Vaccine compositions of combinations of herpesvirus envelope proteins that induce immune responses - Patents.com

Abstract

To provide novel and improved antigen compositions for enhancing immune responses to HHV.SOLUTION: Provided is an antigenic composition comprising at least two of the following human herpesvirus polypeptides or one or more nucleic acids encoding the at least two human herpesvirus polypeptides: a glycoprotein B (gB) polypeptide comprising an extracellular domain of human herpesvirus gB; a glycoprotein 350 (gp350) polypeptide comprising an extracellular domain of human herpesvirus gp350; a glycoprotein L (gL) polypeptide; and a glycoprotein H (gH) polypeptide comprising an extracellular domain of human herpesvirus gH.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of and relies on the filing date of U.S. Provisional Patent Application No. 62 / 451,396, filed January 27, 2017, the entire disclosure of which is incorporated herein by reference.

[0002] [Government interests] This invention was made with Government support under Grant No. Q574LJ15 awarded by the Uniformed Services University. The Government has certain rights in this invention.

[0003] [Sequence table] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on January 25, 2018, is named HMJ-153-PCT_SL.txt and is 207545 bytes in size. [Background technology]

[0004] Human herpesviruses are a group of enveloped DNA viruses that cause significant morbidity and mortality in humans worldwide (Non-Patent Document 1). There are eight known types of human herpesviruses (HHVs), including (i) human herpesvirus type 1 (HHV-1), which is herpes simplex virus 1 (HSV-1), (ii) HHV-2, which is herpes simplex virus 2 (HSV-2), (iii) HHV-3, which is varicella zoster virus (VZV), (iv) HHV-4, which is Epstein-Barr virus (EBV), (v) HHV-5, which is human cytomegalovirus (HCMV), (vi) HHV-6, (vii) HHV-7, and (viii) HHV-8, which is Kaposi's sarcoma-associated herpesvirus (KSHV).

[0005] In humans, these viruses are known to cause the following diseases: HSV-1 causes oral herpes, HSV-2 causes genital herpes, and VZV causes chicken pox and shingles. EBV causes infectious mononucleosis and is strongly associated with some B-cell lymphomas, nasopharyngeal carcinomas, and gastric adenocarcinomas. HCMV causes severe infections in immunosuppressed patients and is the leading non-genetic cause of hearing loss. HHV-6 and HHV-7 cause roseola (sixth disease) in children, and HVV-8 causes Kaposi's sarcoma in several clinical settings, including in patients infected with the human immunodeficiency virus (HIV).

[0006] EBV mainly infects B cells and nasopharyngeal epithelial cells. EBV infection of B cells is initiated by the binding of the EBV envelope protein gp350 to the complement receptor CR2 / CD21 (Non-Patent Document 2 and Non-Patent Document 3). By binding to B cell CR2, EBV gp42 interacts with cell surface MHC-II receptors, resulting in its association with the heterodimeric EBV gH / gL protein. The heterodimeric gH / gL then undergoes a conformational change by binding gp42, resulting in the activation of the EBV fusion protein gB, which directly mediates virus-host cell membrane fusion (Non-Patent Document 4). Like EBV, the binding, fusion and host cell entry of other HHV family members are primarily mediated by the gB, gH and gL polypeptides, together with other accessory proteins that typically bind to various receptors on the host cell surface.

[0007] Currently, there is no prophylactic EBV vaccine in clinical use. Studies in non-human primates using a gp350-based vaccination strategy have shown protection against EBV-induced lymphoma and EBV replication (Non-Patent Document 5). A phase II clinical trial conducted in EBV-seronegative young adults using recombinant monomeric gp350 protein versus placebo suggested a partial protective effect of gp350 vaccination against the development of infectious mononucleosis (IM) (Non-Patent Document 6 and Non-Patent Document 7). However, the vaccine did not prevent asymptomatic EBV infection. A phase I trial of recombinant monomeric gp350 protein given to children with chronic kidney disease showed that only a few subjects developed detectable neutralizing serum anti-gp350 titers (Non-Patent Document 8).

