JP2010248194A - Peptide structures and their use in the diagnosis of herpes simplex virus type 2 - Google Patents

Abstract

Provided is a peptide structure useful for diagnosis of herpes simplex virus type 2 antibody.
A following peptide structure: [(X ¹⁾ _p -16 amino acid sequence _{^{- (X 2) q -Sp]}} n - core, wherein "16 amino acid sequence",: GluGluPheGluGlyAlaGlyAspGlyGluProProGluAspAspAsp; indicates, and wherein X ¹ and X ² , which may be the same or different, represent 1 to 6 non-interfering amino acid residues which may be the same or different; Sp is a spacer extending outwardly from the core N is at least 4; p is 0 or 1; q is 0 or 1; and the linkage between the core and the spacer group is chemically or physically Possible peptide structure.
[Selection figure] None

Description

(Background of the Invention)
(1. Field of the Invention)
The present invention relates to the diagnosis of herpes simplex virus type 2 antibodies in samples, particularly serum samples taken from patients suspected of being infected with herpes simplex virus type 2 (HSV-2). The present invention provides multiple shown peptide structures useful for this purpose, and methods and kits for diagnosis using these peptide structures, and for the preparation of multiple shown peptides Useful monomeric peptides are also provided.

(Details of related technology)
Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are two closely related viruses that infect humans. Most people over the age of 15 have antibodies to HSV-1 or HSV-2, indicating that they are infected and carry these viruses. Both viruses produce orogenital lesions and can also infect the eyes, skin and nervous system. HSV-1 is primarily responsible for oral lesions, while HSV-2 is primarily responsible for genital lesions. Following a primary infection, the virus can enter a latent period and cause reinfection in neural tissue, where it can be periodically activated. Reactivation is often asymptomatic because the virus can be dispersible and can be infected with a lack of obvious clinical lesions.

  The need for a simple and inexpensive serodiagnostic test that could distinguish HSV-1 antibodies from HSV-2 antibodies was recognized at a recent meeting of WHO (1996). The determination of HSV serotypes is important in HSV vaccine attempts to establish individual immunological conditions prior to vaccination. It is desirable to prevent neonatal herpes by screening pregnant women and their sexual partners for asymptomatic HSV-2 infection, and an appropriate recommended prevention design to prevent infection ,desired.

  Advances to HSV-2 specific serodiagnostic reagents were facilitated by the identification of an HSV-2 glycoprotein called 92K or gG2 more than 10 years ago (Marsden et al., 1978, 1984; Roizman et al., 1984). Subsequently, gV1, the counterpart of HSV-1, was identified. Determination and comparison of the DNA sequences of the genes encoding gG1 and gG2 showed that the two proteins were significantly different. The presence of gG1 and gG2 serotype-specific epitopes (Ackerman et al., 1986; Frame et al., 1986; Marsden et al., 1984; Roizman et al., 1984) may allow the use of gG1 and gG2 as serotype-specific diagnostic reagents. Produced.

  Various serological tests for HSV-2 specific antibodies have been developed using gG2. These tests include ELISA using serotype specific monoclonal antibodies, immunodot blot and western blot assays and serum blocking assays. For these purposes, gG2 is expressed in various cells including mammalian cells infected with HSV-2, transformed mammalian cells, insect cells infected with recombinant baculovirus and Escherichia coli using several expression vectors. Obtained from the source. E. E. coli was also used to express a fragment of gG2 containing a type-specific region and a slightly shortened, nearly full-length fragment of gG2 fused to a maltose binding protein. Antigens from these sources were used either as crude extracts or purified by various subsequent procedures (including ion exchange chromatography, monoclonal antibody-affinity chromatography and lectin-affinity chromatography).

  Through serological testing with gG2, Western blots are considered the most reliable (WHO Conference, Copenhagen, 1995). However, this method is cumbersome, relatively expensive, and not suitable for general screening purposes outside of a well-equipped diagnostic laboratory. There is a need for a reliable serodiagnostic test that is simple, inexpensive and can be used like an ELISA (Enzyme-Linked Immunosorbent Assay) in a microtiter plate.

