WO2019044925A1 - 改変型HSV gDタンパク質及びこれを含むワクチン - Google Patents
改変型HSV gDタンパク質及びこれを含むワクチン Download PDFInfo
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Definitions
- the present invention relates to modified HSV gD proteins and vaccines comprising the same.
- HSV Human herpes simplex virus
- HSV Human herpes simplex virus
- HSV which is a dsDNA virus, belongs to the alpha herpesvirus subfamily, and there are two serotypes, HSV-1 and HSV-2.
- HSV causes various diseases such as encephalitis, meningitis, herpes labialis, genital herpes, skin disease, corneal herpes, systemic neonatal herpes etc. in humans.
- HSV is an extremely important virus in medical hygiene, and in fact, effective antiviral agents such as acyclovir and valacyclovir have been developed.
- the anti-HSV agents developed so far inhibit the replication of viral DNA growing in infected cells, they are effective against HSV which is latently infected inside the ganglia in the state of DNA. Not shown. That is, when infected with HSV, removal of HSV from the latent infection site is impossible with existing anti-HSV agents. In order to overcome such a situation, it is necessary to develop new vaccines effective for both primary infection protection and relapse protection.
- Pathogens that cause infections are broadly divided into Class I pathogens that can obtain sufficient effects with conventional vaccines and Class II pathogens that can not obtain sufficient protective immunity with conventional vaccines and pathogen infection history. .
- the clever immune escape mechanisms that they possess have been pointed out as the reasons for the difficulty in protecting class II pathogens.
- HSV is classified into Class II pathogens, which are thought to be because HSV has an immune evasion mechanism and cleverly bypasses the host's immune response.
- studies using attenuated live vaccines and adjuvant inactivated vaccines have been attempted so far, but both responses to T cell immunity and B cell immunity are both inadequate and can be obtained after natural infection There was not much difference with the level of inadequate immune response.
- HSV achieves entry into the host cell through steps such as cell surface adsorption, association with the viral receptor, and membrane fusion of the cell membrane and the viral envelope. This system is triggered by the association of multiple viral envelope proteins with host cell membrane proteins. So far, envelope glycoprotein B (gB), envelope glycoprotein C (gC), envelope glycoprotein D (gD), envelope glycoprotein H (gH) and envelope glycoprotein L (gL) have been known. Among viral envelope proteins, gD initially forms a complex with the host cell membrane receptor herpesvirus entry mediator (HVEM), Nectin-1, or Nectin-2. Crystal structures have been reported for complexes (Non-Patent Documents 1 and 2).
- HVEM herpesvirus entry mediator
- Nectin-1 Nectin-1
- Nectin-2 Nectin-2
- gD interacts with gH / gL, and activated gH / gL further activates gB (Non-patent Document 3).
- gB is considered to be responsible for the main function of membrane fusion through the receptor 3-O-sulfonated heparan sulfate (3-OS HS) or PILR ⁇ (Non-patent Documents 4 and 5).
- 3-OS HS 3-O-sulfonated heparan sulfate
- PILR ⁇ Non-patent Documents 4 and 5
- stampfer et al. “Stuck in the middle: structural insights into the role of gH / gL heterodimer in herpesvirus entry”, Curr Opin Virol, Vol. 3, No. 1, Feb. 2013, pages 13-19 E. Trybala et al., "Herpes Simplex Virus Types 1 and 2 Differ in Their Interaction with Heparan Sulfate", JOURNAL OF VIROLOGY, Vol. 74, No. 19, Oct. 2000, pages 9106-9114, ISSN: 0022-538X J.
- antiviral agents such as acyclovir are used for the treatment of HSV.
- these antiviral agents can not completely remove the virus, and the virus reactivates when taking the drug. Therefore, it is desirable to develop a preventive vaccine that protects HSV infection itself or a therapeutic vaccine that alleviates or alleviates recurrent symptoms, but there is currently no effective vaccine and its unmet needs are high.
- the present invention can induce an immune serum having a high content of neutralizing antibodies exhibiting high neutralizing activity against HSV gD in induction of immunity as compared to wild type HSV gD, and is used for the prevention and / or treatment of HSV infection It is an object of the present invention to provide a modified HSV gD protein and a vaccine comprising the same.
- the present inventors conducted comprehensive B cell epitope analysis and T cell epitope analysis on gD proteins known as one of the major protective antigens of HSV, and used beneficial epitopes or no benefit or harmful effects in protective activity expression. It tried to classify into various epitopes. And, by de-epitoping useless or harmful epitopes and immunologically highlighting useful epitopes, or by adding beneficial epitopes such as promiscuous T cell epitopes, Immune refocusing (Immune refocusing) It has led to the completion of a modified HSV gD vaccine with enhanced ability to induce neutralizing antibodies and cellular immunity, and as a result, enhanced ability to protect against infection.
- the present invention relates to the following inventions.
- a modified protein modified HSV gD protein of envelope glycoprotein D (gD) of herpes simplex virus (HSV), which is a receptor binding domain (RBD) in the ectodomain of wild type HSV gD
- a modified HSV gD protein which has been modified such that at least one of B cell epitopes (decotopes) with low or no neutralizing antibody inducing activity as compared to existing B cell epitopes does not function as an epitope.
- the B cell epitope present in RBD is selected from the group consisting of the 134th arginine residue, the 139th aspartic acid residue, and the 222nd arginine residue in the amino acid sequence set forth in SEQ ID NO: 1
- the modified HSV gD protein of (1) which is an epitope comprising an amino acid residue corresponding to at least one amino acid residue.
- the modified HSV gD protein of (1) or (2) which is a B cell epitope present in the N-terminal proline rich region (PRR) of the gD ectodomain.
- the decotope Or an epitope comprising an amino acid residue corresponding to the 50th proline residue in the amino acid sequence of SEQ ID NO: 1 of the ectodomain of wild type HSV gD, or At the surface of the crystal structure of wild type HSV gD ectodomain, it is an epitope comprising at least one amino acid residue present in a region of 1.5 nm or less from the amino acid corresponding to the 50th proline residue, (3) Modified HSV gD protein. (5) The modification of the decotope is carried out by glycosylated introduction by substitution of amino acid residues, deletion of amino acid residues, and / or substitution or deletion of amino acid residues, any of (1) to (4) Some modified HSV gD proteins.
- the modification of the decotope is a modification of the decotope by glycosylation of the ectodomain of wild type HSV gD to the amino acid residue corresponding to the 50th proline residue in the amino acid sequence of SEQ ID NO: 1 (5) a modified HSV gD protein, which comprises (7)
- the modification of the decotope includes the modification of the ectodomain of wild type HSV gD by glycosylation to the amino acid residue corresponding to proline 74 in the amino acid sequence of SEQ ID NO: 1 (5 Or (6) modified HSV gD protein.
- the modification of the decotope includes the modification of the ectodomain of wild type HSV gD by glycosylation to the amino acid residue corresponding to the arginine at position 186 in the amino acid sequence of SEQ ID NO: 1 (5
- the modified HSV gD protein according to any of the above-(7) (9)
- the ectodomain of wild type HSV gD consists of the amino acid sequence set forth in SEQ ID NO: 1,
- the modification of the decoration is By substituting the 50th proline residue in the amino acid sequence shown in SEQ ID NO: 1 with an asparagine residue and replacing the 51st proline residue with an amino acid residue other than a proline residue Modifications to be made, A modification which is carried out by glycosylated by replacing the 74th proline residue in the amino acid sequence set forth in SEQ ID NO: 1 with an asparagine residue and replacing the 76th glutamic acid residue with a serine residue, and A modification which is carried out by glycation by replacing the
- the modified HSV gD protein according to any one of (1) to (10), wherein at least one promiscuous T cell epitope is linked to the C-terminal side of the ectodomain of HSV gD. gD protein.
- the promiscuous T cell epitope is a promiscuous T cell epitope consisting of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 Modified HSV gD protein.
- the modified HSV gD protein further comprises a deletion of at least a portion of the amino acid residues corresponding to the amino acid residues 251 to 315 in the amino acid sequence of SEQ ID NO: 1 of wild type HSV gD ( 1)
- the modified HSV gD protein of any one of (1) to (13).
- the amino acid residue corresponding to the valine residue at position 231 in the amino acid sequence of SEQ ID NO: 1 in the ectodomain of wild type HSV gD is substituted with another amino acid residue
- An HSV vaccine comprising the modified HSV gD protein of any of (1) to (16).
- a modified protein (modified HSV gD protein) of envelope glycoprotein D (gD) of herpes simplex virus (HSV), which is an N-terminal proline of gD ectodomain in the ectodomain of wild type HSV gD
- HSV herpes simplex virus
- PRR rich region
- the B cell epitope present in PRR is Or an epitope comprising an amino acid residue corresponding to the 50th proline residue in the amino acid sequence of SEQ ID NO: 1 of the ectodomain of wild type HSV gD, or At the surface of the crystal structure of wild type HSV gD ectodomain, it is an epitope comprising at least one amino acid residue present in a region of 1.5 nm or less from the amino acid corresponding to the 50th proline residue, (18) The modified HSV gD protein. (20) The modified HSV gD protein according to (18) or (19), wherein the modification is carried out by glycosylation by substitution or deletion of amino acid residues.
- the modification includes modification of the ectodomain of wild type HSV gD by glycosylation to the amino acid residue corresponding to the 50th proline residue in the amino acid sequence of SEQ ID NO: 1 (20 Modified HSV gD protein.
- the modification includes modification of the ectodomain of wild type HSV gD by glycosylation to an amino acid residue corresponding to the 74th proline residue in the amino acid sequence of SEQ ID NO: 1 (20 Or (21) modified HSV gD protein.
- the modification includes modification of the ectodomain of wild type HSV gD by glycosylation to an amino acid residue corresponding to arginine at position 186 in the amino acid sequence of SEQ ID NO: 1, (20) Any of the modified HSV gD proteins of (22).
- the ectodomain of wild type HSV gD consists of the amino acid sequence set forth in SEQ ID NO: 1, Modification is By substituting the 50th proline residue in the amino acid sequence shown in SEQ ID NO: 1 with an asparagine residue and replacing the 51st proline residue with an amino acid residue other than a proline residue Modifications to be made, A modification which is carried out by glycosylated by replacing the 74th proline residue in the amino acid sequence set forth in SEQ ID NO: 1 with an asparagine residue and replacing the 76th glutamic acid residue with a serine residue, and A modification which is carried out by glycation by replacing the arginine residue at position 186 in the amino acid sequence set forth in SEQ ID NO: 1 with an asparagine residue, Comprising at least one modification selected from the group consisting of (20) The modified HSV gD protein.
- the modified HSV gD protein according to any one of (19) to (24), wherein at least one promiscuous T cell epitope is linked to the C-terminal side of the ectodomain of HSV gD. gD protein.
- the promiscuous T cell epitope is a promiscuous T cell epitope consisting of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 (25
- the serum contains a relatively large amount of neutralizing antibody having high neutralizing activity, as compared to when it is induced by wild type HSV gD. obtain. That is, the modified HSV gD protein of the present invention and a vaccine containing the same can induce immunorefocussing and provide a strong protective effect against HSV. Therefore, high preventive and therapeutic effects can be expected against HSV infection.
- FIG. 1 shows a schematic representation of the primary structure of HSV gD.
- FIG. 1 shows MOE diagrams of the gD receptor binding domain (RBD) peripheral region and the P50 peripheral region on the HSV gD crystal structure.
- FIG. 6 shows the survival rate after prophylactic administration of the anti-gD antibody of Example 5 to mice.
- FIG. 6 shows symptom scores after prophylactic administration of the anti-gD antibody of Example 5 to mice.
- FIG. 7 shows the survival rate after therapeutic administration of the anti-gD antibody of Example 5 to mice.
- FIG. 6 shows symptom scores after therapeutic administration of the anti-gD antibody of Example 5 to mice.
- FIG. 6 shows symptom scores after prophylactic administration of the anti-gD antibody of Example 6 to guinea pigs.
- FIG. 6 shows the symptom score after therapeutic administration of the anti-gD antibody of Example 6 to guinea pigs.
- Figure 7 shows HSV release in vaginal swab following therapeutic administration of the anti-gD antibody of Example 6 to guinea pigs. It is a figure which shows T cell stimulation activity analysis of the synthetic peptide of Example 7 of a mouse, (A) shows the result of IFN-gamma production ELISpot analysis, (B) shows the result of IL-2 production ELISpot analysis.
- FIG. 6 shows symptom scores after therapeutic administration of the anti-gD antibody of Example 5 to mice.
- FIG. 6 shows symptom scores after prophylactic administration of the anti-gD antibody of Example 6 to guinea pigs.
- FIG. 6 shows the
- FIG. 1 shows a schematic diagram of a design strategy for a modified gD protein.
- FIG. 10 shows the reactivities of various gD variants produced in Example 9 with HVEM.
- FIG. It is a figure which shows the analysis result of the neutralizing antibody induction activity using the mouse
- FIG. It is a figure which shows the analysis result of the neutralizing antibody induction activity using the mouse
- FIG. It is a figure which shows the analysis result of the neutralizing antibody induction activity using the mouse
- FIG. It is a figure which shows the analysis result of the neutralizing antibody induction activity using the mouse
- FIG. It is a figure which shows the analysis result of the neutralizing antibody induction activity using the mouse
- FIG. It is a figure which shows the analysis result of the neutralizing antibody in
- FIG. 9 It is a figure which shows the analysis result of the neutralizing antibody induction activity using the mouse
- FIG. It is a figure which shows the analysis result of anti-gD binding antibody induction activity by the ELISA method of Example 9.
- FIG. It is a figure which shows the analysis result of anti-gD binding antibody induction activity by the ELISA method of Example 9.
- FIG. It is a figure which shows the analysis result of anti-gD binding antibody induction activity by the ELISA method of Example 9.
- FIG. It is a figure which shows the analysis result of anti-gD binding antibody induction activity by the ELISA method of Example 9.
- FIG. It is a figure which shows the analysis result of anti-gD binding antibody induction activity by the ELISA method of Example 9.
- FIG. It is a figure which shows the analysis result of anti-gD binding antibody induction activity by the ELISA method of Example 9.
- FIG. FIG. FIG.