[0008] Nor is there a commercially available prophylactic HCMV vaccine today. Although some efficacy was demonstrated in overt HCMV disease in high-risk recipient-donor+renal transplant recipients, earlier clinical trials using live attenuated Towne or AD169 HCMV virus vaccines, both of which lack expression of the pentameric complex (gH / gL / UL128 / UL130 / UL131A), proved ineffective in preventing HCMV infection in either healthy volunteers or renal transplant recipients (Non-Patent Document 9). New HCMV virus strains engineered to express the pentameric complex are currently being evaluated, but safety concerns remain with this approach. A phase II clinical trial using recombinant HCMV gB protein derived from the Towne strain of HCMV (Non-Patent Document 10) demonstrated 50% efficacy in preventing HCMV infection in HCMV-seronegative women (Non-Patent Document 11) and 50% efficacy in preventing HCMV viremia in solid organ transplant patients. The HCMV gB protein used in the Phase II clinical trial was designed to be cloned into gB was modified to remove the nucleotide sequence 144 of the HCMV IgG1 gene. Thus, gB was not in its native trimeric conformation (Non-Patent Document 12). Although these two studies contributed to the further evaluation of gB as a prophylactic HCMV vaccine, they demonstrate a compelling need for more effective prophylactic vaccine formulations.

[0009] Patent Documents 1 and 2 describe strategies for enhancing immunity, including multimerizing antigens. For example, Patent Document 1 describes a fusion protein comprising at least two antigens and an oligomerization domain separated by a linker sequence, including multimeric HHV antigens such as gp350, gB, gH and gL. Patent Document 2 describes a modified herpesvirus gB obtained by inserting a peptide linker into the furin cleavage site of the herpesvirus gB polypeptide extracellular domain. The insertion of the peptide linker removes the furin recognition sequence, such that expression of the modified herpesvirus gB results in the production of a homotrimeric gB complex that provides enhanced immunogenicity.

[0010] Combining multiple antigens in a vaccine does not necessarily result in enhanced immunity or even additive effects. Indeed, when multiple antigens are co-administered as part of a multicomponent vaccine or as part of a sequential immunization schedule, the antibody response to one or more antigens may be reduced or attenuated by vaccination or immune interference (Non-Patent Document 13). Similarly, when certain haptens are combined with carrier proteins, antibody responses to the hapten are often inhibited if the recipient has been pre-immunized with the carrier protein. This phenomenon has been termed carrier-induced epitope suppression and has been demonstrated to occur with many peptide-carrier protein conjugates (Non-Patent Document 14). Also, when certain saccharides are combined with carrier proteins, they may occur, especially when the recipient is primed with a high dose of the carrier protein (i.e., a dose high enough to induce an antibody response against the carrier protein) (Non-Patent Document 14). Thus, often, when two or more antigens are administered to a subject, the antibody response to one or more antigens is attenuated by immune interference. Thus, when certain haptens are combined with carrier proteins as part of a vaccination or immunization schedule, antibody responses to the hapten are often inhibited if the recipient has been pre-immunized with the carrier protein. This phenomenon has been termed carrier-induced epitope suppression and has been demonstrated to occur with many peptide-carrier protein conjugates (Non-Patent Document 14). Also, when certain saccharides are combined with carrier proteins, they may occur, especially when the recipient is pre-primed with a high dose of the carrier protein (i.e., a dose high enough to induce an antibody response against the carrier protein) (Non-Patent Document 14). Thus, often, when two or more antigens are administered to a subject, antibody responses to one or more antigens are attenuated by immune interference. When administering multiple proteins in a single dose, it is important to carefully evaluate the interactions between the proteins and how those interactions may affect the immune system response.