ここでＸ³は、アミノ酸残基であり、そして「ｐｅｐｓｅｑ」は、ペプチド（Ｘ¹）_p−１６アミノ酸ペプチド−（Ｘ²）_qの配列である、
構造。
（項目５） ｎが、４から１６である、項目１、２、３または４に従う構造。
（項目６） ｐおよびｑが、共に１である、項目１、２、３、４または５に従う構造。
（項目７） Ｘ¹がＰｒｏであり、そしてＸ²がＳｅｒである、項目６に従う構造。
（項目８） 前記スペーサー基Ｓｐが、４〜６のグリシン残基の長さと等価な長さを有する、項目５、６または７に従う構造。
（項目９） 単純ヘルペスウイルス２型に対する抗体について試験するための方法であって、結合反応が、該抗体と前記項目のいずれかに記載の構造との間で行なわれ、そして該結合の発生または程度を、それぞれ検出または測定する、方法。
（項目１０） 前記結合反応が、ＥＬＩＳＡとして行なわれる、項目９に従う方法。
（項目１１） 患者から得た血清のサンプルに対して実行する、項目９または１０に従う方法。
（項目１２） 患者の体液を、そこに存在する抗体について試験するため
のキットであって、（１）項目１、２、３、４、５、６、７または８に記載の複数示されたペプチド構造；および（２）該ペプチド構造物への該抗体の結合を検出または測定するための標識された抗ヒト免疫グロブリン抗体を含む、キット。
（項目１３） 以下の一般式のペプチド
（Ｘ¹）_p−１６アミノ酸配列−（Ｘ²）_q−（Ｓｐ）_r
ここで、Ｘ¹、Ｘ²、ｐ、ｑおよび「１６アミノ酸配列」が、項目１、６または７において定義され、ｒが、０または１である、
ペプチドならびにそれらのアミノ基誘導体およびカルボキシル基誘導体。
（発明の要旨）
驚くべきことに、各分岐が、コアにペプチド配列を含む別々の複数の分岐として内在する場合、１６アミノ酸ペプチド、より好ましくは１８アミノ酸ペプチドが、ＨＳＶ−２抗体に対して高い特異性を有し、そして特に偽陽性または偽陰性をほとんど与えないことが、見出された。結果的に、このようなペプチド構造は、ＨＳＶ−２抗体の試験において、特に患者からの体液サンプルでの、および最も特に、血清サンプルでの試験に役立つ。１６アミノ酸ペプチドは、ｇＧ２のアミノ酸５６２〜５７７に由来し、かつ明細書内で後に示す配列番号１の配列を有する。さらに、このペプチドのいずれかの末端あるいは両端に少数のさらなるアミノ酸を含むかまたはわずかに短縮することが可能である。ｇＧ２の５６１〜５７８アミノ酸由来の１８アミノ酸ペプチド（明細書で後に示す配列番号５５を有する）は、最も好ましい。 The present invention provides the following:
(Item 1) Multiple peptide structures represented by the formula (1): [(X ¹ ) _p -16 amino acid sequence- (X ² ) _q -Sp] _n -core, where the “16 amino acid sequence” SEQ ID NO: 68):
Glu Glu Phe Glu Gly Ala Gly Asp Gly
1 5
Glu Pro Pro Glu Asp Asp Asp;
10 15
Indicate
And where X ¹ and X ² , which may be the same or different, represent 1 to 6 non-interfering amino acid residues that may be the same or different; Sp is outward from the core Indicates an extended spacer group;
n is at least 4;
p is 0 or 1;
q is 0 or 1;
And the linkage between the core and the spacer group can be chemical or physical,
Peptide structure.
(Item 2) The structure according to item 1, wherein the connection between the core and the spacer group is a covalent bond.
(Item 3) A structure according to item 1 or 2 which is in the form of a peptide as a whole.
(Item 4) A structure according to Item 1, 2 or 3, wherein the following formula (4)

Where X ³ is an amino acid residue and “pepseq” is the sequence of peptide (X ¹ ) _p- 16 amino acid peptide- (X ² ) _q .
Construction.
(Item 5) A structure according to items 1, 2, 3 or 4 wherein n is 4 to 16.
(Item 6) A structure according to items 1, 2, 3, 4 or 5 wherein p and q are both 1.
(Item 7) A structure according to item 6, wherein X ¹ is Pro and X ² is Ser.
(Item 8) The structure according to item 5, 6 or 7, wherein the spacer group Sp has a length equivalent to the length of 4 to 6 glycine residues.
(Item 9) A method for testing for an antibody against herpes simplex virus type 2, wherein a binding reaction is performed between the antibody and the structure according to any of the preceding items, and the occurrence of the binding or A method of detecting or measuring the degree, respectively.
(Item 10) The method according to item 9, wherein the binding reaction is performed as an ELISA.
(Item 11) A method according to item 9 or 10, which is performed on a sample of serum obtained from a patient.
(Item 12) A kit for testing a patient's bodily fluid for an antibody present therein, wherein (1) a plurality of items according to items 1, 2, 3, 4, 5, 6, 7 or 8 are indicated A kit comprising: a peptide structure; and (2) a labeled anti-human immunoglobulin antibody for detecting or measuring binding of the antibody to the peptide structure.
(Item 13) The following general formula of the peptide (X ¹⁾ _p -16 amino acid sequence _{^{- (X 2) q - (}} Sp) r
Wherein X ¹ , X ² , p, q and “16 amino acid sequence” are defined in item 1, 6 or 7, and r is 0 or 1;
Peptides and their amino group derivatives and carboxyl group derivatives.
(Summary of the Invention)
Surprisingly, a 16 amino acid peptide, more preferably an 18 amino acid peptide, has a high specificity for the HSV-2 antibody when each branch is inherent as a separate plurality of branches containing the peptide sequence in the core. And in particular it has been found to give few false positives or false negatives. Consequently, such peptide structures are useful in testing HSV-2 antibodies, particularly in body fluid samples from patients, and most particularly in serum samples. The 16 amino acid peptide is derived from amino acids 562-577 of gG2 and has the sequence of SEQ ID NO: 1 as shown later in the specification. In addition, a small number of additional amino acids can be included or slightly shortened at either end or both ends of the peptide. Most preferred is an 18 amino acid peptide derived from 561-578 amino acids of gG2 (having SEQ ID NO: 55 shown later in the specification).