- FIG. 16 shows the results of cell-mediated immunity (T cell immunity) inducing activity of Example 9. It is a figure which shows survival rate of experiment 1 of the mouse infection protection test of the modified
- FIG. It is a figure which shows survival rate of experiment 1 of the mouse infection protection test of the modified
- FIG. It is a figure which shows survival rate of experiment 1 of the mouse infection protection test of the modified
- FIG. It is a figure which shows survival rate of experiment 2 of the mouse infection protection test of the modified
- FIG. 16 shows the analysis results of antibody population in immune serum of Example 9.
- FIG. 16 shows the results of inhibition of gD-HVEM interaction by the modified gD immune serum of Example 9.
- FIG. 6 shows the comparison result of multiple alignment of the amino acid sequence of HSV-1 derived gD (SEQ ID NO: 2) and the amino acid sequence of HSV-2 derived gD (SEQ ID NO: 3); Indicates the 50th to 54th amino acid residues (P50 to P54) of gD.
- the modified HSV gD protein of the present invention is a modified protein of envelope glycoprotein D (gD) of herpes simplex virus (HSV), which is located in the receptor binding domain (RBD) in the ectodomain of wild type HSV gD.
- HSV herpes simplex virus
- the present invention is based on the hypothesis proposed by the present inventors, that the “decoy region” is present in the HSV gD antigen.
- the “decoy region” is derived from the English language “Decoy” and is considered to be one of the immune escape mechanisms by which the pathogen escapes from the host's immune response.
- the “decoy region” is an antigen region that induces an antibody with no or low neutralizing antibody activity, and this deceptive inprinting (also referred to as “immune bias”) prevents generation of a neutralizing antibody, or It is believed that the pathogen is a mechanism to escape the host's immune response so that production is low.
- the present inventors conducted detailed epitope mapping analysis on anti-gD monoclonal antibodies obtained by exhaustive search of anti-HSV gD antibodies performed using a human antibody library. As a result, by classifying the B cell epitopes of HSV gD into useless or harmful epitopes and useful epitopes that can induce neutralizing antibodies, the existence of decoy regions where useful or harmful epitopes are concentrated is clarified. I made it. Then, by de-epitoping the futile or harmful epitope of the decoy region and immunologically emphasizing the beneficial epitope, a modified HSV gD protein capable of inducing an antibody with high neutralizing activity has been obtained.
- wild-type HSV gD refers to an HSV-1 derived envelope glycoprotein D (gD) having the amino acid sequence set forth in SEQ ID NO: 2 or a HSV-2 derived gD (SEQ ID NO: 3) having the amino acid sequence set forth in GenBank accession number: ABU 45433. 1) refers to the full length, and as a result of multiple alignment and comparison of the two, the sequence identity is 84% (FIG. 35).
- the three-dimensional structure of gD has also been analyzed, for example, the intracellular domain having amino acid residues 316 to 339, the transmembrane domain having amino acid residues 340 to 369, and It is known to consist of an ectodomain having amino acid residue 315.
- Wild-type HSV gD ectodomain refers to the soluble, antigenic, extracellular region of wild-type HSV gD.
- An example of the wild type HSV gD ectodomain is a wild type gD ectodomain 1-315 derived from HSV-2 strain 333 consisting of the amino acid sequence set forth in SEQ ID NO: 1.
- modified HSV gD protein (also referred to as “modified protein of HSV gD” or “variant”) means that at least one amino acid residue or continuous amino acid residue region relative to wild type HSV gD is It refers to a protein which has been substituted, deleted or added, and also includes a protein which is not present in the wild type, such as a glycosylated protein by substitution or deletion of amino acid residues.
- Neutralizing antibody inducing activity refers to the ability to induce neutralizing antibodies of an antigen protein, which is evaluated by neutralizing antibody titer in immune serum obtained by inoculating an antigen protein test animal. It can be done.
- Negtralizing antibody refers to an antibody capable of losing the infectivity of viral particles, for example, at the concentration of antibody required to reduce the number of plaques of the test virus by 50% (NT50) Assess the height of the neutralizing activity.
- a B cell epitope having high neutralizing antibody inducing activity is referred to as “a useful epitope”.
- Useful epitopes in the ectodomain of wild type HSV gD are typically B cell epitopes present in the receptor binding domain (RBD).
- RBD receptor binding domain
- epitopes containing residues are typically epitopes present in the receptor binding domain (RBD).
- “Decotepe” refers to a B cell epitope having low or no neutralizing antibody inducing activity as compared to the B cell epitope present in RBD in the ectodomain of wild type HSV gD. Classified as "useless or harmful epitope”.
- the “decotope” is selected from the group consisting of the arginine residue at position 134, the aspartic acid residue at position 139, and the arginine residue at position 222 in the amino acid sequence of SEQ ID NO: 1 in the ectodomain of wild type HSV gD
- the B cell epitope has low or no neutralizing antibody-inducing activity as compared to a B cell epitope containing an amino acid residue corresponding to at least one amino acid residue.
- Examples of “decotopes” in the ectodomain of HSV gD in this embodiment include epitopes present in the N-terminal proline rich region (PRR) of gD ectodomain.
- PRR N-terminal proline rich region
- PRR of HSV gD is a decoy region.
- PRR is located opposite to RBD in the crystal structure of the ectodomain of wild type HSV gD ( Figure 3).
- the P50 peripheral region in PRR is a particularly typical decoy region.
- the “P50 peripheral region” refers to the 50th proline of HSV-2 derived wild type gD ectodomain 1-315 consisting of the amino acid sequence set forth in SEQ ID NO: 1 on the surface of the crystal structure of the ectodomain of wild type HSV gD It refers to a region where the distance from the amino acid residue corresponding to the residue is 1.5 nm or less.
- distance from amino acid residue refers to the linear distance from the amino acid residue corresponding to the 50th proline residue regardless of the shape of the surface of the crystal structure of the ectodomain of wild type HSV gD. .
- decotope is an amino acid residue corresponding to the 50th proline residue in HSV-2 derived wild type gD ectodomain 1-315 consisting of the amino acid sequence set forth in SEQ ID NO: 1 of the ectodomain of wild type HSV gD Is an epitope comprising
- the "corresponding" amino acid residue is a multiple alignment of the amino acid sequence of the wild type gD ectodomain derived from HSV-2 consisting of the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence of gD having other similarities.
- sequence alignment multiple it means the amino acid residue of gD having other similarities at the position corresponding to the amino acid residue shown in the predetermined SEQ ID NO: 1 in the aligned sequences.
- decopepe is the 50th proline residue in HSV-2 derived wild-type gD ectodomain 1-315 consisting of the amino acid sequence set forth in SEQ ID NO: 1 on the surface of the crystal structure of HSV gD ectodomain It is an epitope comprising at least one amino acid residue which is present in a region of 1.5 nm or less in distance from the corresponding amino acid (ie, P50 peripheral region).
- the decopepe can be identified from the crystal structure of wild type HSV gD. The distance is preferably 1 nm or less.
- the production ratio of useless or harmful antibodies can be reduced when used for induction of immunity, and the production ratio of highly neutralizing antibodies with high neutralizing activity can be increased by highlighting useful epitopes. it can.
- de-epitope refers to modification of a site that has contributed to antibody production as an epitope in wild-type HSV gD so as not to function as an epitope, and is also referred to as masking of an epitope.
- a method of de-epitope there is a method of replacing an amino acid residue at the site of the epitope with another amino acid residue, a method of deleting (deleting) an amino acid residue at the site of the epitope, and a site of the epitope.
- transducing a sugar chain by substitution or deletion of an amino acid residue are mentioned.
- a method of introducing a sugar chain particularly a method of introducing an N-type sugar chain (N-glycosidic linked sugar chain) is preferable.
- This is effective in that not only the portion into which the sugar chain has been introduced but also its surrounding decote can be masked simultaneously by its bulkiness.
- proteins such as antibodies and receptors that interact with gD
- an interaction network in terms of surface and area in which a wide range of amino acids form a bond cooperatively is formed.
- Glycosylation is considered to be a method of de-epitope effective to hide surrounding residues extensively due to its bulkiness and at the same time inhibit access of the binding partner.
- sugar chains are difficult to induce anti-sugar chain antibodies (Non-patent Document 10), and it is considered that the possibility of appearance of new immunogenicity by modification can be reduced.
- the sugar chain introduction to an amino acid residue refers to a sugar chain introduction to three consecutive amino acid residues including the position of the amino acid residue by deletion, substitution or addition of an amino acid at the position of the amino acid residue.
- the method for introducing a sugar chain is not particularly limited as long as it is a conventional method, but when introducing an N-type sugar chain, for example, the amino acid sequence of the wild type gD protein ectodomain (SEQ ID NO: 1) is used as a template Primers are designed such that three consecutive amino acid sequences at the target site for introducing a sugar chain are N—X—S / T (X is any amino acid other than proline), and mutations are introduced by PCR.
- the gD variant can be obtained by cloning the nucleic acid sequence of the mutant gD protein of interest, and further, if necessary, a tag such as 6 ⁇ His, etc., linked to a suitable vector, and expressing it. Then, an N-type sugar chain is added to asparagine at the target site of the gD variant by a conventional method.
- the N-type sugar chain for example, high mannose type based on GlcNAc, hybrid type, composite type and the like can be mentioned.
- Deepitope that is, modification of the decotope
- the ectodomain of wild-type HSV gD consists of the amino acid sequence set forth in SEQ ID NO: 1, and modification of the decotope, replacing the 50th proline residue in the amino acid sequence set forth in SEQ ID NO: 1 with an asparagine residue A modification that is carried out by being glycosylated by replacing the proline residue at position th with an amino acid residue other than the proline residue, the proline residue at position 74 in the amino acid sequence set forth in SEQ ID NO: 1 to an asparagine residue A modification that is performed by substitution and glycosylation by substituting the 76th glutamic acid residue with a serine residue, and an asparagine residue as the 186th arginine residue in the amino acid sequence set forth in SEQ ID NO: 1 Selected from the group consisting of modifications that are carried out by being glycosylated by substitution with More preferably contains at least one modification.
- the modified HSV gD protein further comprises a deletion of at least a portion of the amino acid residues corresponding to the amino acid residues 251 to 315 in the amino acid sequence of SEQ ID NO: 1 of wild type HSV gD.
- Amino acid residues corresponding to the 251st to 315th amino acid residues in the amino acid sequence of SEQ ID NO: 1 form a C-terminal function region 3 (FR3) in the ectodomain of wild type HSV gD. Deletion of at least part of this part is also effective to induce immunorefocussing to a beneficial epitope.
- FR3 and N-terminal sequence FR1 are bound to be wound on the same surface of core beta sheet structure FR2 in gD molecule. It is suggested that it is possible. Since FR1 and FR3 interfere with each other, only one of them can be coupled to FR2. Usually, on the viral envelope, FR3 is bound, and upon binding to the receptor, it is assumed that the FR3 is detached, the conformation changes so that FR1 is bound, and the receptor binding region is exposed. The neutralizing antibody no. From the epitope analysis of 82, antibody no.
- 82 is known to bind to the Nectin-1 binding region, to decrease the reactivity with the FR1 deficiency (gD34-315), and to inhibit the binding of gD to HVEM or Nectin-1. That is, antibody no.
- gD34-315 the FR1 deficiency
- Nectin-1 That is, antibody no.
- it is considered effective to delete FR3 or to inhibit binding of FR3 to FR2.
- Deletion of at least part of FR3 is deletion of full length of FR3 or deletion of continuous or non-consecutive sequences of part of FR3 and substitution of some amino acid residues for other amino acid residues include.
- the partial deletion of FR3 is, for example, deletion of a portion corresponding to the amino acid residue at positions 276-315 in wild-type gD ectodomain 1-315 derived from HSV-2 consisting of the amino acid sequence set forth in SEQ ID NO: 1 Is preferred, and may be a deletion of all or part of the amino acid residues at positions 276-315.
- the modified HSV gD protein further comprises at least one promiscuous T cell epitope linked to the C-terminus of the ectodomain of HSV gD.
- T cell epitopes also exist in the transmembrane domain and the intracellular domain, considering the use as a vaccine, it is preferable to design in a secretory expression type composed of the extracellular domain, so Linking T cell epitopes is preferable because T cell epitopes not contained in the extracellular region can be effectively used.
- the promiscuous T cell epitope is preferably a promiscuous T cell epitope consisting of the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.
- those having promiscuous stimulating activity for both mouse and human T cells are actually three peptides of DP2, DP3 and DP5.
- DP5 consisting of the amino acid sequence set forth in SEQ ID NO: 8 is particularly preferred.
- promiscuous T cell epitopes may be linked. Specifically, for example, in the ligation of promiscuous T cell epitopes, two or more promiscuous T cell epitopes consisting of the amino acid sequence set forth in SEQ ID NO: 8 are linked to the C-terminus of wild type HSV gD ectodomain May be included.
- the HSV gD protein comprises an amino acid residue corresponding to the valine residue at position 231 in HSV-2 derived wild type gD ectodomain 1-315 consisting of the amino acid sequence set forth in SEQ ID NO: 1 It further includes substitution for amino acid residues (especially tryptophan residues). Since the V231W mutation is suggested to inhibit the binding of FR3 to FR2 (Non-patent Document 9), a variant containing this additional mutation is preferred from the viewpoint of inhibiting the binding of FR3 to FR2. This mutation makes the B cell epitope present in the receptor binding domain more prominent, and can increase the production rate of neutralizing antibody upon induction of immunity.
- the modified HSV gD protein of the present invention can be produced by genetic engineering techniques.
- the production method is not particularly limited. For example, using a cDNA of a wild-type gD protein (SEQ ID NO: 4) as a template, a primer for introducing a target mutation is designed to obtain a nucleic acid into which a mutation has been introduced by PCR. They can be obtained by operatively linking to an expression promoter, optionally linking a tag, and introducing and expressing in an appropriate expression vector. Further, in the case of a modified form by sugar chain introduction, it can be obtained as described above.
- the vector and promoter are not particularly limited, and examples include pCAG vector and CAG promoter.
- the modified HSV gD protein produced may be optionally purified.
- the purification method is not particularly limited, and purification by affinity chromatography column, gel filtration chromatography column, ion exchange chromatography column and the like can be mentioned.
- HSV infections include infections due to HSV-1 and HSV-2, and include, for example, herpes labialis, corneal herpes, genital herpes, generalized neonatal herpes, and stomatitis due to HSV, skin disease, encephalitis, meninges And inflammation and myelitis.
- the HSV vaccine of the invention comprises the modified HSV gD protein of the invention.