Prior technical literature

[0011]

Patent Document 1

Patent document 2

Non-licensed literature

[0012] [Non-licensed document 1] Eisenberg et al., Viruses, 4:800-32, 2012 [Non-licensed document 2] Hutt-Fletcher, J. Virol., 81:7825-32, 2007 [Non-licensed document 3] Shannon-Lowe et al., Curr. Opin. Virol., 4:78-84, 2014

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

[0013] There is a need for new and improved antigen compositions for enhancing the immune response to HHV. [Means for solving the problem]

[0014] Human herpes viruses share a common strategy for infecting host cells. Specifically, the viral envelope membrane fuses with the plasma membrane of the host cell and then enters the cytoplasm, or the viral envelope membrane fuses with the endosomal membrane after the virus is invaginated into the plasma membrane and then enters the cytoplasm of the host cell. The core HHV envelope proteins involved in the fusion process are the conserved glycoprotein B (gB), glycoprotein H (gH) and glycoprotein L (gL). The gH and gL proteins typically form a non-covalently associated heterodimeric complex during the fusion process.

[0015] As disclosed in the present application, immunization with a combination of two or more of these HHV proteins involved in mediating HHV binding, fusion and entry into host cells, such as gp350, gH, gL and gB, results in additive or synergistic antibody responses. These robust results are particularly unexpected, given the challenges of vaccine or immune interference commonly observed when administering multiple antigens as part of a multicomponent vaccine or sequential vaccination schedule, as recognized in the art. Without intending to be bound by any theory, it appears that the combination of two or more HHV polypeptides induces high titer neutralizing antibody responses that provide improved protection against HHV infection in vivo by blocking various steps of the virus-host cell fusion process.

[0016] Recently, a strategy of multimerizing HHV proteins to enhance immunogenicity has been reported (see, for example, Patent Documents 1 and 2), but the present inventors have discovered that unexpected additive and synergistic antibody responses can be obtained by combining HHV fusion and host cell entry proteins in monomeric or multimeric forms. Thus, in certain embodiments, one or more of the HHV fusion and host cell entry proteins are monomeric and / or multimeric. The HHV fusion and host cell entry proteins may be recombinant or native proteins. In certain embodiments, the HHV fusion and host cell entry proteins are modified and are not naturally occurring proteins. For example, the proteins may be truncated, multimeric, or combined in a fusion protein.

[0017] Although typically administered as a polypeptide, nucleic acids encoding the HHV fusion and host cell entry proteins can also be administered as DNA, RNA, or viral vector vaccines, or virus-like particles expressing the HHV fusion and host cell entry proteins can also be administered.

[0018] The present disclosure also discloses for the first time that high titer anti-HHV antibodies, such as those generated in response to the combination of HHV proteins disclosed herein, can passively transfer immunity to protect against HHV infection. This aspect encompasses methods for identifying biological samples containing high titer anti-HHV antibodies, as well as methods for passively transferring immunity to a subject in need thereof by collecting antibodies and / or immune cells from individuals who are highly seropositive to HHV antigens and / or who have been administered an antigenic composition disclosed herein, and administering those antibodies and / or immune cells to the subject, thereby protecting the subject from HHV infection, particularly in individuals who are immunocompromised or at risk of developing HHV infection.

[0019] In a first aspect, the disclosure provides antigenic compositions comprising at least two of the following antigenic human herpesvirus polypeptides (or one or more nucleic acids encoding same): a glycoprotein B (gB) polypeptide comprising the extracellular domain of human herpesvirus gB; a glycoprotein 350 (gp350) polypeptide comprising the extracellular domain of human herpesvirus gp350; a glycoprotein L (gL) polypeptide; and a glycoprotein H (gH) polypeptide comprising the extracellular domain of human herpesvirus gH. Such compositions may optionally include adjuvants and / or excipients common in the field of vaccine development.

[0020] The human herpesvirus from which the polypeptide is derived may be human cytomegalovirus (HCMV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV6), human herpesvirus 7 (HHV7), and / or Kaposi's sarcoma-associated herpesvirus (HSHV). In one embodiment, the polypeptide is an EBV polypeptide.