  The present invention is particularly surprising because this is a region of HSV-2 glycoprotein gG2 (McGeoch et al., (1987)) that has high homology with HSV-1 glycoprotein gG1, and the peptides of the present invention are: It is highly specific for the HSV-2 antiserum and shows very few false positives with the HSV-1 antiserum.

In one aspect, the present invention provides multiple shown peptide structures of formula (1): [(X ¹ ) _p- (16 amino acid sequence)-(X ² ) _q -Sp] _n -core, where The “16 amino acid sequence” is the following sequence (SEQ ID NO: 68)
Glu Glu Phe Glu Gly Ala Gly Asp Gly
1 5
Glu Pro Pro Glu Asp Asp Asp
10 15
Where X ¹ and X ² , which may be the same or different, represent 1 to 6 non-interfering amino acid residues that may be the same or different;
Sp represents a spacer group extending outward from the core;
n is at least 4;
p is 0 or 1;
q is 0 or 1;
And the linkage between the core and the spacer group can be chemical or physical.

  The present invention includes the use of the peptide constructs of the present invention in the diagnosis of antibodies against HSV-2. In particular, the present invention provides a method for testing an antibody against herpes simplex virus type 2 wherein a binding reaction is performed between the antibody and the peptide construct of the present invention, and the presence or degree of binding is detected or measured, respectively. Is done.

  The present invention further includes a kit for testing a patient's body fluid for antibodies present therein, including: (1) a plurality of shown peptide structures of the present invention; and (2) a peptide structure. A labeled anti-human immunoglobulin antibody for detection or measurement of antibody binding to.

The invention also provides the peptide of formula (2) itself:
(X ¹ ) _p -16 amino acid sequence- (X ² ) _q- (Sp) _r , wherein “16 amino acid sequence”, X ¹ , X ² , p and q are as defined above, and r is 0 or 1 and includes their N-terminal and C-terminal functional derivatives.

FIG. 1 shows the detection of HSV-2 antisera and HSV-1 antisera by the method of the invention: Panels A, C, E = HSV-2: Panels B, D and F = HSV-1. FIG. 2 shows screening by ELISA of sera from individuals not infected with HSV.

(Description of preferred embodiments)
The multiple shown peptide structures of the present invention are preferably based on a branched lysine core. Lysine is a dibasic amino acid that provides two sites for peptide bonds. These lysine core structures are well known and are described in Tam (1988) and Tam et al. (1989), US Pat. PCT publication WO 92/18528 (Medical Research Council). These can be used in the present invention. Preferably, this structure is the entire peptide of formula (3):
[(X ¹⁾ _p -16 amino acid sequence _{^{- (X 2) q -Sp]}} n - (Lys) n-1 - (X 3) m (3)
Where n is 2 ^a (a is a number from 2 to 5) (ie, n = 4, 8, 16, or 32) and the lysine residue is branched (Lys) _n-1 X ³ represents 1 to 4 amino acid residues which may be the same or different, m is 0 or 1, and the other letters are defined above. Preferably, p is 0 or 1, q is 0 or 1, and Sp is a spacer of a length equivalent to the length of 4-6 glycine residues (and most preferably Sp is actually 4 Represents -6 glycine residues), and m is 0 or 1. Most preferably, the peptide structure of the present invention is of the following formula (4):

ここで、Ｘ³は、アミノ酸残基であり、そして「ｐｅｐｓｅｑ」は、上記で定義したペプチド配列（Ｘ¹）_p−１６アミノ酸配列−（Ｘ²）_qである。
・分岐したリジンコアを用いることは、本質的なことではない。コアは、多くの形態を取り得る。例えば、分子の残りが物理的に吸着されるかまたは化学的に結合される（通常は、共有結合によって）固体不活性原料であり得る。それは、例えば、直鎖状または分岐したポリマーであり得、それには例えば、トリメチロールプロパンのポリエステルまたは低分子量のポリアミドもしくはポリアクリルアミドあるいは樹状の分岐を含むポリマー（例えば、米国特許第４，５０７，４６６号（Ｔｏｍａｌｉａら、１９８５））が挙げられる。それは、プリントされた回路基板またはシリコンチップのアレイであり得、それによって試験される抗体が、ペプチド構造物に結合する場合、電荷または電流の変化が生じ、これが検出され得る。コアへの好ましい連結は、共有結合を介してである。

Here, X ³ is an amino acid residue, and “pepseq” is the peptide sequence (X ¹ ) _p- 16 amino acid sequence − (X ² ) _q defined above.
• It is not essential to use a branched lysine core. The core can take many forms. For example, the rest of the molecule can be a solid inert source that is physically adsorbed or chemically bound (usually by a covalent bond). It can be, for example, a linear or branched polymer, such as a polyester of trimethylolpropane or a low molecular weight polyamide or polyacrylamide or a polymer containing dendritic branches (eg, US Pat. No. 4,507, 466 (Tomalia et al., 1985)). It can be an array of printed circuit boards or silicon chips, whereby when the antibody being tested binds to the peptide structure, a change in charge or current occurs, which can be detected. A preferred linkage to the core is through a covalent bond.