- the dosage form of the HSV vaccine of the present embodiment may be, for example, liquid, powder (lyophilized powder, dry powder), capsule, tablet, frozen state.
- the HSV vaccine of the present embodiment may include a pharmaceutically acceptable carrier.
- a pharmaceutically acceptable carrier carriers commonly used for vaccine production can be used without limitation, and specifically, saline, buffered saline, dextrose, water, glycerol, isotonic aqueous buffer and combinations thereof may be mentioned.
- Be The vaccine may further contain an emulsifying agent, a preservative (for example, thimerosal), an isotonicity agent, a pH adjusting agent, and the like as appropriate.
- Adjuvants include, for example, aluminum adjuvants or oil-in-water emulsion adjuvants including squalene (AS03, MF59 etc.), CpG and Toll-like acceptance such as 3-O-deacylated-4'-monophosphoryl lipid A (MPL)
- MPL 3-O-deacylated-4'-monophosphoryl lipid A
- body ligands saponin adjuvants
- polymer adjuvants such as poly ⁇ -glutamic acid
- polysaccharides such as chitosan and inulin.
- the HSV vaccine of the present embodiment can be obtained by mixing the modified HSV gD protein of the present invention and, if necessary, a carrier, an adjuvant and the like.
- the adjuvant may be mixed at the time of use.
- the administration route of the HSV vaccine of this embodiment is, for example, transdermal administration, sublingual administration, eye drop administration, intradermal administration, intramuscular administration, oral administration, enteral administration, nasal administration, intravenous administration, subcutaneous administration, Intraperitoneal administration may be by oral administration via the mouth.
- the administration method of the HSV vaccine of this embodiment is, for example, a method of administering by a syringe, a transdermal patch, a microneedle, an implantable sustained-release device, a syringe with a microneedle, a needleless device, a spray It is also good.
- various antibodies that bind to various epitopes on gD2 were obtained by biopanning with gD2.
- gD2 gD1-315 protein which is a wild type soluble ectodomain derived from 333 strain of HSV-2 having the amino acid sequence set forth in SEQ ID NO: 1 was used.
- the gD1-315 protein was expressed in host cells and purified.
- scFv-phage display library prepared using human VH and VL cDNAs prepared from mRNA derived from human B cell was used.
- the scFv-phage display library is an scFv having reactivity to HSV-2 gD by screening a scFv-phage display library prepared using human VH and VL cDNAs from mRNA derived from human B cells. -I got phage.
- scFv-phage was expressed.
- scFv-phage With respect to the expressed scFv-phage, the reactivity to gD1-315 was confirmed by phage ELISA. After 100 ⁇ L of gD2 (2 ⁇ g / mL PBS) is immobilized on a 96-well microtiter plate (MaxiSorp plate, NUNC) overnight at 4 ° C., each well is washed 3 times with PBS and 300 ⁇ L of 1% BSA / PBS for 1 hour at room temperature. Each well was washed 3 times with PBS-T (0.05% Tween / PBS), scFv-phage was diluted 10-fold with 1% BSA / PBS, added in 100 ⁇ L, and allowed to react at 37 ° C. for 1 hour.
- PBS-T 0.05% Tween / PBS
- HRP-labeled antibody HRP-conjugated anti-M13 antibody diluted in 1% BSA PBS: anti-M13 / HRP / 1% BSA PBS
- TMB enzyme substrate
- ScFv-hFc was prepared based on the seven types of scFv-phage obtained.
- the variable region of the isolated scFv gene was linked to a human Fc gene, cloned into a pCAG vector, and a scFv-hFc expression plasmid was constructed.
- Each expression plasmid was expressed using the Expi 293 expression system (Life technology). After 4 to 6 days of culture, the supernatant was purified by Protain A affinity chromatography columns (HiTrap Protein A HP Columns, GE Healthcare) and dialyzed against PBS. The purity was confirmed by size exclusion chromatography (Superdex 200 5/150 GL, GE Healthcare) and SDS-PAGE.
- Nectin-1 Recombinant Human Nectin-1 Protein, R & D SYSTEMS
- scFv-phage or Nectin-1 Recombinant Human Nectin-1 Protein, R & D SYSTEMS
- 1% BSA PBS 1% BSA PBS at any dilution ratio, added in 100 ⁇ L, and brought to 37 ° C. It was allowed to react for one hour.
- HRP-labeled antibody anti-M13 / HRP / 1% BSA PBS, or HRP-added anti-His-tag antibody diluted in 1% BSA PBS: anti-His-tag / HRP / 1% BSA PBS
- HRP-labeled antibody anti-M13 / HRP / 1% BSA PBS, or HRP-added anti-His-tag antibody diluted in 1% BSA PBS: anti-His-tag / HRP / 1% BSA PBS
- Example 2 Epitope Mapping of Anti-gD2 Binding Antibody Western blotting using gD1-315 in a denatured state was used to analyze whether the epitope of each antibody was a conformational epitope or a linear epitope.
- membranes were reacted with each scFv-hFc at a concentration of 1 ⁇ g / mL 2% skimmed milk-PBS-T for 30 minutes at room temperature. After washing again, it was reacted with anti-hFc / HRP / 2% skimmed milk-PBS-T and developed with Immobilon Western Detection Regent (Millipore).
- the antibodies that reacted with the denaturing agent and gD1-315 denatured by heat shock were no. 1 and No. 1 Two of thirteen, these epitopes were suggested to be linear. All other antibodies were only reactive with non-denatured gD1-315, suggesting a conformational epitope.
- FIG. 1 shows a schematic view of the primary structure of gD, and FR1 (K1 to H39), FR2 (I55 to R184), and FR3 (T251 to G315) are shown, respectively.
- the sugar chain originally added to gD is linked to N94, N121 and N262.
- cDNA SEQ ID NO: 33
- wild type gD protein derived from 333 strain of HSV-2 was used as a template. Since the N-linked sugar chain binds to asparagine of N-X-S / T (X is any amino acid other than proline), when introducing a sugar chain, the following primers are used, and the amino acid at the target site It was mutated by PCR such that the sequence was NXT or NXS (X is any amino acid except proline). Fw is Forward, Re is Reverse. The underlined part is the mutated part.
- SC-A-Fw 5'-CGGGCC AAT GCCTCCTGCAAGTACGCT-3 '(SEQ ID NO: 15)
- SC-A-Re 5'-GGAGGC ATT GGCCCGGTGCTCCAGGAT-3 '(SEQ ID NO: 16)
- SC-B-Fw 5'-GGGTGG AAT GGC ACC AAGCCCCCTACTAC ACC AGC-3 '(SEQ ID NO: 17)
- SC-B-Re 5'-GGGCTT GGT GCC ATT CCACC CGGCGATTTTTAA-3 '(SEQ ID NO: 18)
- SC-D-Fw 5'-CATGCC AAT TCG ACC GCCCCCCAGATCGTGCGC-3 '(SEQ ID NO: 19)
- the completed variant sequence was cloned into pCAGGS1-dhfr-neo vector to obtain a plasmid for expression.
- Each expression plasmid was expressed using the Expi 293 expression system.
- the supernatant was purified by Ni-NTA affinity chromatography column (TALON superflow Metal Affinity Resin, TaKaRa) and dialyzed against PBS. The purity was confirmed by size exclusion chromatography and SDS-PAGE.
- Reactivity analysis of each antibody was carried out by competitive ELISA against these glycosylated mutants.
- 100 ⁇ L of gD variant (2 ⁇ g / mL PBS) was immobilized on a 96 well microtiter plate (MaxiSorp plate, NUNC) at 4 ° C. overnight.
- Each well was washed 3 times with PBS and blocked with 300 ⁇ L of 1% BSA PBS for 1 hour at room temperature.
- Each well is washed 3 times with PBS-T (0.05% Tween PBS), each scFv-hFc is diluted in 1% BSA PBS at an arbitrary dilution ratio, added in 100 ⁇ L, and allowed to react at 37 ° C.
- C-terminal (FR3) deficient body gD1-275 and gD25-253 have antibody No. The reactivity of 1 disappeared. From the above analysis, antibody no. 1 is suggested to have a linear epitope, so antibody Nos. One epitope was considered to be present.
- gD254-275 is a flexible region that corresponds to the "root" of FR3, depending on the structure of the entire FR3, antibody no. 72 and antibody no. It is presumed that the strength of the bond of 75 changes. That is, in FR1 deficient gD34-315, FR3 was bound to FR2, and as a result, the epitope in the vicinity of P50 (SC-F) and the epitope in gD254-275 were separated and the reactivity decreased. It is thought that it is not. Similarly, antibody no. 75 and the epitope in the vicinity of P50 (SC-F) and the epitope in gD254-275 should be far apart. Since 75 strongly binds to gD254-275, it is speculated that it was not susceptible to this effect.
- Antibody No. The reactivity of the 82 antibody with an alanine substituted gD variant was analyzed by a competition ELISA. The results are shown in FIG. Antibody No. In the reactivity analysis of 82, the reactivity was significantly reduced in the five mutants of R134A, D139A, T213M, S216N and R222A, and the reactivity was reduced in R196A. It is reported that these amino acids are all amino acids located in close proximity to each other at the same interface, and in particular, for S216 and R222, the binding site of Nectin-1. From this, antibody no. It was again suggested that 82 binds to the Nectin-1 binding domain.
- D30A is considered to be a part of the epitope present in 25-33 amino acids, but in the vicinity of R222 and D30, when FR1 is bound to FR2, FR1 is present on the same interface due to interference I can not do it. For this reason, antibody no.
- 82 binds it is thought that the reaction proceeds while the bonds between FR1 and FR2 are released.
- antibody no With regard to antibodies other than 82, in the reactivity analysis using SC-F peripheral alanine substitution products, a tendency to almost agree with the results of reactivity of various anti-gD2 antibodies and various gD variants shown in Table 3 is seen The Antibody No. The reactivity of 78 decreased at P51A and I55A. Antibody No. No. 72 for V57A and E256A, antibody no. At 75, in addition to the two, a decrease in reactivity was observed with I55A and D259A. SC-F and all these residues are located in the vicinity on the same interface. 72 and antibody no. Results support the hypothesis that some of the 75 epitopes are between 254-275. Antibody No. As for No. 5, antibody no.
- the three alanine substitutes having reduced reactivity with 5 are I55, E76, and I80. 72, antibody no. 75, antibody no. The 78 binding regions were scattered in directions different by about 120 ° around I55.
- Antibody No. The epitope of 13 was unknown.
- Antibody No. The 13 epitopes are suggested to be Linear, which is considered to be because the mutants so far could not be mutated directly.
- FIG. 3 shows a MOE view of the gD receptor binding domain (RBD) peripheral region and the P50 peripheral region on the HSV gD structure.
- RBD gD receptor binding domain
- Example 3 Neutralizing Activity of Anti-gD2 Antibody The in vitro HSV neutralizing activity analysis of seven antibody clones was carried out in a plaque number reduction test and a cell to cell infection spread suppression test.
- the target viruses used were 2 types of Human herpesvirus 2 (HSV-2) MS strain (VR-540) and Human herpesvirus 1 (HSV-1) KOS strain (VR-1493) purchased from ATCC.
- Vero cells CCL. 81) purchased from ATCC were used for virus culture, infectivity titer measurement, and neutralizing antibody titer measurement. Vero cells are cultured at 37 ° C., 5% CO 2 conditions. At the time of expansion, maintenance, and analysis plate preparation, MEM medium containing 10% FBS was used, and at the time of infectivity measurement and neutralizing antibody titer measurement, MEM medium containing 2% FBS was used.
- the virus bank used for the neutralization test and the below-mentioned infection defense ability analysis was prepared by the following method.
- the HSV-2 MS strain and the HSV-1 KOS strain were m. o.
- the recovered infected cell culture bottle was freeze thawed three times to disrupt the cells, and then centrifuged at 3500 rpm in a TOMY centrifuge at room temperature for 10 minutes, and the supernatant was used as an HSV-2 virus bank and an HSV-1 virus bank.
- ⁇ Neutralization test> (Plaque number reduction test) In the plaque reduction activity measurement, a test antibody was prepared to a predetermined concentration, mixed with about 100 PFU of HSV-2 MS strain or HSV-1 KOS strain, and then reacted at 37 ° C. for 1 hour. The reaction solution is seeded on a full sheet of Vero cells in a 48-well plate, adsorbed at 30 ° C. for 1 hour, cultured for 24 hours in 1% methylcellulose-containing MEM (2% FBS) medium, and methanol / ethanol 1 to 1 In 50% methanol / 50% ethanol ( ⁇ 20 ° C.) mixed for 30 minutes at ⁇ 20 ° C.
- an anti-HSV gD monoclonal antibody is reacted at 37 ° C. for 1 hour, immunostained with anti-mouse IgG-HRP (Dako P0447) and TMBH, and an image of each well is taken in with an ELISPOT analyzer (ImmunoSpot S6 Analyzer, CTL company) The number of plaques was counted using analysis software (BioSpot CTL).
- Vero cells in full sheet in a 48-well plate were inoculated with about 100 PFU of HSV-2 MS strain or HSV-1 KOS strain, and adsorbed at 30 ° C. for 1 hour. Thereafter, 1% methylcellulose-containing MEM (2% FBS) medium (antibody concentrations 5 ⁇ g / mL, 25 ⁇ g / mL and 125 ⁇ g / mL) containing a predetermined concentration of the test antibody is added, and the HSV-2 MS strain is about 40 hours After culture, the HSV-1 KOS strain was cultured for about 48 hours and then inactivated and fixed at -20.degree. C.
- the self-prepared anti-HSV gD monoclonal antibody is reacted at 37 ° C. for 1 hour, immunostained with anti-mouse IgG-HRP and TMBH, the image of each well is taken in with ELISPOT analyzer, and the average plaque size in analysis software was analyzed.
- antibody No. 1 did not show any plaque number reducing activity against both strains.
- the neutralizing activities of 13 against HSV-1 and HSV-2 were both relatively weak, and showed a pattern of partial inhibition against HSV-1.
- "partial inhibition” refers to a case where the neutralizing activity is relatively weak, and a dose-dependent decrease has not been clearly confirmed.
- the antibody No. 75 was significantly weaker in neutralizing activity against HSV-2 than HSV-1 in any case. 72 showed a partial inhibition pattern for HSV-2.