[0021] In certain embodiments, the gB, gp350, gL, and / or gH polypeptides, when present in an antigenic composition, each further comprise a corresponding intracellular domain. The extracellular domain of a selected polypeptide may be fused to the intracellular domain via a polypeptide linker sequence, for example, about 6 to about 70 amino acids in length, or in particular about 15 amino acids in length. In other embodiments, at least two, and optionally three, human herpesvirus polypeptides form a fusion protein, which optionally includes a polypeptide linker sequence covalently linking the polypeptides.

[0022] In further embodiments, the antigenic composition comprises a gB polypeptide, gp350, gL and In various embodiments mentioned herein, the gB polypeptide may be monomeric or multimeric (e.g., dimeric, trimeric, tetrameric, etc.). In certain embodiments, the antigenic composition comprises a gB polypeptide, a gH polypeptide, and a gL polypeptide. The gL and gH polypeptides may optionally exist as heterodimers. In certain embodiments, the heterodimer is a fusion protein. In other embodiments, the heterodimer is a non-covalently associated protein complex. In one embodiment, the gB polypeptide is monomeric, dimeric, or trimeric, and the gL and gH polypeptides form a heterodimer. In another embodiment, the gB polypeptide is monomeric, and the gL and gH polypeptides form a monomeric heterodimer.

[0023] HCMV embodiments of the antigenic compositions contemplate combinations of at least the following: gB, gH and gL polypeptides. In one embodiment, the gB polypeptide is monomeric, dimeric or trimeric, and the gL and gH polypeptides form a heterodimer, which may be monomeric or multimeric (e.g., monomeric, dimeric, trimeric or tetrameric). In another embodiment, the gB polypeptide is monomeric or trimeric, and the gL and gH polypeptides form a monomeric or trimeric heterodimer. These antigenic compositions may further comprise an HCMV glycoprotein O (gO) polypeptide or an HCMV unique long 128 (UL128) polypeptide, an HCMV unique long 130 (UL130) polypeptide, and an HCMV unique long 131A (UL131A) polypeptide, and optionally an HCMV glycoprotein M polypeptide and / or an HCMV glycoprotein N polypeptide.

[0024] In EBV embodiments of the antigenic composition, at least the following combinations are contemplated: (a) a combination of a gp350 polypeptide with a gB polypeptide, where the gp350 polypeptide is a monomeric or tetrameric gp350 and the gB polypeptide is a trimer gB; (b) a combination of a gp350 polypeptide with a gH polypeptide and a gL polypeptide, where (i) the polypeptides are monomeric, or (ii) the gp350 polypeptide is a tetramer and the gH and gL polypeptides are trimers. (c) a combination of a gB polypeptide, a gH polypeptide, and a gL polypeptide, wherein the gB polypeptide is a trimer gB, and both the gH and gL polypeptides are monomers or trimers; and (d) a combination of a monomeric gp350 polypeptide, a monomeric gH polypeptide, a monomeric gL polypeptide, and a trimer gB polypeptide, wherein the gp350 polypeptide is a tetramer, the gH and gL polypeptides are monomers or trimers, and the gB polypeptide is a trimer. The EBV antigen composition may also optionally include a human EBV glycoprotein 42 (gp42) polypeptide, a BDLF2 polypeptide, and / or a human EBV BamH1-M right reading frame 2 (BMRF-2) polypeptide.

[0025] In the HSV-1 and / or HSV-2 embodiments of the antigenic compositions, at least the following combinations are contemplated: a gH polypeptide, a gL polypeptide, and a gB polypeptide, each of which may be monomeric or multimeric, and optionally, the gH and gL polypeptides form a gH / gL heterodimer. In certain embodiments, the gH / gL heterodimer is monomeric, dimeric, trimer, or tetrameric, and the gB polypeptide is monomeric, dimeric, or trimer. In one embodiment, the combination comprises a monomeric or trimer gH / gL heterodimer and a monomeric or trimer gB polypeptide. These antigenic compositions may also optionally comprise a monomeric, dimeric, trimer, or tetrameric form of HSV-1 or HSV-2 glycoprotein D (gD) polypeptide.