  It should be clear from the above that it is not necessary that the entire molecule of the peptide structure is composed of amino acids. A non-peptide core or non-peptide spacer or non-peptide bond between one branch and another may be mentioned. However, whole peptides or whole amino acid structures are very convenient from a synthetic point of view.

  The spacer arm extends outward from the core of the structure and includes any compatible residues. Most preferably, the spacer arm molecule comprises an amino acid residue. Preferably, the spacer arm comprises a glycine residue (which is an uncharged polar amino acid) or a majority of non-polar amino acids. The term “non-polar amino acid” as used herein includes alanine, valine, leucine and isoleucine. Preferred spacer arm lengths are at least 4 glycine residues, but shorter spacers (eg, length equivalent to one glycine residue) have a small effect. Having a length equivalent to more than 6 glycine residues would not be very advantageous. This is because the possibility of interfering with each other between such highly flexible branches may be too high.

The 16 amino acid sequence SEQ ID NO: 68 shows several amino acid residues that do not inhibit its ability to bind to HSV-2 antibodies with high specificity either on the left side (N-terminus) or on the right side (C-terminus) or both. Can be used to elongate. These can be in the “native” gG2 sequence. Thus, X ¹ is, Glu His Arg Gly Gly Pro (SEQ ID NO: 69), His Arg Gly Gly Pro (SEQ ID NO: 70), Arg Gly Gly Pro (SEQ ID NO: 71), 3 amino acid sequence Gly Gly Pro, 2 amino acid sequence Gly Pro or can be a single amino acid Pro and X ² is Ser Ala Thr Gly Leu Phe (SEQ ID NO: 72), Ser Ala Thr Gly Leu (SEQ ID NO: 73), Ser Ala Thr Gly (SEQ ID NO: 74), 3 It can be the amino acid sequence Ser Ala Thr, the two amino acid sequence Ser Ala or the single amino acid Ser. Alternatively, X ¹ and X ² may contain other amino acids, in which case they are preferably nonpolar amino acids or glycine or serine. Most preferably, the 16 amino acid sequence is extended by one amino acid at one or both ends, in particular by one or both of the adjacent amino acids of the natural sequence where the N-terminus is Pro and the C-terminus is Ser. The Thus, the following 17 and 18 amino acid sequences are particularly preferred:
Pro Glu Glu Phe Glu Gly Ala Gly Asp
1 5
Gly Glu Pro Pro Glu Asp Asp Asp
10 15
(SEQ ID NO: 75)
Glu Glu Phe Glu Gly Ala Gly Asp Gly
1 5
Glu Pro Pro Glu Asp Asp Asp Ser
10 15
(SEQ ID NO: 76)
Pro Glu Glu Phe Glu Gly Ala Gly Asp
1 5
Gly Glu Pro Pro Glu Asp Asp Asp Ser
10 15
(SEQ ID NO: 55)
Preliminary results indicate that the peptide structure having a peptide sequence extended to the C-terminus or N-terminus is slightly more inferior than some of the “positive peptide structures 55” containing 18 amino acid SEQ ID NO: 55. Indicates that it gives poor diagnostic results.

  If the peptide structure is entirely a peptide, the peptide from which the peptide sequence is derived and in fact the peptide sequence itself is a peptide structure can be made by any well-known synthetic method described above, in particular The Fmoc method, which is a continuous flow version, is particularly convenient.

  The present invention may be practiced in any method suitable for the detection of antibodies (especially any suitable fluid, especially body fluids containing antibodies. Serum is a normal body fluid but contains immunoglobulins). Other fluids (eg, tears, saliva or milk) can be assayed.

  In routine testing, this assay is performed heterogeneously by attaching the peptide structure of the invention to an insoluble carrier, such as a plastic surface of a microplate, but the invention is limited to such assays. Not. In a sandwich assay, the antiserum is incubated with the peptide structure for an appropriate period to allow binding, and with the antibody against the immunoglobulin for an appropriate period. Thus, for example, assuming that the patient is human and the antibody is an IgG class antibody, add an appropriately labeled anti-human IgG antibody, wash the plate, and label attached to the plate The amount (through the antibody and peptide construct) is measured. The present invention can be applied to the detection of IgM antibodies using anti-human IgM as the second antibody. Alternatively, in a competitive assay, a known amount of labeled antibody can be performed that can compete with serum or displace from its binding. However, such assays are somewhat unsuitable for detecting low concentrations of antibody. Any common label, such as a radioactive label and an enzyme label, can be used (particularly peroxidase and alkaline phosphatase). Indirect labels such as biotin can also be used in binding with a suitable directly labeled binding partner (eg, labeled streptavidin or labeled avidin). A radioactive label can be assayed with a radioactive count and an enzyme labeled with, for example, a colorimetric, fluorescent or chemiluminescent signal. Norddisk Diagnostics Ltd. requires alkaline phosphatase enzyme labeling. Amplification methods of detection, such as “Ampak” ®, can be used to further sensitize the assay.

  The amount of peptide construct to use per assay (eg per well of the plate) is then usually less than 1 μg, preferably less than 0.1 μg, and preferably 0.05-0.5 μg. is there. In any particular case, the most appropriate amount will depend on the nature of the relevant peptide structure.