- the cell-to-cell infection spread inhibitory activity was examined in duplicate by setting the final concentration of each antibody to 20 ⁇ g / mL against MS strain (HSV-2). While 82 showed clear inhibitory activity, the other 6 antibodies did not show clear activity. This activity is considered to be an important activity that can lead to an effect of suppressing the spread of infection or therapeutic effect on recurrent symptoms in therapeutic administration under conditions where viral infection has already been established in the living body.
- antibody no. 82 not only has potent plaque number reduction activity against both HSV-1 and HSV-2 strains, but also has cell to cell infection spread suppression activity that can lead to therapeutic effects, etc. Is considered to be a unique antibody with superior features that are essentially different from the anti-gD binding antibody of the present invention, and its superiority is related to the presence of the epitope region on the gD receptor binding domain (RBD) It was considered a thing.
- Example 4 Anti-gD2 Antibody No. Detailed analysis of 82 virus neutralization activity Anti-gD2 antibody No. In addition to the 82 human antibodies scFv-hFc, human-mouse chimeric IgG whose Fc region was made mouse type and human-guinea pig chimeric IgG whose Fc region was made guinea pig type were prepared, respectively HSV-2 (MS strain ) And HSV-1 (KOS strain) were examined for neutralizing activity (plaque number reduction activity).
- HSV-2 MS strain
- HSV-1 KOS strain
- VH region of the isolated scFv gene was ligated with a heavy chain constant region gene (CH1-CH2-CH3) derived from mouse IgG2a, and cloned into a pCAG vector to construct a heavy chain expression plasmid.
- VL region of the scFv gene was linked to a mouse CL gene, and cloned into a pCAG vector to construct an L chain expression plasmid.
- the Expi293 expression system was used. The expression plasmid was transfected into cells, and the culture supernatant was recovered in 4 to 6 days.
- the culture supernatant was purified using Hi Trap Protein A HP Column (GE Healthcare) and dialyzed against PBS. The purity was confirmed by size exclusion chromatography and SDS-PAGE. Human-mouse chimeric IgG2a was obtained by the above method.
- the VH region of the isolated scFv gene was ligated with the H chain constant region gene (CH1-CH2-CH3) derived from guinea pig IgG2, and cloned into pCAG vector.
- the VL region of the scFv gene was linked to the guinea pig CK gene and cloned into the pCAG vector.
- the cloned antibody gene was amplified by PCR and cloned into a pXC vector (Lonza) to construct H-chain and L-chain expression plasmids. Thereafter, both plasmids were ligated into one expression plasmid.
- CHO cells were used for expression.
- the expression plasmid was stably introduced into CHO cells, and antibody overexpressing CHO cells were obtained using GS Xceed expression system (Lonza).
- the high expressing cells were cultured for 12 days in Fed-Batch, and the culture supernatant was recovered.
- the culture supernatant was purified using rProteinA sepharose Fast Flow (Cat # 17127903 / GE Healthcare) to obtain human-guinea pig chimeric IgG2 kappa antibody.
- Anti-gD2 antibody No. Using 82 scFv-hFc, human-mouse chimeric IgG and human-guinea pig chimeric IgG, virus neutralization activity (plaque number reduction activity) was analyzed in the same manner as in Example 4. The results are shown in Table 6.
- Antibody No. The 82 scFv-hFc, human-mouse chimeric IgG, and human-guinea pig chimeric IgG are all 0.05 ⁇ g / mL or more against both MS (HSV-2) and KOS (HSV-1) strains. The concentration showed 50% plaque number reduction activity.
- Example 5 Anti-gD2 Antibody No. 82 Evaluation of mouse infection protective ability Using the mouse genital herpes infection model, anti-gD2 antibody no. The protective ability at 82 prophylactic and therapeutic administration was evaluated.
- ⁇ Mouse infection protection test> A murine genital herpes infection model was used to perform an infection protection test in the prophylactic and therapeutic administration of anti-HSV gD2 monoclonal antibodies.
- BALB / c mice (5 weeks old, female) were used.
- a predetermined amount of antibody is dissolved in saline for injection (saline), and 200 ⁇ L / animal, 24 hours before virus inoculation for prophylactic administration and 48 hours after virus inoculation for therapeutic administration.
- saline saline
- 200 ⁇ L / animal 200 ⁇ L / animal, 24 hours before virus inoculation for prophylactic administration and 48 hours after virus inoculation for therapeutic administration.
- was administered intraperitoneally at a volume of The number of cases of N 10 was set per group.
- Depo-Provera was subcutaneously inoculated at 2 mg / animal 6 days before virus inoculation. Under anesthesia, 5 ⁇ 10 5 PFU / 20 ⁇ L of HSV-2 MS strain was intravaginally inoculated, and 21 days follow-up was performed. The ability to protect against infection was evaluated using survival days (survival rate) and symptom scores as indices. The symptom score defined the score by the presence or absence and the degree of the vaginal lesion symptom, and showed the average value in each group.
- the scoring method was 0: no change, 1: partial erythema / swelling, 2: extensive swelling / edema, 3: ulcer / bleeding, 4: death. If there are serious systemic symptoms with no prospect of recovery (pilation, paralysis, tremors, convulsions, etc.), the score is set to 3.5 on that day, sacrificed for death, and treated as death the next day, the score is 4 And
- Table 7 shows survival days by dose, survival rate in FIG. 4 and symptom score in FIG. 5 in the prophylactic administration.
- Table 8 shows survival days by dose, survival rate in FIG. 6, and symptom score in FIG. 7 in therapeutic administration.
- Non-patent Document 8 there is a report that HSV migrates to ganglia within 48 hours after invading the body from locally infected sites.
- antibody no In the case of therapeutic administration of 82, the 10 mg / kg and 3 mg / kg two doses showed significant improvement in survival rate and symptom score, almost the same as in the case of prophylactic administration. From the above, antibody no. It has been confirmed that 82 exhibits a strong protective effect against not only prophylactic administration but also therapeutic administration.
- Example 6 Anti-gD2 Antibody No. 82 guinea pig infection protective ability evaluation The guinea pig genital herpes infection model (acute stage) is used, anti-gD2 antibody No. The protective ability at 82 prophylactic and therapeutic administration was evaluated.
- ⁇ Guinea pig infection protection test> Using the guinea pig genital herpes infection model, anti-gD2 antibody no. Infection protection testing was performed on prophylactic and therapeutic administration of 82 (human-guinea pig chimeric IgG2 ⁇ ). Hartley guinea pigs (3-5 weeks old, female) purchased from SLC were used. A predetermined amount of antibody is dissolved in saline for injection (saline), and in the case of prophylactic administration, 1 mg to 30 mg each 24 hours before virus inoculation and in the case of therapeutic administration 4 days after virus inoculation. It was administered intraperitoneally at a volume of / kg / animal.
- vaginal swab vaginal swab
- the vaginal Swab was collected by wiping the mucous membrane on the inner wall of the vagina after inserting a swab moistened with MEM medium into the vagina.
- vaginal swabs were suspended in MEM medium dispensed in 1 mL aliquots in siliconized tubes and stored frozen until use.
- Vaginal swab was diluted with stock solution, 10 times, 100 times, 1000 times, and seeded at 100 uL / well in 90-well or 48-well full-sheet Vero cells.
- virus adsorption was performed at 37 ° C. for 1 hour, and after culturing for 24 to 72 hours in 2% FBS MEM medium containing 1% methyl cellulose, the number of plaques was counted by a predetermined method.
- Symptom scores for prophylactic administration are shown in FIG. 9 .
- Symptom scores for the results of therapeutic administration are shown in FIG. 9 and HSV release in vaginal swab is shown in FIG.
- saline administration group set as negative control group at 30 mg / kg, 10 mg / kg, 3 mg / kg out of set doses (30 mg / kg, 10 mg / kg, 3 mg / kg, 1 mg / kg) It showed a significant improvement in symptom score compared to.
- Example 7 Comprehensive analysis of T cell epitopes present on HSV gD ⁇ Search for HLA Class II restricted promiscuous T cell epitope cluster sequences present on HSV gD 2> Regarding the gD2 full-length amino acid sequence (ABU 45433.1; SEQ ID NO: 3) of HSV-2 333 strain published in GenBank, HLA Class II restricted promiscuous T cell epitope cluster using EpiVax's algorithm (EpiMatrix) The sequence was searched.
- the cluster sequences are classified into eight major HLA DR super types analyzed by EpiMatrix (DRB1 * 0101, DRB1 * 0301, DRB1 * 0401, DRB1 * 0701, DRB1 * 0801, DRB1 * 1101, DRB1 * 1301, DRB1.
- Peptide synthesis was performed on these 5 sequences.
- the peptide was modified with acetylation at the N-terminal side and amidated modification at the C-terminal side. Aliquots of 2.5 mg / tube were made at a synthetic purity of 95% or more, and the lyophilized peptide was stored at -20.degree. When using lyophilized peptide, it was dissolved or suspended in DMSO (Sigma D2650) to 10 mM. The synthesized peptides were subjected to human and mouse T cell stimulation activity analysis.
- HLA DR super types incorporated into the EpiVax algorithm (DRB1 * 0101, DRB1 * 0301, DRB1 * 0401, DRB1 * 0701, DRB1 * 0801, DRB1 * 1101, DRB1 * 1301, DRB1 * 1501 2.) Frozen samples of donor PBMCs with homozygous or heterozygous of any of the genes and having a history of HSV exposure were purchased. In addition, PBMCs from anti-HSV antibody negative donors were also purchased to analyze nonspecific responses (Donor 14, 67).
- C. T. Frozen cells were thawed and washed with Thawing Medium (CTL WashTM Medium) according to the protocol of L company, adjusted to a predetermined concentration with a medium (CTL TestTM Medium), and subjected to human IFN- ⁇ ELISpot analysis.
- Cells are seeded at 100 ⁇ L each at a concentration of 0.5 and 1 ⁇ 10 7 cells / mL in ELISpot dedicated 96 well plates, and 100 ⁇ L of each peptide solution prepared to 20 ⁇ M is added (final concentration: 10 ⁇ M / 0.1% DMSO included)
- the medium was then cultured at 37 ° C. in a CO 2 incubator for 5 days.
- Table 10 (A) shows the average number of spots of the number of IFN- ⁇ producing cells, and gray shading indicates that the number of spots was too small to be determined.
- Table 10 (B) performs determination of the presence or absence of T cell stimulation activity, using the stimulation index (SI: Stimulation Index) with respect to a negative control (None) as an index.
- SI Stimulation Index
- Ni negative control
- T cell stimulatory activity analysis was performed by mouse immunogenicity studies of synthetic peptides.
- a stock solution was prepared by dissolving or suspending synthetic peptide in DMSO at 10 mM.
- 10% HCO-60 / saline (saline for injection) was prepared using NIKKOL HCO-60 (manufactured by Nikko Chemicals Co., Ltd.) as a solvent for administration.
- 100 ⁇ g of the synthetic peptide was mixed in a solvent with 10 ⁇ g of CpG and 10 ⁇ g of MPLA, adjusted to a volume of 210 ⁇ L / animal, and administered subcutaneously to the back of a mouse (4-5 weeks old, female).
- mice Three strains of BALB / c (I-Ad / I-Ed), C57BL / 6 (I-Ab) and C3H / HeN (I-Ak / IEk) were used as mice. Twenty-one days after the primary immunization, a booster was given, and two weeks after that, spleens were collected and spleen cells were prepared and subjected to the following cytokine production response analysis (ELISpot analysis). The prepared splenocytes were seeded at 1 ⁇ 10 6 cells / well in PVDF membrane 96 wells (MSIPS 4 W 10 Millipore), and cultured with 10 ⁇ M of various peptides for 20 hours.
- ELISpot analysis cytokine production response analysis
- the culture was carried out using RPMI 1640 medium in the presence of 10% FBS in an incubator set at 37 ° C. and a CO 2 concentration of 5%.
- the Con A addition group was set as a positive control.
- Detection of IFN- ⁇ producing cells is mouse IFN gamma ELISPOT Ready-SET-Go! It carried out using (trademark, ebioscience 88-7384-88). Images were captured with ELISpot analyzer (CTL Immunospot S5 versa analyzer) and the number of spots was counted with immunospot software.
- Each of 5 peptides of DP1 to DP5 was a mouse of 3 strains of BALB / c (I-Ad / I-Ed), C57BL / 6 (I-Ab) and C3H / HeN (I-Ak / IEk) (each group n After immunizing (2), spleens were harvested, splenocytes were prepared, and T cell responsiveness (IFN- ⁇ and IL-2 production stimulating activity) to each peptide was analyzed by ELISpot. All measurements of each sample were performed in 2 wells. The experimental results are shown in FIG. In any of IFN- ⁇ production ELISpot analysis (FIG. 11 (A)) and IL-2 production ELISpot analysis (FIG. 11 (B)), four peptides other than DP1 can stimulate mouse spleen T cells of two or more strains. confirmed.
- Example 8 Population Analysis of Anti-gD2 Antibodies Contained in Human Serum Fraction (Immunoglobulin)
- Benilon a human gamma globulin cocktail
- a competition test with the obtained antibody was carried out using (Blood Don Benil (registered trademark) -I for intravenous injection, Institute of Chemistry and Serum Therapy Research Institute).
- As a competitive test monoclonal antibodies were reacted after reacting gD1-315 with benilon. In this system, it is considered that the binding amount of the monoclonal antibody (test antibody) decreases and the competition rate increases as the amount of the competitive antibody contained in the Benilon increases.
- SPR Biacore
- the chip surface was treated twice with 350 mM EDTA for 10 seconds, and washed with Buffer for 10 seconds. Immobilization and regeneration were performed in the same manner each time a new sample was measured.
- the competition rate was calculated as follows. Apply the antibody to be examined at a concentration of 20 ⁇ g / mL for 120 seconds at a flow rate of 20 ⁇ L / min, increase RU to (1), continue applying Benilon at a concentration of 150 ⁇ g / mL for 120 seconds at a flow rate of 20 ⁇ L / min, and then When 20 ⁇ g / mL antibody was applied for 120 seconds and the increased RU was (2), the competition rate was calculated by the formula of (1- (2) / (1)) ⁇ 100.
- the number of antibodies recognizing an epitope in the P50 peripheral region on the wild-type gD antigen is the number of antibodies recognizing an epitope on or around RBD It turned out that there were more than.
- the P50 peripheral region was considered to be a decoy region that can induce a large amount of low-benefit antibodies although it has high antibody-inducing ability.