[0026] In VZV embodiments of the antigenic composition, at least the following combinations are contemplated: A combination of a gH polypeptide, a gL polypeptide, and a gB polypeptide, each polypeptide being monomeric or multimeric, optionally with the gH and gL polypeptides forming a gH / gL heterodimer. In certain embodiments, the gH / gL heterodimer is monomeric, dimeric, trimer, or tetrameric, and the gB polypeptide is monomeric, dimeric, or trimer. In one embodiment, the combination comprises a monomeric or trimer gH / gL heterodimer and a monomeric or trimer gB polypeptide. These antigenic compositions may also optionally comprise one or more of a human VZV glycoprotein C (gC) polypeptide, a human VZV glycoprotein E (gE) polypeptide, and / or a human VZV glycoprotein I (gI) polypeptide.

[0027] In HHV-6 or HHV-7 embodiments of the antigenic composition, at least the following combinations are contemplated: a gH polypeptide, a gL polypeptide, and a gB polypeptide, where each polypeptide is monomeric or multimeric, and optionally, the gH and gL polypeptides form a gH / gL heterodimer. In certain embodiments, the gH / gL heterodimer is monomeric, dimeric, trimer, or tetrameric, and the gB polypeptide is monomeric, dimeric, or trimer. In one embodiment, the combination comprises a monomeric or trimer gH / gL heterodimer and a monomeric or trimer gB polypeptide.

[0028] In KSHV embodiments of the antigenic compositions, at least the following combinations are contemplated: a gH polypeptide, a gL polypeptide, and a gB polypeptide, each polypeptide being monomeric or multimeric, and optionally, the gH and gL polypeptides forming a gH / gL heterodimer. In certain embodiments, the gH / gL heterodimer is monomeric, dimeric, trimer, or tetrameric, and the gB polypeptide is monomeric, dimeric, or trimer. In one embodiment, the combination comprises a monomeric or trimer gH / gL heterodimer and a monomeric or trimer gB polypeptide. These antigenic compositions may also optionally comprise one or more of a human KSHV glycoprotein M (gM) polypeptide, a human KSHV glycoprotein N (gN) polypeptide, a human KSHV open reading frame 68 (ORF68) polypeptide, and / or a human KSHV K8.1 polypeptide.

[0029] In antigenic compositions comprising a nucleic acid, the nucleic acid may be in a viral vector capable of expressing a human herpesvirus polypeptide.

[0030] Also provided are methods of preventing or treating human herpes virus infection in a subject by administering a therapeutically effective amount of two or more HHV polypeptides that make up one or more of the disclosed antigenic compositions. Additionally provided are methods of inducing immunity to human herpes virus in a subject by administering a therapeutically effective amount of two or more HHV fusion and host cell entry proteins that make up one or more of the disclosed antigenic compositions. The two or more HHV fusion and host cell entry proteins may be administered simultaneously or separately.

[0031] The subject to be treated is at risk for developing post-transplantation lymphoproliferative disorder (PTLD) following hematopoietic stem cell or solid organ transplantation. The subject may be a patient suffering from a primary immune deficiency syndrome (PAS), and / or a patient suffering from a primary immune deficiency syndrome (PAS). In the disclosed methods, the antigenic compositions may be administered sequentially or simultaneously.

[0032] Also disclosed are recombinant nucleic acid constructs that express the HHV polypeptides or protein complexes, along with their corresponding encoded polypeptides.