  In kits for performing this assay, the basic components are most often the peptide construct of the present invention and an anti-human immunoglobulin labeled antibody. These are of course usually provided in separate containers within the kit.

  Since the present invention is useful as an intermediate in the production of the peptide structure of the present invention, it includes the peptide of formula (2) itself. For example, it may be convenient to provide a peptide that lacks a spacer prepared for attachment to the rest of the molecule (core and spacer) (ie, r is 0). This is particularly useful when obtaining a core and spacer from a supplier, especially where the core and spacer contain a pendant group such as an aminoethyl group prepared for attachment to the C-terminal peptide by a peptide bond. It is particularly useful when it is an amino acid polymer. Alternatively, the peptide can be synthesized using a spacer (r = 1 in formula (2)) prepared to bind to the core. Such a preferred peptide is most preferably SEQ ID NO: 55 of the 18 amino acid peptide sequence as described above.

  The present invention also includes a peptide having a carboxy protecting group and an amino protecting group, particularly where the amino acid side chain is protected with a t-butyloxycarbonyl group and the N-terminus is protected with 9-fluorenylmethoxycarbonyl (Fmoc). Derivatives of such peptides, particularly including such peptides are included. The invention further includes peptides having “reactive” C-terminal groups, such as esters, particularly pentafluorophenyl esters, especially for peptides lacking spacers (r = 0). “Reactive” means capable of reacting with the free amino group of the spacer to form an amide bond with them. Other such reactive derivatives are well known in the art and are included within the scope of the present invention.

  The following examples illustrate the invention.

(Example)
(Materials and methods)
(Serum and preliminary serology)
A total of 174 serum specimens were collected from 118 individuals. Of these, 155 sera were collected from 100 patients. Paired sera were collected from 55 of these patients. An additional 18 sera were collected from 18 children with various conditions not associated with HSV. The titer of HSV-specific antibodies in serum collected in Edinburgh was determined by complement binding assay (Smith et al., 1967), while the titer in serum collected in Glasgow was determined using an anti-HSV IgG ELISA kit (“Enzgnost”). [Registered trademark], Behring Diagnostics). HSV-infected patients from whom sera were collected had an average adult age of approximately 27 years and approximately the same number of men and women.

(Virus isolation and typing)
All virus isolates were obtained from genital lesions, with one being an isolate isolated from the patient's lips. Viruses were typed by indirect fluorescence using a panel of type-specific monoclonal antibodies (“Syva Micro Track” ®, Behringer Diagnostics).

(Peptide synthesis)
A series of 67 peptides (mostly 18 amino acids long overlapping 10 amino acids) spanning amino acids 21-699 of the predicted open reading frame of gG2 were synthesized by continuous flow Fmoc chemistry. Residues 1-20 were not synthesized because they were thought to contain a signal sequence that was excised from the mature protein. This peptide was made as multiple presented peptide structures of the present invention. Each structure consists of four copies of each amino acid sequence delimited on a branched lysine core (Tam, 1988), but each such amino acid sequence has four glycine residues to increase sensitivity. It is separated from the core by a group. Such a peptide structure is represented by the formula (5):

によって表され得る。ここで、「ｐｅｐｓｅｑ」は、処方物内の各存在における同じアミノ酸配列を示し、そして以下の表１および配列識別番号１〜６７の１文字表記で示されるペプチドの配列から選択される。それらを、製造業者によって記載される標準的なＦｍｏｃ手順および製造業者の指示に従うカップリング試薬としてＨＢＴＵ［２−（１Ｈ−ベンゾトリアゾール−１−イル）−１，１，３，３−テトラメチルウロニウム ヘキサフルオロホスフェート］またはＴＢＴＵ［２−（１Ｈ−ベンゾトリアゾール−１−イル）−１，１，３，３−テトラメチルウロニウム テトラフルオロボレート］のいずれかを使用して、Ｓｈｉｍａｄｚｕ ＰＳＳＭ−８ペプチド合成機で作製する。ペプチドを、逆相ＨＰＬＣによって分析し、そして少なくとも８０％純粋であることを判断する。

Can be represented by: Here, “pepseq” indicates the same amino acid sequence at each occurrence in the formulation, and is selected from the peptide sequences shown in Table 1 below and the one-letter code of SEQ ID NOs: 1-67. They are used as coupling reagents according to the standard Fmoc procedure described by the manufacturer and the manufacturer's instructions as HBTU [2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluro Shimadzu PSSM-8 peptide using either [Nium Hexafluorophosphate] or TBTU [2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate] Prepare with a synthesizer. The peptide is analyzed by reverse phase HPLC and determined to be at least 80% pure.