- Example 9 Design of HSV gD Variants for Vaccine Antigens
- the RBD as an epitope region for which neutralizing antibody epitopes are present, and the P50 peripheral region where epitopes of less useful antibodies are present.
- a gD protein variant capable of inducing more neutralizing antibodies by using FR3 as the decoy region highlighting useful epitopes, de-epitoping the epitopes in the decoy region by glycosylation or deletion mutation, etc.
- gD modification as described below from the three viewpoints of being able to efficiently and effectively elicit both humoral immune responses and cellular immune responses by linking promiscuous T cell epitope cluster peptides.
- the body was designed and its inducing activity of neutralizing antibody was examined.
- FR3 and the N-terminal sequence FR1 are within gD molecule. It has been suggested that it can be wound together on the very same side of the core beta sheet structure FR2. Since FR1 and FR3 interfere with each other, only one of them can be coupled to FR2. Usually, on the viral envelope, FR3 is bound, and upon binding to the receptor, it is assumed that the FR3 is detached, the conformation changes so that FR1 is bound, and the receptor binding region is exposed.
- Antibody No. From the epitope analysis of 82, antibody no.
- 82 is known to bind to the Nectin-1 binding region, to decrease the reactivity with the FR1 deficiency (gD34-315), and to inhibit the binding of gD to HVEM or Nectin-1. That is, antibody no. It was considered effective to delete FR3 or to inhibit the binding of FR3 to FR2 in order to highlight 82 epitopes and induce immunorefocussing to the relevant region.
- the FR3 deficiency was based on gD1-275 reported in the literature (Non-patent document 12). In addition, antibody no. Since the presence of one epitope, it was expected that deletion of FR3 would suppress the induction of some non-neutralizing antibodies.
- Non-patent Document 9 the V231 W mutant has been suggested to inhibit the binding of FR3 to FR2.
- antibody no Although it can not be expected to suppress the induction of non-neutralizing antibodies such as 1st class, antibody no. It was thought possible to highlight 82 epitopes.
- T cell epitope peptide of (3) With regard to the linkage of the T cell epitope peptide of (3), a T cell immune response can be induced by linking the predicted T cell epitope sequence to the C terminus of the variant.
- T cell epitopes also exist in the transmembrane domain and the intracellular domain, considering the use as a vaccine, it is necessary to design a secretory expression type composed of the extracellular domain, so the transmembrane domain and the intracellular domain By linking T cell epitopes present in the region, T cell epitopes not included in the extracellular region can be effectively used.
- FIG. 1 The schematic diagram of the design strategy of the above modified gD is shown in FIG. Moreover, in the three-dimensional structural model of HSV gD2 monomer, glycan introduction site and predicted antibody No. 1 are expected. The addition of 82 epitopes etc. is shown in FIG.
- variants based on gD1-275 were prepared. As these are FR3 deficient variants, the V231W mutations affecting FR3 were not examined. Although there was no change in the properties only with sugar chain introduction (gD1-275v3 and gD1-275v5), the presence of aggregates was confirmed in gD1-275v3-55 among the variants linked with DP5. Since the difference from gD1-275v5-55, which had good properties, was whether or not R186 (SC-A) was introduced, the three-dimensional structure of R186 (SC-A) existing near 275 aa in structure is DP5. It is inferred that the structural change that triggers aggregation, such as adsorption to the main part of the substance, has been prevented. On the other hand, eight variants were prepared based on gD1-315, and none of them was lower than gD1-315, which is the drug substance, in terms of both the characteristics and the expression amount.
- FIG. 13 shows antibody No. of each gD variant when the reactivity with gD1-315 (wild type) is “1”. The relative value of reactivity with 82 is shown.
- Antibody No. Although 82 bound to all the gD variants produced, the strength of its reactivity varied.
- Antibody No. to gD1-315 Based on the reactivity of 82, in most variants, antibody no. The reactivity with 82 was enhanced.
- the reactivity with 82 was enhanced, and in particular, gD1-315v5-55 exhibited about 12 times as much reactivity as gD1-315 bulk.
- the tandem linkage of DP5 to the C-terminus is thought to play a role in that the sequence of DP5 itself inhibits the binding of FR3 to the gD receptor binding domain.
- antibody No. 1 is compared with the mutant without mutation (gD1-315v3-55, gD1-315v5-55).
- the reactivity of 82 slightly decreases.
- the literature non-patent document 9 in which V231W was introduced, it was reported that it had the effect of inhibiting the binding of FR3. It is thought that there is an effect of changing the
- each variant has a structure in which FR1 is bound to the receptor binding region. Therefore, the reactivity of HVEM and each variant was measured by competitive ELISA.
- HVEM can bind to gD only when FR1 binds to the receptor binding region and adopts the characteristic hairpin structure. All interaction sites of HVEM were shown to be present in FR1, and the improvement of reactivity with HVEM was as described above.
- the product with the highest reactivity with HVEM as compared with the original drug gD1-315 is gD1-315v5-55, then gD1-315v5-55V or gD1-275v5-55, and further gD1-315v3-
- the reaction with HVEM in the order of 55, gD1-315 and gD1-315v5 showed almost no reaction.
- FR3-deleted gD1-275v5-55 promotes the binding of FR1 followed by the binding of HVEM over the other gD1-315 based variants in which FR3 is present.
- Antibody No. Since 1-275v5 and 1-315v5 showed almost the same reactivity in the reactivity analysis of 82, it can be inferred that the effect of FR3 deficiency is almost the same as the effect of R186 (SC-A) introduction.
- ⁇ Mouse immunogenicity test of modified gD> The wild-type gD antigen gD1-315 (WT) was used as a positive control and the saline as a negative control, and an immunogenicity test of the modified gD antigen was performed.
- a predetermined amount of antigen was dissolved in saline for injection (Saline) together with MPLA (10 ⁇ g / animal) and CpG (1 ⁇ g / animal), and mice were immunized with a volume of 200 ⁇ L / animal.
- the analysis results on the neutralizing antibody induction activity are shown in FIG. 15 to FIG.
- the data for modified gD is shown as a solid black line
- the data for wild type gD is shown as a gray dotted line
- the high dose administration group (3 ⁇ g / animal) is a closed circle
- the middle dose administration group (0.3 ⁇ g / animal) is a black triangle
- the low dose administration group (0.1 ⁇ g / animal) is indicated by a black square.
- n 4 animal cases in each group, their mean values were plotted, and ⁇ SE error bars were added.
- the symbols and broken lines in each graph were the same as in the above-mentioned neutralizing antibody induction activity graph. Contrary to the results regarding the neutralizing antibody activity, 11 types of gD1-315v5 of (C), gD1-315V of (F), and gD1-275 of (J) among the 14 types of modified gD used for evaluation
- the anti-gD binding antibody activity in the immune serum tended to show a relatively low value as compared to that of the wild type gD. That is, it was found that modified gD induces high neutralizing antibody activity at a lower binding antibody titer (amount of antibody) than wild-type gD.
- T cell-mediated immunity inducing activity
- mice 3 ⁇ g / mouse administration group
- mice 3 ⁇ g / mouse administration group
- gD1-315v3-55 wild type gD
- wild type gD wild type gD
- the IFN- ⁇ production response activity (recall activity) of splenocytes (pool samples containing 4 cases) was compared and analyzed.
- 5-week-old BALB / c mice were immunized 3 times with 2 ⁇ g of antigen on the back subcutaneously at 2 week intervals, and spleen cells were collected 2 weeks after the final immunization.
- mice genital herpes infection infection model the protective ability of various modified gD was evaluated by prophylactic administration.
- the experiment was performed a total of 4 times (Experiments 1 to 4), and all 0.1 ⁇ g / animal / dose, 0.03 ⁇ g / animal / dose, 0.01 ⁇ g / dose for positive control, negative control, and test modified gD antigen.
- Four doses of animal / dose and 0.003 ⁇ g / animal / dose were set.
- Depo-Provera was subcutaneously inoculated at 2 mg / animal 6 days before virus inoculation. Under anesthesia, 5 ⁇ 10 5 PFU / 20 ⁇ L of HSV-2 MS strain was intravaginally inoculated, and 21 days follow-up was performed. The infection protective ability was evaluated using the survival days (survival rate) as an index. The average values were plotted in the graph. Kaplan-Meier test for significant difference against negative control group, and the minimum effective dose based on the test result is "++", one dose stronger than the wild-type gD by two times more than common dose Was determined to be "+” and the equivalent was " ⁇ ".
- ⁇ Antibody population analysis in modified gD immune serum> Of the series of modified gDs that were found to be superior to wild-type gD (gD1-315) in the mouse immunogenicity test and mouse infection protection test, gD1-315v3-55 and gD1-315v5-55 Analysis of immune serum was performed to investigate the mechanism. Target antibody No. It was investigated whether efficient induction of 82-like antibodies was actually triggered. In order to make the antibody amount uniform, IgG was purified from serum and analyzed by a competition method using SPR (Biacore).
- Anti-gD2 antibody competition test using SPR Anti-gD2 antibody competition studies were performed using Biacore 3000 (GE Healthcare), HBS-EP buffer (GE Healthcare) was used in all experiments, temperature was set at 25 ° C, flow rate was 20 ⁇ L min.
- the Sensor chip used CM5 sensor chip (GE Healthcare), and experiments were performed based on the recommended protocol. Approximately 300 RU of gD1-315-His was immobilized on the chip surface and washed twice for 30 seconds at a flow rate of 30 ⁇ L / min using 0.1 M and pH 2.0 glycine-hydrochloride buffer for chip regeneration. . Procedure of competition, calculation of competition rate was performed as follows.
- HVEM Recombinant Human HVEM / TNFRSF14 Fc Chimera Protein, R & D SYSTEMS
- HVEM Recombinant Human HVEM / TNFRSF14 Fc Chimera Protein, R & D SYSTEMS
- HRP-labeled antibody anti-hFc / HRP / 1% BSA PBS
- the results are shown in FIG.
- the antibody that inhibits the gD-HVEM interaction is more abundant in the modified gD immune serum than in the wild type gD immune serum, and in particular, the gD1-315v5-55 immune serum showed a strong inhibitory action. From the above results, it is suggested that the modified gD may provide a stronger protective effect by inducing a high quality antibody capable of inhibiting the interaction between gD and HVEM more efficiently than the wild type gD.
- the modified HSV gD protein of the present invention can be used to produce a vaccine effective for the prevention and treatment of HSV infection.
