eg , Poly (α-hydroxy acid), polyhydroxybutyric acid, polyorthoester, polyanhydride, polycaprolactone, and the like, and poly (lactide-co-glycolide)), which is poly (lactide-co-glycolide). -Glycolide) is preferred and optionally treated to have a negatively charged surface (eg with SDS) or a positively charged surface (eg a cationic surfactant (eg CTAB)).

(H. liposome)
Examples of liposome formulations suitable for use as adjuvants are described in US Pat. Nos. 6,090,406, 5,916,588, and EP0626169.

(I. Polyoxyethylene ether formulation and polyoxyethylene ester formulation)
Adjuvants suitable for use in the present invention include polyoxyethylene ethers and polyoxyethylene esters (Ref. 32). Such formulations include polyoxyethylene sorbitan ester surfactants combined with octoxynol (ref. 33) and at least one additional nonionic surfactant (eg, octoxynol (ref. 34)). Further included are polyoxyethylene alkyl ether or ester surfactants in combination with.

  Preferred polyoxyethylene esters are the following groups: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steolyl ether, polyoxyethylene-8-steolyl ether, polyoxyethylene-4- Selected from lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.

(J. Polyphosphazene (PCPP))
PCPP formulations are described, for example, in references 35 and 36.

(K. muramyl peptide)
Examples of muramyl peptides suitable for use as adjuvants of the present invention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl- D-isoglutamine (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy ) -Ethylamine MTP-PE).

(L. imidazoquinolone compound)
Examples of imidazoquinolone compounds suitable for use as adjuvants of the invention include imiquamod and homologs thereof, which are further described in refs. 37 and 38.

  The present invention can also include aspects of the combination of one or more of the above identified adjuvants. For example, the following adjuvant composition can be used in the present invention.

(1) Saponins and oil-in-water emulsions (Reference 39);
(2) saponins (eg QS21) + non-toxic LPS derivatives (eg 3dMPL) (see WO 94/00153);
(3) saponin (eg QS21) + non-toxic LPS derivative (eg 3dMPL) + cholesterol;
(4) Saponin (eg QS21) + 3dMPL + IL-12 (optional + sterol) (Reference 40);
(5) For example, a combination of QS21 and / or an oil-in-water emulsion and 3dMPL (reference document 41);
(6) Microfluidized into 10% squalene, 0.4% Tween 80, 5% pluronic block polymer L121, and submicron emulsions or vortexed to make larger particle size emulsions. SAF including any thr-MDP.

(7) Group consisting of 2% Squalene, 0.2% Tween 80, and the following: monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS) (preferably MPL + CWS (Detox ^™ )) Ribi ^TM adjuvant system (RAS) (Ribi Immunochem) containing one or more bacterial cell wall components from; and (8) one or more inorganic salts (eg, aluminum salts) + non-toxin derivatives of LPS (eg, 3dPML).

  Aluminum salt and MF59 are preferred adjuvants for parenteral immunization. Mutant bacterial toxins are preferred mucosal adjuvants.

  The composition may contain antibiotics.

(Further antigen)
The compositions of the present invention may further contain one or more additional non-GBS antigens, including additional bacterial, viral or parasitic antigens.

  In another embodiment, the combination of GBS antigens of the invention is combined with one or more additional non-GBS antigens suitable for use in vaccines designed to protect the elderly or non-immune individuals. . For example, the combination of GBS antigens is Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermis, Pseudomonas aeruginosa, influenza A Can be.