(Example 1: Enzyme-linked immunosorbent assay (ELISA))
Serum was tested for reactivity using a truncated version of HSV-2 glycoprotein D (gD2t) and a peptide construct containing the gG2 peptide sequence. HSV-1 glycoprotein D (gD1) and HSV-2 glycoprotein (gD2) are very homologous to many epitopes recognized by antibodies raised by both HSV-1 and HSV-2. . Glycoprotein D is also an immunodominant protein. The properties of these two gDs make gD sensitive and useful reagents for the diagnosis of HSV specific antibodies. The truncated form contains the first 326 amino acids of gD but lacks the carboxy-terminal transmembrane domain and is consequently secreted from the cell. This property is advantageous because the secreted protein can be more easily purified from the cell culture medium than the entire gD is purified from the cell membrane. However, this property does not distinguish between HSV-1 and HSV-2 antibodies. gD2t (purified to homogeneity after expression from DNA in Chinese hamster ovary cells) was provided by SmithKline Beecham Biologicals (Rixensart, Belgium) at a concentration of 660 μg / ml. This was diluted in phosphate buffered saline (PBS) and microtiter wells (Immunolon 1, Dynatech) were used to coat 0.5 μg or 0.25 μg in 50 μl.

  Peptide structures (see Table 1) were dissolved in water whenever possible. Water-insoluble peptide structure is obtained in 30% acetic acid (peptide structure having the sequence of SEQ ID NO: 7, 8, 12, 14, 15, 21, 25, 26, 62) or by aqueous suspension of peptide structure Solubilized either by aeration of a small amount of ammonia vapor (2 ml). At that time, the peptide structure formed a clear solution (peptide structures having the sequences of SEQ ID NOs: 16, 17 and 18). The solubilized peptide structure was then diluted in PBS to the concentration indicated in the text and 50 μl was added to the wells. Both gD2t and the peptide construct were adsorbed to the plate overnight at 4 ° C. The antigen solution was removed from the plate and unoccupied binding sites on the plate were blocked with PBS containing 1% BSA for 1.5-2 hours. Wells were washed 6 times with 150 mM NaCl (“NaCl-Tween”) containing 0.05% “Tween 20” (Tween is a registered trademark) and with 50 μl of serum diluted as indicated in the text Incubate for 1.5 hours at room temperature on an orbital shaker. Serum was removed and the wells were washed 6 more times with “NaCl-Tween”. In order to detect antibody binding, the wells were in turn with 50 μl biotinylated sheep anti-human IgG (Amersham, 1/1000 dilution) and 50 μl streptavidin-conjugated horseradish peroxidase (Amersham, 1/1000 dilution), Each was incubated for 1.5 hours at 37 ° C. and then washed 6 times with NaCl- “Tween”. In citrate phosphate buffer (pH 4.0) containing 0.01% hydrogen peroxide (50 μl), the chromogenic substrate o-phenylenediamine dihydrochloride (OPDA) was added, and after 10 minutes, 50 μl of Color development was stopped by adding 2N sulfuric acid. The plate was read on a “Titertek” multi-scan plate reader at 492 nm.

(result)
(Preliminary screening of all peptides against a subset of sera)
In this initial experiment, all peptide constructs were screened against a set of 24 sera. Four sera from patients who isolated the virus were found to be type 1 and were antibody positive and CF titers ranged from 16-256. Fifteen sera from patients who isolated the virus were found to be type 2 and were antibody positive and CF titers ranged from 16-256. Three sera were from patients who had no experimental evidence of HSV infection in virus isolation and were antibody negative. Two sera were antibody negative but from the patient from whom the virus was isolated: HSV-1 was cultured from one patient and HSV-2 was cultured from the other patient. This serum was also screened against gD2t (500 ng / well) to confirm the presence of HSV specific antibodies. Wells treated with PBS without any antigen served as controls. In the initial screen, wells were coated with 1.0 μg of peptide and the absorbance value at 492 nm was recorded.

  A peptide specific for HSV-2 should have the following properties: Should react with sera containing antibodies raised by HSV-2 infection. Furthermore, this peptide should not react with sera without HSV-specific antibodies or with sera containing antibodies elicited by HSV-1 infection in the absence of HSV-2 infection.

  In the absence of any previous data on the reactivity of this peptide, this data was analyzed by two methods. In the first method, a cut-off value of 0.25 was selected on the basis that it could serve as an appropriate value to distinguish between HSV antibody positive and negative sera. Only peptide structures containing peptide sequences (SEQ ID NOs: 3, 7, 9, 10, 11, 12, 13, 14, 17, 21, 26, 55, 57, 62, and 66, 67) have 15 HSVs. -2 produced all of the sera and absorbance values greater than 0.25, while only the peptide construct containing the sequence (SEQ ID NO: 39, 50, 51, 55, 56, 63 and 64) produced all 5 antibody negative sera Gave an absorbance value of less than 0.25. And only this peptide structure comprising the sequences of SEQ ID NOs: 38, 50 and 55 gave all four HSV-1 sera and absorbance values less than 0.25. Therefore, only “Peptide Structure 55” (having the sequence of SEQ ID NO: 55) met all of the above defined criteria required for HSV-2 antibody specific reagents. In the second method, the absorbance value of each serum in the control PBS-coated well was subtracted from the absorbance value in the peptide-coated well and a cut-off value of 0.1 was selected. Only peptide constructs comprising the sequences of SEQ ID NOs: 7, 9, 10, 11, 12, 13, 14, 17, 23, 26, 39, 57, 62, 66 and 67 are found in all 15 HSV-2 sera and 0 Only peptide structures containing the sequences SEQ ID NO: 50, 51, 55, 56 and 64 gave all five antibody negative sera and absorbance values less than 0.1, while producing absorbance values greater than .1. . And only the peptide constructs containing the sequences SEQ ID NO: 37, 38, 39, 50, 55 and 64 gave all four HSV-1 sera and absorbance values less than 0.1. Therefore, none of the peptides met all criteria. However, of the four peptide structures comprising the sequences of SEQ ID NOs: 39, 50, 55 and 64 which gave all five antibody negative sera and all four HSV-1 sera and absorbance values less than 0.1, SEQ ID NO: Peptide constructs having 39, 50 and 64 sequences were reactive with 5, 1 and 4 out of 15 HSV-2 sera, respectively. On the other hand, “Peptide Structure 55” was reactive with 14 of 15 sera. Thus, “peptide structure 55” was considered a possible candidate for type-specific serodiagnosis of HSV.