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Abstract
Description
(1)単純ヘルペスウイルス(HSV)のエンベロープ糖タンパク質D(gD)の改変タンパク質(改変型HSV gDタンパク質)であって、野生型HSV gDのエクトドメイン(ectodomain)において、レセプター結合ドメイン(RBD)に存在するB細胞エピトープと比較して、中和抗体誘導活性が低い又は無いB細胞エピトープ(デコトープ)の少なくとも1つがエピトープとして機能しないように改変された、改変型HSV gDタンパク質。
(2)RBDに存在するB細胞エピトープが、配列番号1に記載のアミノ酸配列における134番目のアルギニン残基、139番目のアスパラギン酸残基、及び222番目のアルギニン残基からなる群より選択される少なくとも1つのアミノ酸残基に相当するアミノ酸残基を含むエピトープである、(1)の改変型HSV gDタンパク質。
(3)デコトープは、gDエクトドメインのN末端プロリン・リッチ領域(PRR)に存在するB細胞エピトープである、(1)又は(2)の改変型HSV gDタンパク質。
(4)デコトープは、
野生型HSV gDのエクトドメインの、配列番号1に記載のアミノ酸配列における50番目のプロリン残基に相当するアミノ酸残基を含むエピトープ、又は、
野生型HSV gDのエクトドメインの結晶構造の表面において、上記50番目のプロリン残基に相当するアミノ酸からの距離が1.5nm以下の領域に存在する少なくとも1つのアミノ酸残基を含むエピトープ
である、(3)の改変型HSV gDタンパク質。
(5)デコトープの改変は、アミノ酸残基の置換、アミノ酸残基の欠損、及び/又はアミノ酸残基の置換又は欠損によって糖鎖導入されることによって行われる、(1)~(4)のいずれかの改変型HSV gDタンパク質。
(6)デコトープの改変は、デコトープの改変は、野生型HSV gDのエクトドメインの、配列番号1に記載のアミノ酸配列における50番目のプロリン残基に相当するアミノ酸残基への糖鎖導入よって行われる改変を含む、(5)の改変型HSV gDタンパク質。
(7)デコトープの改変は、野生型HSV gDのエクトドメインの、配列番号1に記載のアミノ酸配列における74番目のプロリンに相当するアミノ酸残基への糖鎖導入によって行われる改変を含む、(5)又は(6)の改変型HSV gDタンパク質。
(8)デコトープの改変は、野生型HSV gDのエクトドメインの、配列番号1に記載のアミノ酸配列における186番目のアルギニンに相当するアミノ酸残基への糖鎖導入によって行われる改変を含む、(5)~(7)のいずれかの改変型HSV gDタンパク質。
(9)野生型HSV gDのエクトドメインは、配列番号1に記載のアミノ酸配列からなり、
デコトープの改変は、
配列番号1に記載のアミノ酸配列における50番目のプロリン残基をアスパラギン残基に置換し、51番目のプロリン残基をプロリン残基以外のアミノ酸残基に置換することによって糖鎖導入されることによって行われる改変、
配列番号1に記載のアミノ酸配列における74番目のプロリン残基をアスパラギン残基に置換し、76番目のグルタミン酸残基をセリン残基に置換することによって糖鎖導入されることによって行われる改変、及び
配列番号1に記載のアミノ酸配列における186番目のアルギニン残基をアスパラギン残基に置換することによって糖鎖導入されることによって行われる改変、
からなる群より選択される少なくとも1つの改変を含む、(5)の改変型HSV gDタンパク質。
(10)糖鎖がN型糖鎖である、(5)~(9)のいずれかの改変型HSV gDタンパク質。
(11)改変型HSV gDタンパク質はさらに、HSV gDのエクトドメインのC末端側に少なくとも1つのプロミスキュアスT細胞エピトープが連結されている、(1)~(10)のいずれかの改変型HSV gDタンパク質。
(12)プロミスキュアスT細胞エピトープは、配列番号4、配列番号5、配列番号6、配列番号7、又は、配列番号8に記載のアミノ酸配列からなるプロミスキュアスT細胞エピトープである、(11)の改変型HSV gDタンパク質。
(13)プロミスキュアスT細胞エピトープは、配列番号8に記載のアミノ酸配列からなるプロミスキュアスT細胞エピトープである、(12)の改変型HSV gDタンパク質。
(14)改変型HSV gDタンパク質はさらに、野生型HSV gDの、配列番号1に記載のアミノ酸配列における251~315番目のアミノ酸残基に相当するアミノ酸残基の少なくとも一部の欠損を含む、(1)~(13)のいずれかの改変型HSV gDタンパク質。
(15)改変型HSV gDタンパク質はさらに、野生型HSV gDのエクトドメインの、配列番号1に記載のアミノ酸配列における231番目のバリン残基に相当するアミノ酸残基が、他のアミノ酸残基に置換されることによって行われる改変を含む、(1)~(14)のいずれかの改変型HSV gDタンパク質。
(16)HSVが、HSV-1又はHSV-2である、(1)~(15)のいずれかの改変型HSV gDタンパク質。
(17)(1)~(16)のいずれかの改変型HSV gDタンパク質を含む、HSVワクチン。
(18)単純ヘルペスウイルス(HSV)のエンベロープ糖タンパク質D(gD)の改変タンパク質(改変型HSV gDタンパク質)であって、野生型HSV gDのエクトドメイン(ectodomain)において、gDエクトドメインのN末端プロリン・リッチ領域(PRR)に存在するB細胞エピトープの少なくとも1つがエピトープとして機能しないように改変された、改変型HSV gDタンパク質。
(19)PRRに存在するB細胞エピトープは、
野生型HSV gDのエクトドメインの、配列番号1に記載のアミノ酸配列における50番目のプロリン残基に相当するアミノ酸残基を含むエピトープ、又は、
野生型HSV gDのエクトドメインの結晶構造の表面において、上記50番目のプロリン残基に相当するアミノ酸からの距離が1.5nm以下の領域に存在する少なくとも1つのアミノ酸残基を含むエピトープ
である、(18)の改変型HSV gDタンパク質。
(20)改変は、アミノ酸残基の置換又は欠損によって糖鎖導入されることによって行われる、(18)又は(19)の改変型HSV gDタンパク質。
(21)改変は、野生型HSV gDのエクトドメインの、配列番号1に記載のアミノ酸配列における50番目のプロリン残基に相当するアミノ酸残基への糖鎖導入よって行われる改変を含む、(20)の改変型HSV gDタンパク質。
(22)改変は、野生型HSV gDのエクトドメインの、配列番号1に記載のアミノ酸配列における74番目のプロリン残基に相当するアミノ酸残基への糖鎖導入によって行われる改変を含む、(20)又は(21)の改変型HSV gDタンパク質。
(23)改変は、野生型HSV gDのエクトドメインの、配列番号1に記載のアミノ酸配列における186番目のアルギニンに相当するアミノ酸残基への糖鎖導入によって行われる改変を含む、(20)~(22)のいずれかの改変型HSV gDタンパク質。
(24)野生型HSV gDのエクトドメインは、配列番号1に記載のアミノ酸配列からなり、
改変は、
配列番号1に記載のアミノ酸配列における50番目のプロリン残基をアスパラギン残基に置換し、51番目のプロリン残基をプロリン残基以外のアミノ酸残基に置換することによって糖鎖導入されることによって行われる改変、
配列番号1に記載のアミノ酸配列における74番目のプロリン残基をアスパラギン残基に置換し、76番目のグルタミン酸残基をセリン残基に置換することによって糖鎖導入されることによって行われる改変、及び
配列番号1に記載のアミノ酸配列における186番目のアルギニン残基をアスパラギン残基に置換することによって糖鎖導入されることによって行われる改変、
からなる群より選択される少なくとも1つの改変を含む、
(20)の改変型HSV gDタンパク質。
(25)改変型HSV gDタンパク質はさらに、HSV gDのエクトドメインのC末端側に少なくとも1つのプロミスキュアスT細胞エピトープが連結されている、(19)~(24)のいずれかの改変型HSV gDタンパク質。
(26)プロミスキュアスT細胞エピトープは、配列番号4、配列番号5、配列番号6、配列番号7、又は、配列番号8に記載のアミノ酸配列からなるプロミスキュアスT細胞エピトープである、(25)に記載の改変型HSV gDタンパク質。
(27)プロミスキュアスT細胞エピトープは、配列番号8に記載のアミノ酸配列からなるプロミスキュアスT細胞エピトープである、(26)の改変型HSV gDタンパク質。
<scFv-phageの取得>
HSV-2 gD(gD2)の網羅的エピトープ解析を実施するため、gD2を対象としたバイオパンニングによってgD2上の様々なエピトープと結合する様々な抗体を取得した。gD2としては、配列番号1に記載のアミノ酸配列を有する、HSV-2の333株に由来する野生型の可溶性エクトドメイン(ectodomain)であるgD1-315タンパク質を用いた。gD1-315タンパク質は宿主細胞にて発現させ、精製した。ライブラリーとしてはヒトB細胞由来のmRNAから作製されたヒトVH及びVL cDNAを使用して調製されたscFv-phageディスプレイライブラリーを用いた。scFv-phageディスプレイライブラリーは、ヒトB細胞由来のmRNAからヒトVH及びVL cDNAを使用して調製されたscFv-phageディスプレイライブラリーをスクリーニングすることによって、HSV-2 gDへの反応性を有するscFv-phageを得た。
パンニング実施後、単離したscFv-phageを発現させた。各scFv遺伝子がクローニングされているファージミドベクターを有する大腸菌TG1株を2×YTCG培地(37℃)で培養し、M13K07ヘルパーファージをmoi=20で感染させた後、2×YTCK培地(25℃)、オーバーナイトでファージの発現を行った。得られたscFv-phageは20%-PEG-2.5M塩酸ナトリウムによる濃縮を行った。
取得された7種類のscFv-phageを基にscFv-hFcを作製した。単離したscFv遺伝子の可変領域をヒトFc遺伝子と連結し、pCAGベクターにクローニングし、scFv-hFc発現プラスミドを構築した。各発現用プラスミドはExpi293発現システム(Life technology)を用いて発現した。4~6日間の培養の後、その上清をProtain A アフィニティクロマトグラフィーカラム(HiTrap Protein A HP Columns、GE Healthcare)によって精製し、PBSで透析を行った。その純度についてはサイズ排除クロマトグラフィー(Superdex 200 5/150 GL、GE Healthcare)とSDS-PAGEによって確認した。
作製したscFv-hFcについて、gDのレセプターであるNectin-1を使用した競合阻害試験を実施した。
結果を表2に示す。競合のパターンにより7種類のscFv-phageを3つのグループに分類した。グループAに属する抗体No.82はNectin-1と強く競合したため、Nectin-1結合領域にエピトープが存在すると示唆された。グループBにはNo.1のみ属し、他の抗体及びレセプターNectin-1と競合しなかった。グループCは残りの5クローン(No.5、No.13、No.72、No.75、No.78)から成り、互いに競合する一方で、レセプターNectin-1とは競合しなかった。
重鎖CDR1:GYAIN (配列番号9)
重鎖CDR2:GIMPIFGTSNYAQKFQ (配列番号10)
重鎖CDR3:DWGAPLEKGAGSPFDV (配列番号11)
軽鎖CDR1:RASQSVSSSYLA (配列番号12)
軽鎖CDR2:GASSRAT (配列番号13)
軽鎖CDR3:QQYGSSPRS (配列番号14)
<変性によるエピトープ解析>
変性状態のgD1-315を用いたWestern blottingによって、各抗体のエピトープがコンフォメーション(conformational)エピトープであるかリニア(linear)エピトープであるかを解析した。
N-結合型糖鎖の導入によるgD改変体を用いたエピトープマスキング法を検討した。糖鎖の導入部位の選定にあたっては既に報告されている結晶構造(非特許文献1、2及び6)に基づき、結晶構造表面に露出しており、二次構造を取っていないループ部位を対象とした。図1にgDの一次構造の模式図を示しており、FR1(K1~H39)、FR2(I55~R184)、FR3(T251~G315)がそれぞれ示されている。また、本来gDに付加されている糖鎖は、N94、N121及びN262に結合している。糖鎖導入部位として、P50(SC-F)、P54(SC-K)、P74(SC-D)、S85(SC-L)、G103(SC-E)、D172(SC-M)、R186(SC-A)、L195(SC-H)、及びH242(SC-B)の9つを選定した。
SC-A-Fw: 5’-CGGGCCAATGCCTCCTGCAAGTACGCT-3’(配列番号15)
SC-A-Re: 5’-GGAGGCATTGGCCCGGTGCTCCAGGAT-3’(配列番号16)
SC-B-Fw: 5’-GGGTGGAATGGCACCAAGCCCCCGTACACCAGC-3’(配列番号17)
SC-B-Re: 5’-GGGCTTGGTGCCATTCCACCCGGCGATTTTTAA-3’(配列番号18)
SC-D-Fw: 5’-CATGCCAATTCGACCGCCCCCCAGATCGTGCGC-3’(配列番号19)
SC-D-Re: 5’-GGGGGCGGTCGAATTGGCATGTAGGAGCACGCT-3’(配列番号20)
SC-E-Fw: 5’-CGCATGAATGACACCTGCGCTATCCCCATCACG-3’(配列番号21)
SC-E-Re: 5’-AGCGCAGGTGTCATTCATGCGATACCAGGCGAT-3’(配列番号22)
SC-F-Fw: 5’-TTCCAGAATGCAAGCATCCCGATCACTGTGTAC-3’(配列番号23)
SC-F-Re: 5’-CGGGATGCTTGCATTCTGGAACGGGTCCTCCAG-3’(配列番号24)
SC-H-Fw: 5’-CTCCCCAATCGCACGCCCCCGGCAGCGTGCCTC-3’(配列番号25)
SC-H-Re: 5’-CGGGGGCGTGCGATTGGGGAGAGCGTACTTGCA-3’(配列番号26)
SC-K-Fw: 5’-AGCATCAATATCACTGTGTACTACGCA-3’(配列番号27)
SC-K-Re: 5’-AGTGATATTGATGCTGGGGGGCTGGAA-3’(配列番号28)
SC-L-Fw: 5’-GGGGCTAATGACACCGCCCGAAAGCACACGTAC-3’(配列番号29)
SC-L-Re: 5’-TCGGGCGGTGTCATTAGCCCCGCGCACGATCTG-3’(配列番号30)
SC-M-Fw: 5’-ATAAACAATTGGACGGAGATCACACAA-3’(配列番号31)
SC-M-Re: 5’-CGTCCAATTGTTTATCTTCACTAGCCG-3’(配列番号32)
gD1-275、gD25-253、gD34-315という3種類の欠損改変体を全合成によって作製した(図1)。
上記で得られた情報を基に、アラニン置換体を用いて、抗体No.82のエピトープが存在すると予測されたNectin-1結合領域であるレセプター結合ドメイン(RBD)、及びP50(SC-F)の周辺領域をアラニンスキャニングした。構造上表面に露出していると考えられるアミノ酸を中心に選出し、アミノ酸をアラニンに置換した変異体14個、中和抗体LP2のブロッキング変異体であるT213M、S216Nを作製した。各アラニンに改変した遺伝子をPCRによって構築し、pCAGGS1-dhfr-neoにクローニングした。発現には、FreeStyle293又はExpi293発現システムを用いた。
7つの抗体クローンのin vitroでのHSV中和活性解析を、プラーク数減少(プラークリダクション)試験及びCell to cell感染拡大抑制試験にて実施した。