If a saccharide antigen or carbohydrate antigen is used, it is preferably conjugated to a carrier protein to enhance immunogenicity {eg refs. 42-51}. Preferred carrier proteins are bacterial toxins or bacterial toxoids (eg diphtheria toxoid or tetanus toxoid). CRM ₁₉₇ diphtheria toxoid is particularly preferred {52}. Other carrier polypeptides include N.I. meningitidis outer membrane protein {53}, synthetic peptide {54, 55}, heat shock protein {56, 57}, pertussis protein {58, 59}, H. et al. Influenzae-derived protein D {60}, cytokine {61}, lymphokine, hormone, growth factor, C.I. difficile-derived toxin A or toxin B {62}, iron uptake protein {63}, and the like. If the mixture contains capsular saccharides from both serotype A and serotype C, the ratio of MenA saccharide: MenC saccharide (w / w) is greater than 1 (eg 2: 1, 3: 1, 4 1: 5: 1, 10: 1 or more). Different saccharides can be bound to the same or different types of carrier proteins. Any suitable conjugation reaction can be used, which has any suitable linker, if necessary.

  Toxic protein antigens can be detoxified by chemical and / or genetic means when necessary (eg, in the case of detoxification of pertussis toxin).

  When a diphtheria antigen is included in the above composition, it is also preferred to include a tetanus antigen and a pertussis antigen. Similarly, where a tetanus antigen is included, it is also preferred to include diphtheria and pertussis antigens. Similarly, where a pertussis antigen is included, it is also preferred to include diphtheria and tetanus antigens.

  The antigens in the above composition are typically present at a concentration of at least 1 μg / ml, respectively. In general, the concentration of any given antigen is sufficient to elicit an immune response against that antigen.

  As an alternative to using protein antigens in the compositions of the invention, nucleic acids encoding the antigens can be used {eg refs. 64-72}. Thus, the protein component of the composition of the invention can be replaced by a nucleic acid encoding the protein, preferably DNA (eg, in the form of a plasmid).

(Definition)
The term “comprising” means “including” as well as “consisting” (eg, a composition “comprising” X may consist solely of or has been added). (Eg, X + Y)).

  The term “about” for the numerical value x means, for example, x ± 10%.

A criterion for the percentage of sequence identity between two amino acid sequences, when aligned, means that the percentage of amino acids is the same comparing the two sequences. This alignment and percent homology or sequence identity can be determined using software programs known in the art (eg, those described in Section 7.7.18 of ref. 73). The preferred alignment is determined by the Smith-Waterman homology search algorithm using a similarity gap search, with a gap start penalty of 12, a gap extension penalty of 2, 62 BLOSUM matrix. This Smith-Waterman homology search algorithm is disclosed in reference 74.

Claims (12)