[0033] In one embodiment, the recombinant nucleic acid construct comprises a first nucleic acid molecule encoding an HHV gL polypeptide, a second nucleic acid molecule encoding an HHV gH polypeptide, and an HHV U and a third nucleic acid molecule encoding an L128 polypeptide, a fourth nucleic acid molecule encoding an HHV UL130 polypeptide, and a fifth nucleic acid molecule encoding an HHV UL131A polypeptide. In certain embodiments, when the polypeptide is expressed from the nucleic acid construct in a host cell, a pentameric gH / gL / UL128 / UL130 / UL131A protein complex is formed. The polypeptide optionally does not include a transmembrane domain and / or an intracellular domain. In one embodiment, the recombinant nucleic acid construct further comprises a first promoter operably linked to the first nucleic acid and a second promoter operably linked to the third nucleic acid molecule. The nucleic acid construct also optionally comprises a first internal ribosome entry site (IRES) located between the first nucleic acid molecule and the second nucleic acid molecule, a second IRES located between the third nucleic acid molecule and the fourth nucleic acid molecule, and a third IRES located between the fourth nucleic acid molecule and the fifth nucleic acid molecule. Also optionally, the nucleic acid construct comprises a first, a second, a third, a fourth, and a fifth nucleotide sequence encoding an IgG kappa light chain leader peptide, wherein the first, the second, the third, the fourth, and the fifth nucleotide sequence encoding an IgG kappa light chain leader peptide are in frame with the first, the second, the third, the fourth, and the fifth nucleic acid molecule, respectively. In certain embodiments, the HHV is HCMV, EBV, HSV-1, HSV-2, VZV, KSHV.

[0034] In another embodiment, the recombinant nucleic acid construct comprises a first nucleic acid molecule encoding an HHV gL polypeptide, a second nucleic acid molecule encoding an HHV gH polypeptide, and a third nucleic acid molecule encoding an HHV gO polypeptide. In certain embodiments, a trimeric gL / gH / gO protein complex is formed when the polypeptides are expressed from the nucleic acid construct in a host cell. In certain embodiments, the HHV is HCMV, EBV, HSV-1, HSV-2, VZV, or KSHV.

[0035] Also disclosed are methods of passively transferring immunity against Epstein-Barr Virus (EBV). These methods are accomplished by administering to a subject in need thereof immune cells or high titer anti-EBV immunoglobulins obtained from one or more blood, plasma or serum samples, optionally human blood, plasma or serum samples, selected for high titer anti-EBV immunoglobulins. In these embodiments, the titer of the high titer anti-EBV immunoglobulins may be up to 25-fold, 4-fold to 25-fold, or 10-fold to 20-fold higher than the average titer of anti-EBV immunoglobulins obtained from unselected blood, plasma or serum samples. The blood, plasma or serum samples may be obtained from donors immunized with two or more EBV fusion and host cell entry proteins. Alternatively, blood, plasma or serum samples can be obtained from donors immunized with a single multimeric EBV protein involved in mediating EBV binding, fusion and entry into host cells, including, but not limited to, tetrameric gp350, trimeric gH / gL or trimeric gB. The subject in need of treatment may be at risk of developing post-transplant lymphoproliferative disorder (PTLD) after hematopoietic stem cell or solid organ transplantation, or have or be at risk of developing nasopharyngeal carcinoma (NPC), Burkitt's lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastric cancer, severe infectious mononucleosis, chronic active EBV infection, multiple sclerosis, systemic lupus erythematosus or rheumatoid arthritis. In certain embodiments, the subject is seronegative for EBV.

[0036] In one embodiment, the method of passively transferring immunity to EBV is performed on a subject who is simultaneously receiving one or more of the following: anti-CD20 antibody administration, antiviral therapy, interferon alpha administration, radiation therapy, and chemotherapy.

[0037] In another embodiment of the passive transfer method, the method comprises the steps of: (i) identifying a blood, plasma or serum sample obtained from one or more human subjects with high EBV neutralizing activity; and / or (ii) isolating a high titer anti-EBV antibody from the blood, plasma or serum sample with high EBV neutralizing activity. In this embodiment and related method embodiments, the identifying step optionally includes subjecting the blood, plasma or serum sample to a Raji B cell neutralization assay and / or a HeLa cell neutralization assay. In this embodiment, the HeLa cell neutralization assay includes the steps of infecting HeLa cells with GFP-labeled EBV to generate EBV-infected HeLa cells, incubating the blood, plasma or serum sample with the EBV-infected HeLa cells, analyzing the neutralizing activity of the blood, plasma or serum sample by flow cytometry or ELISpot assay, and optionally analyzing the IC of the blood, plasma or serum sample. 50 Also in this embodiment, the blood, plasma or serum sample is selected so that the blood, plasma or serum sample is calculated based on the mean IC 50 IC40001 ... 50 If the antibody has the above function, it is identified as having high EBV neutralizing activity.