Example 2: Optimization of the amount of peptide and serum used
Referring to FIG. 1 of the drawings, the wells were coated with four different amounts of the peptide structure 55 of the present invention: 5 μg (square), 1 μg (diamond), 100 ng (circle) and 10 ng (triangle). Seven sera were diluted 20-fold and then diluted 6-fold twice more. Wells lacking peptide structures and / or serum served as controls (four square grids). The results for three HSV-2 sera (CF titers 16, 256, and 16) and for three HCV-1 sera (CF titers less than 8, 256, and 16) are shown in FIG. 1 (Panel A). , C, E = HSV-2; B, D and F = HSV-1). All HSV-2 sera showed greater reactivity with wells coated with peptide structure 55, whereas HSV-1 sera exceeded the background levels seen with PBS control, and peptide structure 55 Did not react. A total of four concentrations of peptide construct 55 were tested, and the reactivity with serum was very similar. When diluted at 1:40 or less, absorbance greater than 1 was produced or less and appeared to reach a plateau. In contrast, both sera C and E showed lower reactivity, and the curve shape suggested that higher reactivity could be achieved with less diluted serum. To test whether even lower peptide amounts could be used, wells were coated with different amounts ranging from 100 ng to 5 pg and tested for reactivity with serum A and E. Below 5 ng / well, a significant decrease in signal was observed, and below 100 pg no response was observed (data not shown).

Example 3
Based on these observations, all sera were then screened on wells coated with 100 ng and 10 ng of peptide construct 55 and with gD2t: the amount of gD2t was reduced to 250 ng / well and approximately , Reduced to the signal seen with 100 ng peptide construct. Serum was tested at 5-, 10-, and 20-fold dilutions. Wells containing neither peptide structure nor protein were included in all serum dilutions.

(Establishment of cut-off value)
The 21 sera used as HSV negative controls consisted of 3 from Edinburgh and 18 from Glasgow. They were judged negative by showing no clinical signs associated with HSV and lack of reactivity in an ELISA or complement binding assay (see method in Example 1). Serum was diluted 5-fold and screened by ELISA on wells coated with gD2t (250 ng) or peptide construct 55 (either 100 ng or 10 ng) or no antigen (PBS control). Data for gD2t and 100 ng of peptide structure 55 are shown in FIG. Cut-off values (0.166 for gD2t and 0.167 for peptide structure 55) corresponding to the mean + 5 × standard deviation were used for subsequent analysis of other sera and are indicated by dotted lines in the figure.

(Serum analysis from HSV positive patients)
All sera were screened against wells coated with both 100 ng and 10 ng of peptide 55, 250 ng gD2t and no antigen (PBS control). Most sera were screened with three different dilutions: 5-fold, 10-fold, and 20-fold, but some sera were screened using only 5-fold dilutions. For each serum, the background absorbance observed without antigen was subtracted from the value observed for different antigens. Data sets were constructed with these corrected values.

  Serum was grouped into 5 classes for analysis. Classes 1 and 2 include sera collected from patients with clinical lesions: class 1 sera had a CF titer less than 8, whereas class 2 sera had a CF titer greater than 8. Serum containing class 3 and 4 was collected between 7-20 days (class 3) or later than 20 days (class 4) from the initial lesion presentation. Class 5 sera were derived from patients who did not present primary lesions and whose previous sera had a CF titer of at least 8. Here, primary lesions are defined as clinically apparent lesions having a CF titer of less than 8 or a 4-fold increase in 20 days.

  Serum was diluted 5-fold and screened on wells coated with 100 ng peptide that gave the lowest number of false positives. These results are summarized in Table 2 below. A positive result was recorded when the absorbance exceeded 0.166 (using mean + 5 × standard deviation).