対象とするウイルスはATCCから購入した、Human herpesvirus 2(HSV-2) MS株(VR-540)及びHuman herpesvirus 1(HSV-1)KOS株(VR-1493)の2種を用いた。ウイルスの培養、感染価測定、中和抗体価測定にはATCCから購入したVero細胞(CCL.81)を使用した。Vero細胞は、37℃、5%CO2条件下で培養する。拡張、維持、解析プレート作製時は、10%FBS含有MEM培地を使用し、感染価測定及び中和抗体価測定時は、2%FBS含有MEM培地を使用した。中和試験及び後述の感染防御能解析に用いるウイルスバンクは、以下の方法によって調製した。HSV-2 MS株及びHSV-1 KOS株をm.o.i=0.01~1でフルシートのVero細胞に接種し、2~3日間2%FBS含有MEM培地で培養した。回収した感染細胞培養ボトルを3回凍結融解して細胞を破砕後、TOMY遠心器で室温にて3500rpmで10分遠心し、上清をHSV-2ウイルスバンク及びHSV-1ウイルスバンクとした。
(プラーク数減少試験)
プラークリダクション活性測定は、被験抗体を所定の濃度になるように調製し約100PFUのHSV-2 MS株又はHSV-1 KOS株と混合後、37℃にて1時間反応させた。48ウェルプレートにフルシートになったVero細胞に反応液を播種し、30℃にて1時間吸着後に1%メチルセルロース含有MEM(2%FBS)培地で24時間培養後、メタノールとエタノールを1対1で混合した50%メタノール/50%エタノール(-20℃)で、-20℃にて30分間不活化及び固定を行った。その後、抗HSV gDモノクローナル抗体を37℃にて1時間反応させ、抗マウスIgG-HRP(Dako P0447)とTMBHで免疫染色し、ELISPOTアナライザー(ImmunoSpot S6 Analyzer、CTL社)で各ウェルの画像を取り込み、解析ソフト(BioSpot CTL社)でプラーク数をカウントした。
Cell to Cell感染拡大抑制活性測定は、48ウェルプレートにフルシートになったVero細胞に約100PFUのHSV-2 MS株又はHSV-1 KOS株を接種し、30℃で1時間吸着した。その後、所定濃度の被験抗体を含有した1%メチルセルロース含有MEM(2%FBS)培地(抗体濃度は5μg/mL、25μg/mL及び125μg/mL)を添加し、HSV-2 MS株は約40時間培養後、HSV-1 KOS株は約48時間培養後、50%メタノール/50%エタノール(-20℃)で、-20℃にて30分間不活化及び固定を行った。その後、自家調製した抗HSV gDモノクローナル抗体を37℃にて1時間反応させ、抗マウスIgG-HRPとTMBHで免疫染色し、ELISPOTアナライザーで各ウェルの画像を取り込み、解析ソフトでプラークサイズの平均値を解析した。
実験結果を表5に示す。プラーク数減少活性については、MS株(HSV-2)及びKOS株(HSV-1)のそれぞれに対して各抗体の最終濃度を50μg/mL、10μg/mL及び2μg/mLに設定し、各濃度における測定は2ウェルずつ検出して平均値を取った。その結果、Aグループに分類されgDレセプター結合ドメイン(RBD)上(及びFR1上)にエピトープ領域を有する抗体No.82が、HSV-1及びHSV-2の両株に対して最も顕著なプラーク数減少活性を示した。これに対して、Bグループに分類されFR3にエピトープ領域を有する抗体No.1は両株に対するプラーク数減少活性を全く示さなかった。また、C1グループに分類されP50周辺或いはその他の不明な部分にエピトープ領域を有する抗体No.5及び抗体No.13のHSV-1及びHSV-2に対する中和活性はいずれも相対的に弱く、HSV-1に対してはパーシャル阻害のパターンを示した。ここで、「パーシャル阻害」とは、中和活性が相対的に弱く、また用量依存的な低下が明確に確認されていない場合をいう。さらに、C2グループに分類されP50周辺(及びFR3)にエピトープ領域を有する抗体No.72及び抗体No.75はいずれもHSV-1よりもHSV-2に対する中和活性が顕著に弱く、抗体No.72はHSV―2に対してはパーシャル阻害のパターンを示した。また同じくC2グループに分類される抗体No.78の両株に対する中和活性に然程偏りは認められないが、その強度は抗体No.82に比して見劣りのするものであった。抗体No.78は、用量依存的に阻害するが5倍段階希釈の傾きに対し、阻害効果の傾きがほかのものと比べて、緩やかなパターンを示した。
抗gD2抗体No.82のヒト型抗体scFv-hFcに加えて、Fc領域をマウス型にしたヒト-マウスキメラIgG、及びFc領域をモルモット型にしたヒト-モルモットキメラIgGを調製し、それぞれのHSV-2(MS株)及びHSV-1(KOS株)に対する中和活性(プラーク数減少活性)を検討した。
単離したscFv 遺伝子のVH領域をマウスIgG2aに由来するH鎖定常領域遺伝子(CH1-CH2-CH3)と連結し、pCAGベクターにクローニングし、H鎖発現プラスミドを構築した。また、scFv遺伝子のVL領域をマウスCL遺伝子と連結し、pCAGベクターにクローニングし、L鎖発現プラスミドを構築した。発現には、Expi293発現システムを用いた。発現プラスミドを細胞にトランスフェクションし、4~6日で培養上清を回収した。培養上清をHi Trap ProteinA HP Column(GEヘルスケア)を用いて精製し、PBSで透析を行った。その純度についてはサイズ排除クロマトグラフィーとSDS-PAGEによって確認した。以上の方法によりヒト-マウスキメラIgG2aを得た。
単離したscFv遺伝子のVH領域をモルモットIgG2に由来するH鎖定常領域遺伝子(CH1-CH2-CH3)と連結し、pCAGベクターにクローニングした。また、同様にscFv 遺伝子のVL領域をモルモットCK遺伝子と連結し、pCAGベクターにクローニングした。次に、クローニングされた抗体遺伝子をPCRにより増幅し、pXCベクター(ロンザ社)にクローニングし、H鎖及びL鎖の発現プラスミドを構築した。その後、両プラスミドを連結して、1つの発現プラスミドとした。発現にはCHO細胞を使用した。発現プラスミドをCHO細胞に安定導入させ、GS Xceed expression system(ロンザ社)を利用して、抗体高発現CHO細胞を取得した。高発現細胞を12日間Fed-Batch培養し、培養上清を回収した。培養上清をrProteinA sepharose Fast Flow(Cat#17127903/GEヘルスケア)を用いて精製し、ヒト‐モルモットキメラIgG2κ抗体を得た。
抗gD2抗体No.82のscFv-hFc、ヒト-マウスキメラIgG、及びヒト-モルモットキメラIgGを用いて、実施例4と同様にウイルス中和活性(プラーク数減少活性)について解析した。その結果を表6に示した。抗体No.82のscFv-hFc、ヒト-マウスキメラIgG、及びヒト-モルモットキメラIgGのいずれも、MS(HSV-2)及びKOS(HSV-1)の両株に対して0.05μg/mL又はそれ以上の濃度では50%プラーク数減少活性を示した。
マウス性器ヘルペス感染モデルを用いて、抗gD2抗体No.82の予防的投与及び治療的投与における感染防御能を評価した。
マウス性器ヘルペス感染モデルを用いて、抗HSV gD2モノクローナル抗体の予防的投与及び治療的投与における感染防御試験を実施した。BALB/cマウス(5週齢、メス)を用いた。所定量の抗体を注射用生理食塩水(saline)に溶解し、予防的投与の場合にはウイルス接種24時間前に、また治療的投与の場合にはウイルス接種48時間後に、いずれも200μL/匹の容量にて腹腔内投与した。1群あたりN=10の例数を設定した。ウイルス接種時の感染効率を向上させるために、ウイルス接種6日前にDepo-Proveraを2mg/匹で皮下接種した。麻酔下で5×105PFU/20μLのHSV-2 MS株を経腟接種し、21日間経過観察を行った。生存日数(生存率)及び症状スコアを指標に感染防御能を評価した。症状スコアは、膣病変症状の有無及び程度によってスコアを定義し各群における平均値を示した。スコアの付け方として、0:変化なし、1:部分的な紅斑・腫脹、2:広範囲の腫脹・浮腫、3:潰瘍・出血、4:死亡、とした。回復の見込みのない重篤な全身症状(立毛、麻痺、震戦、痙攣等)が認められた場合、その日はスコアを3.5とし、犠牲死させ、次の日に死亡として扱いスコアを4とした。
予防的投与における、投与量別の生存日数を表7に、生存率を図4に、症状スコアを図5にそれぞれ示す。治療的投与における、投与量別の生存日数を表8に、生存率を図6に、症状スコアを図7にそれぞれ示す。
モルモット性器ヘルペス感染モデル(急性期)を用いて、抗gD2抗体No.82の予防的投与及び治療的投与における感染防御能を評価した。
モルモット性器ヘルペス感染モデルを用いて、抗gD2抗体No.82(ヒト-モルモットキメラIgG2κ)の予防的投与及び治療的投与における感染防御試験を実施した。SLC社から購入したHartleyモルモット(3~5週齢、メス)を用いた。所定量の抗体を注射用生理食塩水(saline)に溶解し、予防的投与の場合にはウイルス接種24時間前に、また治療的投与の場合にはウイルス接種4日間後に、いずれも1mg~30mg/kg/匹の容量にて腹腔内投与した。治療的投与の場合は、投与前に症状観察を行い、膣症状を呈している個体を選別し、各群の平均スコアに偏りが生じないようにランダマイズした。1群あたりN=10~15の例数を設定した。ウイルス接種は麻酔下で5×105PFU/50μLのHSV-2 MS株を経腟接種し、急性期症状を接種後2~3週間観察した。症状スコアは、0:明確な病変なし、0.5-1:紅斑、1.5-2:限局的な水泡、2.5-3:限局的な潰瘍又は痂皮、3-5:広範に及ぶ水泡・潰瘍又は痂皮、3-7:失禁を伴う広範な潰瘍又は痂皮、7.5:重篤な症状による安楽殺、8:死亡とした。また、ウイルス接種後7日目において、膣拭い液(膣swab)を採取し、プラーク法によってウイルス放出量を測定した。膣Swabは、MEM培地で湿潤させた綿棒を膣内に挿入後、膣内壁の粘膜を拭い取るようにして採取した。採取した膣swabは、シリコナイズドチューブに1mLずつ分注したMEM培地にて懸濁し、使用時まで凍結保存した。膣swabを原液、10倍、100倍、1000倍希釈し、100uL/ウェルで96ウェル又は48ウェルにフルシートになったVero細胞に接種した。膣Swab接種後37℃で1時間ウイルス吸着を行い、1%メチルセルロース含有2%FBS MEM培地で24~72時間培養した後に、所定の方法でプラーク数を計測した。
予防的投与における症状スコアを図8に示す。治療的投与の結果における、症状スコアを図9に、膣拭い液中のHSV放出量を図10にそれぞれ示す。
<HSV gD2上に存在するHLA Class II拘束性プロミスキュアスT細胞エピトープクラスター配列の探索>
GenBankに公開されているHSV-2 333株のgD2全長アミノ酸配列(ABU45433.1;配列番号3)に関して、EpiVax社のアルゴリズム(EpiMatrix)を用いて、HLA Class II拘束性プロミスキュアスT細胞エピトープクラスター配列を探索した。当該クラスター配列は、EpiMatrixが解析対象とする8種類の主要なHLA DR super type(DRB1*0101、DRB1*0301、DRB1*0401、DRB1*0701、DRB1*0801、DRB1*1101、DRB1*1301、DRB1*1501)のうちの大部分に対して結合する可能性が高い(Z-Score≧1.64)と予測される15~25アミノ酸からなるペプチド配列である。
C.T.L社から販売されているヒトPBMC提供者の血清を抗HSV IgG抗体検出ELISA kit(デンカ生研)を用いてスクリーニングし、HSV既感染(抗体陽性)ドナー由来のPBMCを購入した。EpiVax社のアルゴリズムに組み込まれている8種類の主要なHLA DR super type(DRB1*0101、DRB1*0301、DRB1*0401、DRB1*0701、DRB1*0801、DRB1*1101、DRB1*1301、DRB1*1501)遺伝子のいずれかのホモ接合体又はヘテロ接合体を有し、HSV暴露歴の有るドナーのPBMCの凍結サンプルを購入した。また、非特異的な応答を解析するために、抗HSV抗体陰性ドナー由来PBMCも購入した(Donor 14、67)。
T細胞刺激活性解析は、合成ペプチドのマウス免疫原性試験によって行われた。合成ペプチドをDMSOにて10mMに溶解又は懸濁したstock solutionを調製した。また投与用の溶媒として、NIKKOL HCO-60(日光ケミカルズ株式会社製)を用いて10%HCO-60/saline(注射用生理食塩水)を調製した。合成ペプチド100μgをCpG10μg及びMPLA10μgと共に溶媒に混合し210μL/匹の容量に調製して、マウス(4~5週齢、雌)の背部皮下に投与した。マウスはBALB/c(I-Ad/I-Ed)、C57BL/6(I-Ab)、C3H/HeN(I-Ak/IEk)の3系統を用いた。初回免疫から21日後に追加免疫を行い、その2週間後に脾臓を回収し脾細胞を調製して、以下のサイトカイン産生応答解析(ELISpot解析)に供した。調製した脾細胞を1×106細胞/ウェルになるようにPVDF膜96ウェル(MSIPS4W10 Millipore)に播種し、各種ペプチド10μMと20時間培養した。培養は、RPMI1640培地を用いて10%FBS存在下で、37℃かつCO2濃度5%に設定したインキュベーターにて実施した。陽性対照としてCon A添加群を設定した。IFN-γ産生細胞の検出はmouse IFN gamma ELISPOT Ready-SET-Go!(登録商標、ebioscience 88-7384-88)を用いて行なった。ELISpot analyzer(CTL Immunospot S5 versa analyzer)で画像を取り込み、スポット数をimmunospot softwareでカウントした。
実際のヒト血清中に含まれる抗HSV gD抗体のポピュレーションを解析するために、ヒトガンマグロブリンカクテルであるベニロン(献血ベニロン(登録商標)-I静注用、一般財団法人化学及血清療法研究所)を用いて、取得された抗体との競合試験を行った。競合試験としてgD1-315とベニロンを反応させた後にモノクローナル抗体を反応させた。この系では、ベニロンに含まれる競合抗体の量が多いほど当該モノクローナル抗体(被検抗体)の結合量が少なくなり、競合率は高くなると考えられる。競合試験にはSPR(Biacore)を用いた。
競合試験はBiacore 3000(GE Healthcare)を使用して行われた。すべての実験においてHBS-EP buffer(GE Healthcare)を使用し、温度は25℃、流速は10μL/分に設定した。Sensor chipはNTA(GE Healthcare)を使用した。推奨されたプロトコルに基づき、0.5mM NiCl2を10秒反応させた。次に、gD1-315-Hisを3μg/mLで60秒反応させ、約300 resonance units(RU)固相化した。Biacoreを表面プラズモン共鳴センサーとして用いて解析を行った場合、得られるシグナル値である「RU」は、1mm2あたり物質が1pg結合したときの単位として表される。また、chipの再生については350mM EDTAで10秒、2回、chip表面を処理し、Bufferで10秒洗浄した。新たな検体を測定するたびに固相化及び再生を同様の方法で実施した。競合率は次のように算出された。濃度20μg/mLの検討する抗体を流速20μL/分で120秒アプライし、増加したRUを(1)とし、濃度150μg/mLのベニロンを流速20μL/分で120秒アプライし続けて、その後、濃度20μg/mL抗体120秒アプライし、増加したRUを(2)とした時、(1-(2)/(1))×100の計算式によって競合率を計算した。すべてのサンプルは一回のみ測定した。