[0038] In another embodiment of the passive transfer method, the method comprises administering to one or more human donor subjects at least two of the following EBV polypeptides: an EBV gp350 polypeptide, an EBV gH / gL heterodimer comprising an EBV gH polypeptide and an EBV gL polypeptide, and an EBV gB polypeptide, in an amount sufficient to produce high titer anti-EBV immunoglobulin, and collecting the high titer anti-EBV immunoglobulin from the one or more human donor subjects prior to administering the high titer anti-EBV immunoglobulin to the subject. In certain embodiments, the EBV gp350 polypeptide is a monomer, dimer, trimer or tetramer, the EBV gB polypeptide is a monomer, dimer or trimer, and the gH / gL heterodimer is a monomer, dimer, trimer or tetramer.

[0039] In a further embodiment, a method for passively transferring immunity against human cytomegalovirus (HCMV) is provided, comprising administering to a subject in need thereof immune cells or high titer anti-HCMV immunoglobulins obtained from one or more blood, plasma or serum samples, optionally human blood, plasma or serum samples, selected for high titer anti-HCMV immunoglobulins. Optionally, the blood, plasma or serum samples are obtained from donors immunized with two or more HCMV fusion and host cell entry proteins. Also, the blood, plasma or serum samples can be obtained from donors immunized with a single multimeric HCMV protein involved in mediating HCMV binding, fusion and entry into host cells, including but not limited to trimeric gH / gL or trimeric gB. In one embodiment of this passive transfer method, the subject at risk of contracting HCMV infection is a pregnant woman, a transplant patient, a patient immunosuppressed during chemotherapy or radiation therapy, or a patient infected with human immunodeficiency virus (HIV).

[0040] In another embodiment of the method of passive HCMV transfer, the method includes one or more of the following steps, performed prior to the step of administering high titer anti-HCMV immunoglobulin to the subject: (i) administering to one or more human donor subjects at least two of an HCMV gB polypeptide, an HCMV gH / gL heterodimer comprising an HCMV gH polypeptide and an HCMV gL polypeptide, an HCMV glycoprotein O (gO) polypeptide, an HCMV UL128 polypeptide, an HCMV UL130 polypeptide, and an HCMV unique UL131A polypeptide, in an amount sufficient to generate a high titer anti-HCMV immunoglobulin response in the subject, and (ii) collecting the high titer anti-HCMV immunoglobulin from the one or more human donor subjects. In certain embodiments, the HCMV gB polypeptide is a monomer, dimer, or trimer, and the gH / gL heterodimer is a monomer, dimer, trimer, or tetramer.

[0041] Also disclosed are methods for passively transferring immunity to herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2). These methods involve administering immune cells or anti-HSV-1 and / or anti-HSV-2 high titer immune globulin to a subject in need thereof. Passive transfer is achieved by administering to a subject receiving the antibody, the immune cells or anti-HSV-1 or anti-HSV-2 hyperimmunoglobulins being obtained from one or more blood, plasma or serum samples, optionally human blood, plasma or serum samples, selected for the anti-HSV-1 or anti-HSV-2 hyperimmunoglobulins. Optionally, the blood, plasma or serum samples are obtained from donors immunized with two or more HSV-1 or HSV-2 fusion and host cell entry proteins. Also, the blood, plasma or serum samples can be obtained from donors immunized with a single multimeric HSV-1 or HSV-2 protein involved in mediating HSV-1 or HSV-2 binding, fusion and entry into host cells, including but not limited to trimeric gH / gL or trimeric gB. In another embodiment of this method, the subject is at risk of developing encephalitis caused by HSV-1 or HSV-2 infection, or the subject is a pregnant woman with active HSV-2 or HSV-1 infection, and / or HSV encephalitis.