表２を参照すると、ペプチド構造物５５は、ＨＳＶ−１血清で偽陽性を与えなかった。第２に、ＨＳＶ−１およびＨＳＶ−２血清の両方とｇＤ２ｔ、ならびにＨＳＶ−２血清とペプチド構造物５５との反応性は、第１の提示の時点（クラス１および２血清）で得られた血清についての反応性について、続いてサンプリングされた血清（クラス３、４および５の血清）の反応性より低かった。全体としては、クラス３、４および５由来のＨＳＶ−１血清の９５％（４１／４３）およびＨＳＶ−２血清の９８％（５０／５１）はｇＤ２ｔと反応したが、一方、ＨＳＶ−２のクラス３、４および５の血清の９２％（４７／５１）は、ペプチド構造物５５と反応した。第３に、全て（２２／２２）のＨＳＶ−２のクラス５血清は、ｇＤ２ｔおよびペプチド構造物５５の両方と反応した。

Referring to Table 2, peptide structure 55 did not give false positives with HSV-1 serum. Second, reactivity of both HSV-1 and HSV-2 sera with gD2t, and HSV-2 serum with peptide structure 55 was obtained at the time of the first presentation (class 1 and 2 sera). Reactivity for sera was lower than that of subsequently sampled sera (class 3, 4 and 5 sera). Overall, 95% (41/43) of HSV-1 sera from classes 3, 4 and 5 and 98% (50/51) of HSV-2 sera reacted with gD2t, whereas HSV-2 sera 92% (47/51) of class 3, 4 and 5 sera reacted with peptide construct 55. Third, all (22/22) HSV-2 class 5 sera reacted with both gD2t and peptide construct 55.

  Thus, peptide 55 has been shown to be completely type-specific for the HSV-2 antibody in human serum, particularly in order to detect the antibody 7 days after the initial presentation of clinical lesions. Were shown to be type-specific.

Example 4
A further peptide structure corresponding to the extension at the N-terminus and C-terminus of “peptide structure 55” comprising the peptide sequence of SEQ ID NO: 55 is synthesized, and having 4-glycine spacer above, 4- of formula (5) Made in a branched configuration. These were tested with gD2t using peptide structures or proteins at (a) 1 μg / well or (b) 100 ng / well. And in each case, 20-fold diluted serum was used. Otherwise, the conditions were the same as in Example 3. The results are shown in Table 3 below. Peptide structure 55 performed best in terms of not giving false positives for HSV-1.

(Example 5)
Two peptide structures of the invention (a peptide structure 55 and a peptide structure comprising a peptide sequence extended by a “native” sequence of two amino acids at each end), the peptide sequence being extended at the N-terminus but C Comparison with terminal truncated (10 amino acids and 8 amino acids, respectively) peptide structures. This comparison shows the criticality of the 16 amino acid core sequence of the present invention. All three peptides were made in the form of 4-branched, four glycine spacers of formula (5). They were all compared with protein gD2t as in Example 3 with a 100 ng / well peptide structure and 250 μg / well protein and serum 5-fold dilutions. The two peptide structures of the present invention perform much better than the peptide structure having the comparative peptide sequence of SEQ ID NO: 86 from all points of view, and the peptide structures of the present invention are false for HSV-1 It was found not to give a positive.

  The following claims define several important aspects of the present invention, but are not intended to include any conceivable aspects, which may require protection in subsequent and foreign patent applications, And it should not be construed as reducing the universality of the inventive concept previously described herein.

（配列表）

(Sequence Listing)

Claims (10)

Formula (1):
  [(X ¹ ) -16 amino acid sequence- (X ² ) -Sp] _n -Branched lysine core
A plurality of shown peptides,
  The “16 amino acid sequence” is the sequence (SEQ ID NO: 68):
  Glu Glu Phe Glu Gly Ala Gly Asp Gly
    1 5

  Glu Pro Pro Glu Asp Asp Asp;
  10 15
Indicate
  X ¹ Is Pro and X ² Is Ser;
  Sp represents a spacer group extending outward from the branched lysine core;
  n is at least 4;
  The linkage between the branched lysine core and the spacer group can be chemical or physical,
peptide. The peptide according to claim 1, wherein the linkage between the branched lysine core and the spacer group is a covalent bond. Formula (4):

The peptide according to claim 1 or 2, wherein
  X ^Three Is an amino acid residue;
  “Pepseq” is a peptide (X ¹ ) -16 amino acid peptide- (X ² )
peptide. The peptide according to claim 1, 2 or 3, wherein n is 4 to 16. The peptide according to claim 4, wherein the spacer group Sp has 4 to 6 glycine residues. A method for assisting in testing for antibodies against herpes simplex virus type 2, comprising:
  A binding reaction is performed between the antibody and the peptide of any of claims 1-5; and
  Detecting or measuring the occurrence or degree of the binding, respectively;
Method. The method of claim 6, wherein the binding reaction is performed as an ELISA. The method according to claim 6 or 7, wherein the method is carried out on a sample of serum obtained from a patient. A kit for testing a patient's bodily fluid for the presence of antibodies in the bodily fluid,
  (1) a plurality of peptides according to claim 1, 2, 3, 4 or 5; and
  (2) a labeled anti-human immunoglobulin antibody for detecting or measuring the binding of the antibody to the peptide;
Including a kit. General formula:
(X ¹ ) -16 amino acid sequence- (X ² )-(Sp) _r
Peptides and derivatives thereof,
The “16 amino acid sequence” is the sequence (SEQ ID NO: 68):
Glu Glu Phe Glu Gly Ala Gly Asp Gly
1 5

Glu Pro Pro Glu Asp Asp Asp;
10 15
Indicate
X ¹ is Pro and X ² is Ser;
Sp represents a spacer group, r is 0 or 1,
The derivative comprises a group selected from the group consisting of a t-butyloxycarbonyl group, 9-fluorenylmethoxycarbonyl (Fmoc), and pentafluorophenyl ester.
peptide.

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