A composition comprising a combination of two or more GBS antigens, wherein the combination is :
i) GBS80 comprising the amino acid sequence of SEQ ID NO: 2 , or an immunogenic fragment thereof comprising at least 8 consecutive amino acids of GBS80 , or an immunogenic polypeptide sequence having 95 % or more sequence identity with GBS80 ;
ii) The following:
GBS91 comprising the amino acid sequence of SEQ ID NO: 11, an immunogenic fragment thereof comprising at least 8 consecutive amino acids of GBS91, or an immunogenic polypeptide sequence having 95% or more sequence identity with GBS91;
GBS104 comprising the amino acid sequence of SEQ ID NO: 19, its immunogenic fragment consisting of at least 8 consecutive amino acids of GBS104, or an immunogenic polypeptide sequence having 95% or more sequence identity with GBS104;
GBS184 comprising the amino acid sequence of SEQ ID NO: 24, an immunogenic fragment thereof consisting of at least 8 consecutive amino acids of GBS184, or an immunogenic polypeptide sequence having 95% or more sequence identity with GBS184,
GBS276 comprising the amino acid sequence of SEQ ID NO: 27, an immunogenic fragment thereof consisting of at least 8 consecutive amino acids of GBS276, or an immunogenic polypeptide sequence having 95% or more sequence identity with GBS276,
GBS305 comprising the amino acid sequence of SEQ ID NO: 32, an immunogenic fragment thereof consisting of at least 8 consecutive amino acids of GBS305, or an immunogenic polypeptide sequence having 95% or more sequence identity with GBS305,
GBS322 comprising the amino acid sequence of SEQ ID NO: 37, an immunogenic fragment thereof consisting of at least 8 consecutive amino acids of GBS322, or an immunogenic polypeptide sequence having 95% or more sequence identity with GBS322,
GBS330 comprising the amino acid sequence of SEQ ID NO: 40, an immunogenic fragment thereof consisting of at least 8 consecutive amino acids of GBS330, or an immunogenic polypeptide sequence having 95% or more sequence identity with GBS330;
GBS338 comprising the amino acid sequence of SEQ ID NO: 42, an immunogenic fragment thereof comprising at least 8 consecutive amino acids of GBS338, or an immunogenic polypeptide sequence having 95% or more sequence identity with GBS338,
GBS361 comprising the amino acid sequence of SEQ ID NO: 45, an immunogenic fragment thereof comprising at least 8 consecutive amino acids of GBS361, or an immunogenic polypeptide sequence having 95% or more sequence identity with GBS361,
GBS404 comprising the amino acid sequence of SEQ ID NO: 48, an immunogenic fragment thereof consisting of at least 8 consecutive amino acids of GBS404, or an immunogenic polypeptide sequence having 95% or more sequence identity with GBS404,
GBS690 comprising the amino acid sequence of SEQ ID NO: 50, an immunogenic fragment thereof comprising at least 8 consecutive amino acids of GBS690, or an immunogenic polypeptide sequence having 95% or more sequence identity with GBS690,
GBS691, comprising the amino acid sequence of SEQ ID NO: 53, an immunogenic fragment thereof consisting of at least 8 consecutive amino acids of GBS691, or an immunogenic polypeptide sequence having 95% or more sequence identity with GBS691
At least one of: and a composition. 2. The composition of claim 1, wherein the GBS antigen combination exhibits improved immunogenicity as measured by an active maternal immunoassay, wherein the active maternal immunoassay comprises an immune schedule. A composition that measures the serum titer of female mice during and the percent survival of pups after challenge. 3. The composition of claim 2, wherein the percent survival of the challenged pup is at least 2 percentage points higher than the percent survival of the challenged pup from a female mouse immunized with a single non-GBS80 antigen. Composition. The composition according to any one of claims 1 to 3 , wherein the combination is GBS80, GBS91, GBS104, GBS184, GBS276, GBS305, GBS322, GBS330, GBS338, GBS361, GBS404, GBS690, and GBS691. are al selection, 2-13 species of GBS antigens, immunogenic fragments of said GBS antigens, or consisting of an immunogenic polypeptide sequence having 95% or more sequence identity to the GBS antigen composition object. 5. The composition of claim 4, wherein the combination consists of 2, 3, 4 or 5 GBS antigens. The composition according to any one of claims 1 to 5 , wherein the GBS80 fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 , 4 , 6, 7, 8, and 9. ,Composition. The composition according to any one of claims 1 to 6 , wherein the combination is GBS80, GBS104 and GBS322 , or an immunogenic fragment of the GBS antigen, or 95% of the GBS antigen. A composition comprising an immunogenic polypeptide sequence having the above sequence identity . The composition according to any one of claims 1 to 7, wherein the GBS80 antigen , an immunogenic fragment of GBS80, or an immunogenic polypeptide having 95% or more identity to GBS80. The sequence has 95% or more identity to GBS91, GBS104, GBS184, GBS276, GBS305, GBS322, GBS330, GBS338, GBS361, GBS404, GBS690 or GBS691, these immunogenic fragments, or the GSB antigen A composition expressed as a fusion polypeptide with at least one GBS antigen selected from immunogenic polypeptide sequences . A composition according to claim 8, wherein the GBS80 antigen and the GBS322 antigen or immunogenic fragments of said GBS antigens, or immunogenic having 95% or more identity the GBS antigen polypeptide Ru sequence or Rana, composition. 10. A composition according to any one of claims 1 to 9 for use in therapy. G BS for the therapeutic or prophylactic treatment of infections, the composition according to any one of claims 1-9. Use of the composition according to any one of claims 1 to 9 for the manufacture of a medicament for the treatment or prevention of GBS infection.