ベニロン競合試験の解析結果を表11に示す。最も高い競合率を示したのは弱中和抗体である抗体No.5であり、次いで抗体No.13、抗体No.75、抗体No.78、抗体No.72とP50周辺領域にエピトープを有する抗体群が上位を占めた。一方、抗体No.82は7クローン中、6番目であり相対的に低い競合率を示し、最も低いものは抗体No.1であった。
本発明者らは、中和抗体のエピトープが存在するRBDが有益なエピトープ領域とし、有益性の低い抗体群のエピトープが存在するP50周辺領域及びFR3をデコイ領域とし、中和抗体をより多く誘導できるgDタンパク質改変体の設計にあたって、有益なエピトープを際立たせること、デコイ領域中のエピトープを糖鎖導入や欠損変異等によって脱エピトープ化すること、さらに、プロミスキュアスT細胞エピトープクラスターペプチドを連結することによって、液性免疫応答、細胞性免疫応答の双方を効率的・効果的に惹起できること、の3つの観点から、以下のようにgD改変体を設計し、その中和抗体の誘導活性を調べた。
野生型HSV gD抗原上に存在する有益なエピトープとして、抗gD2抗体No.82のエピトープであるgDレセプター結合ドメイン(RBD)、及びT細胞エピトープ解析より予測されたプロミスキュアスT細胞エピトープクラスターDP5(gD2 334-350;配列番号8)を想定し、また相対的に有益性の低い抗体群のエピトープが集中しているデコイ領域としてP50周辺領域を想定した。
以上の三つの方針に基づき、gD2の改変を進め、計16種類の改変体を得た(表12)。改変体の作製は、以下に示す方法に従って行った。
gD1-275、gD1-315のプラスミド構築、糖鎖を導入する際に使用したプライマーに関しては、前述の通りである。T細胞エピトープペプチドを導入する場合には、目的のgD配列のC末端残基(D275又はG315)に続けてリンカー(GPGPG)、各T細胞エピトープペプチド、6×His-tagの順で連結するようにDNAを設計し、全長を人工遺伝子合成し、pUC19ベクターにクローニングした。完成した改変体配列をpCAGGS1-dhfr-neoベクターにクローニングし、発現用プラスミドを得た。
(抗体及びgD改変体の発現、精製)
各抗gD2モノクローナル抗体及び各gD改変体はExpi293発現システムを用いて発現した。4~6日間の培養の後、その上清をProtain A アフィニティクロマトグラフィーカラム又はNi-NTA アフィニティクロマトグラフィーカラムによって精製し、PBSで透析を行った。その純度についてはサイズ排除クロマトグラフィーとSDS-PAGEによって確認した。
リン酸緩衝生理食塩水で濃度2μg/mLに調整した100μLのgD改変体を96ウェルマイクロタイタープレートに、4℃にて一晩かけて固相化した。各ウェルをPBSで3回洗浄し、300μLの1%BSA PBSで室温にて1時間ブロッキングした。各ウェルをPBS-T(0.05%Tween PBS)で3回洗浄し、各抗gD2抗体を1%BSA PBSに任意の希釈倍率で希釈し、100μLで加え、37℃にて一時間反応させた。再度各ウェルをPBS-Tで洗浄し、HRP標識抗体(anti-hFc/HRP/1%BSA PBS)を加え、37℃にて一時間反応させた。各ウェルをPBS-Tで洗浄し、TMBで室温にて30分発色させ、1N硫酸で反応を停止させた後、450nm/650nmの吸光度を測定した。
(マウス免疫原性試験)
野生型gD抗原gD1-315(WT)を陽性対照、salineを陰性対照として、改変型gD抗原の免疫原性試験を実施した。MPLA(10μg/匹)及びCpG(1μg/匹)と共に、所定量の抗原を注射用生理食塩水(saline)に溶解し、200μL/匹の容量でマウスに免疫した。試験には、BALB/cマウス(5週齢、メス)を用い、2週間間隔で合計3回、背部皮下に免疫した(1群あたりN=4の例数で実施)。最終免疫(3回目)から2週間後に、個体毎に採血し血清を調製した。調製した血清を段階希釈し、HSV-2に対する中和抗体誘導活性(プラーク数50%減少活性)を評価した。
(マウス感染防御試験)
マウス性器ヘルペス感染モデルを用いて、改変型gD抗原の予防的投与における感染防御試験を実施した。前述のマウス免疫原性試験と同様にして、1群あたりN=10の例数にてマウスを免疫した。最終免疫(3回目)から2週間後に行うウイルス接種時の感染効率を向上させるために、ウイルス接種6日前にDepo-Proveraを2mg/匹で皮下接種した。麻酔下で5×105PFU/20μLのHSV-2 MS株を経腟接種し、21日間経過観察を行った。生存日数(生存率)を指標に感染防御能を評価した。
マウス免疫原性試験及びマウス感染防御試験において野生型gD(gD1-315)に比して優越性が認められた一連の改変型gDのうち、gD1-315v3-55及びgD1-315v5-55について、そのメカニズムを調べるために免疫血清の解析を行った。目的とする抗体No.82様抗体の効率的な誘導が実際に引き起こされているのか否かについて調べた。抗体量を揃えるために、血清からIgGを精製し、SPR(Biacore)を用いた競合法により解析した。
抗gD2抗体競合試験は、Biacore 3000(GE Healthcare)を使用して行い、すべての実験においてHBS-EP buffer (GE Healthcare)を使用し、温度は25℃、流速は20μL分に設定した。Sensor chipはCM5 sensor chip(GE Healthcare)を使用し、推奨されたプロトコルに基づいて実験を行った。chip表面にはgD1-315-Hisを約300 RU固相化し、chipの再生には0.1MかつpH2.0のグリシン-塩酸緩衝液を用い、流速30μL/分で30秒、2回洗浄した。競合の手順、競合率の算出は次のように行った。各種抗gD2抗体10μg/mL又はbufferをchipに結合1分、解離2.5分の条件でアプライし、その後IgG精製した各種免疫血清20μg/mLを結合1分、解離5分の条件でアプライする。bufferアプライ後、免疫血清をアプライしたときに検出されたRUを100%とし、抗gD2抗体アプライによる免疫血清のレスポンスの低下を競合率として算出した。
さらに、改変型gD免疫血清によるgD-HVEM相互作用に対する阻害についても解析した。具体的には、リン酸緩衝生理食塩水で濃度5μg/mLに調整した100μLのgD1-305-cys-strep dimerを96ウェルマイクロタイタープレートに、4℃にて一晩かけて固相化した。その後、上記競合ELISAの方法と同様にブロッキング、洗浄を行い、各免疫血清から精製したIgGを任意の濃度で100μL添加し、室温にて一時間反応させた。その後、1μg/mLのHVEM(Recombinant Human HVEM/TNFRSF14 Fc Chimera Protein、R&D SYSTEMS)を1%BSA PBSに任意の希釈倍率で希釈し、100μLでそれぞれ添加し、さらに室温にて1時間反応させた。再度各ウェルをPBS-Tで洗浄し、HRP標識抗体(anti-hFc/HRP/1%BSA PBS)を反応させた。各ウェルをPBS-Tで洗浄し、発色させ、1N硫酸で反応を停止させた後、450nm/650nmの吸光度を測定した。
Claims (27)
- 単純ヘルペスウイルス(HSV)のエンベロープ糖タンパク質D(gD)の改変タンパク質(改変型HSV gDタンパク質)であって、野生型HSV gDのエクトドメイン(ectodomain)において、レセプター結合ドメイン(RBD)に存在するB細胞エピトープと比較して、中和抗体誘導活性が低い又は無いB細胞エピトープ(デコトープ)の少なくとも1つがエピトープとして機能しないように改変された、改変型HSV gDタンパク質。
- 前記RBDに存在するB細胞エピトープが、配列番号1に記載のアミノ酸配列における134番目のアルギニン残基、139番目のアスパラギン酸残基、及び222番目のアルギニン残基からなる群より選択される少なくとも1つのアミノ酸残基に相当するアミノ酸残基を含むエピトープである、請求項1に記載の改変型HSV gDタンパク質。
- 前記デコトープは、gDエクトドメインのN末端プロリン・リッチ領域(PRR)に存在するB細胞エピトープである、請求項1又は2に記載の改変型HSV gDタンパク質。
- 前記デコトープは、
野生型HSV gDのエクトドメインの、配列番号1に記載のアミノ酸配列における50番目のプロリン残基に相当するアミノ酸残基を含むエピトープ、又は、
野生型HSV gDのエクトドメインの結晶構造の表面において、前記50番目のプロリン残基に相当するアミノ酸からの距離が1.5nm以下の領域に存在する少なくとも1つのアミノ酸残基を含むエピトープ
である、請求項3に記載の改変型HSV gDタンパク質。 - 前記デコトープの改変は、アミノ酸残基の置換、アミノ酸残基の欠損、及び/又はアミノ酸残基の置換又は欠損によって糖鎖導入されることによって行われる、請求項1~4のいずれか一項に記載の改変型HSV gDタンパク質。
- 前記デコトープの改変は、野生型HSV gDのエクトドメインの、配列番号1に記載のアミノ酸配列における50番目のプロリン残基に相当するアミノ酸残基への糖鎖導入よって行われる改変を含む、請求項5に記載の改変型HSV gDタンパク質。
- 前記デコトープの改変は、野生型HSV gDのエクトドメインの、配列番号1に記載のアミノ酸配列における74番目のプロリン残基に相当するアミノ酸残基への糖鎖導入によって行われる改変を含む、請求項5又は6に記載の改変型HSV gDタンパク質。
- 前記デコトープの改変は、野生型HSV gDのエクトドメインの、配列番号1に記載のアミノ酸配列における186番目のアルギニン残基に相当するアミノ酸残基への糖鎖導入によって行われる改変を含む、請求項5~7のいずれか一項に記載の改変型HSV gDタンパク質。
- 前記野生型HSV gDのエクトドメインは、配列番号1に記載のアミノ酸配列からなり、
前記デコトープの改変は、
配列番号1に記載のアミノ酸配列における50番目のプロリン残基をアスパラギン残基に置換し、51番目のプロリン残基をプロリン残基以外のアミノ酸残基に置換することによって糖鎖導入されることによって行われる改変、
配列番号1に記載のアミノ酸配列における74番目のプロリン残基をアスパラギン残基に置換し、76番目のグルタミン酸残基をセリン残基に置換することによって糖鎖導入されることによって行われる改変、及び
配列番号1に記載のアミノ酸配列における186番目のアルギニン残基をアスパラギン残基に置換することによって糖鎖導入されることによって行われる改変、
からなる群より選択される少なくとも1つの改変を含む、
請求項5に記載の改変型HSV gDタンパク質。 - 前記糖鎖がN型糖鎖である、請求項5~9のいずれか一項に記載の改変型HSV gDタンパク質。
- 前記改変型HSV gDタンパク質はさらに、HSV gDのエクトドメインのC末端側に少なくとも1つのプロミスキュアスT細胞エピトープが連結されている、請求項1~10のいずれか一項に記載の改変型HSV gDタンパク質。
- 前記プロミスキュアスT細胞エピトープは、配列番号4、配列番号5、配列番号6、配列番号7、又は、配列番号8に記載のアミノ酸配列からなるプロミスキュアスT細胞エピトープである、請求項11に記載の改変型HSV gDタンパク質。
- 前記プロミスキュアスT細胞エピトープは、配列番号8に記載のアミノ酸配列からなるプロミスキュアスT細胞エピトープである、請求項12に記載の改変型HSV gDタンパク質。
- 前記改変型HSV gDタンパク質はさらに、前記野生型HSV gDの、配列番号1に記載のアミノ酸配列における251~315番目のアミノ酸残基に相当するアミノ酸残基の少なくとも一部の欠損を含む、請求項1~13のいずれか一項に記載の改変型HSV gDタンパク質。
- 前記改変型HSV gDタンパク質はさらに、野生型HSV gDのエクトドメインの、配列番号1に記載のアミノ酸配列における231番目のバリン残基に相当するアミノ酸残基が、他のアミノ酸残基に置換されることによって行われる改変を含む、請求項1~14のいずれか一項に記載の改変型HSV gDタンパク質。
- 前記HSVが、HSV-1又はHSV-2である、請求項1~15のいずれか一項に記載の改変型HSV gDタンパク質。
- 請求項1~16のいずれか一項に記載の改変型HSV gDタンパク質を含む、HSVワクチン。
- 単純ヘルペスウイルス(HSV)のエンベロープ糖タンパク質D(gD)の改変タンパク質(改変型HSV gDタンパク質)であって、野生型HSV gDのエクトドメイン(ectodomain)において、gDエクトドメインのN末端プロリン・リッチ領域(PRR)に存在するB細胞エピトープの少なくとも1つがエピトープとして機能しないように改変された、改変型HSV gDタンパク質。
- 前記PRRに存在するB細胞エピトープは、
野生型HSV gDのエクトドメインの、配列番号1に記載のアミノ酸配列における50番目のプロリン残基に相当するアミノ酸残基を含むエピトープ、又は、
野生型HSV gDのエクトドメインの結晶構造の表面において、前記50番目のプロリン残基に相当するアミノ酸からの距離が1.5nm以下の領域に存在する少なくとも1つのアミノ酸残基を含むエピトープ
である、請求項18に記載の改変型HSV gDタンパク質。 - 前記改変は、アミノ酸残基の置換又は欠損によって糖鎖導入されることによって行われる、請求項18又は19に記載の改変型HSV gDタンパク質。
- 前記改変は、野生型HSV gDのエクトドメインの、配列番号1に記載のアミノ酸配列における50番目のプロリン残基に相当するアミノ酸残基への糖鎖導入よって行われる改変を含む、請求項20に記載の改変型HSV gDタンパク質。
- 前記改変は、野生型HSV gDのエクトドメインの、配列番号1に記載のアミノ酸配列における74番目のプロリン残基に相当するアミノ酸残基への糖鎖導入によって行われる改変を含む、請求項20又は21に記載の改変型HSV gDタンパク質。
- 前記改変は、野生型HSV gDのエクトドメインの、配列番号1に記載のアミノ酸配列における186番目のアルギニンに相当するアミノ酸残基への糖鎖導入によって行われる改変を含む、請求項20~22のいずれか一項に記載の改変型HSV gDタンパク質。
- 前記野生型HSV gDのエクトドメインは、配列番号1に記載のアミノ酸配列からなり、
前記改変は、
配列番号1に記載のアミノ酸配列における50番目のプロリン残基をアスパラギン残基に置換し、51番目のプロリン残基をプロリン残基以外のアミノ酸残基に置換することによって糖鎖導入されることによって行われる改変、
配列番号1に記載のアミノ酸配列における74番目のプロリン残基をアスパラギン残基に置換し、76番目のグルタミン酸残基をセリン残基に置換することによって糖鎖導入されることによって行われる改変、及び
配列番号1に記載のアミノ酸配列における186番目のアルギニン残基をアスパラギン残基に置換することによって糖鎖導入されることによって行われる改変、
からなる群より選択される少なくとも1つの改変を含む、
請求項20に記載の改変型HSV gDタンパク質。 - 前記改変型HSV gDタンパク質はさらに、HSV gDのエクトドメインのC末端側に少なくとも1つのプロミスキュアスT細胞エピトープが連結されている、請求項19~24のいずれか一項に記載の改変型HSV gDタンパク質。
- 前記プロミスキュアスT細胞エピトープは、配列番号4、配列番号5、配列番号6、配列番号7、又は、配列番号8に記載のアミノ酸配列からなるプロミスキュアスT細胞エピトープである、請求項25に記載の改変型HSV gDタンパク質。
- 前記プロミスキュアスT細胞エピトープは、配列番号8に記載のアミノ酸配列からなるプロミスキュアスT細胞エピトープである、請求項26に記載の改変型HSV gDタンパク質。
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