[0042] In another embodiment of the method for passive transfer of HSV-2 or HSV-1, the method comprises one or more of the following steps, performed prior to the step of administering anti-HSV-2 or HSV-1 hyperimmune globulin to the subject: (i) administering to one or more human donor subjects at least two of an HSV-1 or HSV-2 glycoprotein D (gD) polypeptide, an HSV-1 or HSV-2 gH / gL heterodimer comprising an HSV-1 or HSV-2 gH polypeptide and an HSV-1 or HSV-2 gL polypeptide, or an HSV-1 or HSV-2 gB polypeptide, in an amount sufficient to produce anti-HSV-1 or HSV-2 hyperimmune globulin; and / or (ii) collecting anti-HSV-1 and / or anti-HSV-2 hyperimmune globulin from the one or more human donor subjects. In certain embodiments, the HSV-1 or HSV-2 gB polypeptide is a monomer, dimer, or trimer, and the HSV-1 or HSV-2 gH / gL heterodimer is a monomer, dimer, trimer, or tetramer.

[0043] Also disclosed are methods for passively transferring immunity against VZV. These methods achieve passive transfer by administering immune cells or high titer anti-VZV immunoglobulin to a subject in need thereof, the immune cells or high titer anti-VZV immunoglobulin being obtained from one or more blood, plasma or serum samples, optionally human blood, plasma or serum samples, selected for high titer anti-VZV immunoglobulin. Optionally, the blood, plasma or serum sample is obtained from a donor immunized with two or more VZV fusion and host cell entry proteins. Also, the blood, plasma or serum sample can be obtained from a donor immunized with a single multimeric VZV protein involved in mediating VZV binding, fusion and entry into host cells, including but not limited to trimeric gH / gL or trimeric gB. In another embodiment of this method, the subject is at risk of developing herpes zoster (shingles) or chickenpox (varicella).

[0044] In another embodiment of the method for passive transfer of VZV, the method includes one or more of the following steps prior to the step of administering high titer anti-VZV immunoglobulin to the subject: (i) administering to one or more human donor subjects at least two of a VZV gH / gL heterodimer comprising a VZV gH polypeptide and a VZV gL polypeptide, a VZV gB polypeptide, a VZV glycoprotein C (gC) polypeptide, a VZV glycoprotein E (gE) polypeptide, and a VZV glycoprotein I (gI) polypeptide in an amount sufficient to generate high titer anti-VZV immunoglobulin, and / or (ii) collecting high titer anti-VZV immunoglobulin from the one or more human donor subjects. In certain embodiments, the VZV gB polypeptide is a monomer, dimer, or trimer, and the VZV gH / gL heterodimer is a monomer, dimer, trimer, or tetramer.

[0045] In addition, human herpesvirus 6 (HHV-6) or human herpesvirus 7 (HHV- Also disclosed are methods for passively transferring immunity against HHV-6 or HHV-7. These methods achieve passive transfer by administering immune cells or anti-HHV-6 or anti-HHV-7 high titer immunoglobulins to a subject in need thereof, the immune cells or anti-HHV-6 or anti-HHV-7 high titer immunoglobulins being obtained from one or more samples of blood, plasma or serum, optionally a human blood, plasma or serum sample, selected for anti-HHV-6 or anti-HHV-7 high titer immunoglobulins. Optionally, the blood, plasma or serum sample is obtained from a donor immunized with two or more of HHV-6 or HHV-7 fusion and host cell entry proteins. Alternatively, blood, plasma or serum samples can be obtained from donors immunized with a single multimeric HHV-6 or HHV-7 protein involved in mediating HHV-6 or HHV-7 binding, fusion and entry into host cells, including, but not limited to, trimeric gH / gL or trimeric gB. In another embodiment of the method for passive transfer of HHV-6 or HHV-7, the method includes one or more of the following steps performed prior to the step of administering anti-HHV-6 or anti-HHV-7 high titer immunoglobulin to a subject: (i) administering to one or more human donor subjects at least an HHV-6 or HHV-7 gH / gL heterodimer and an HHV-6 or HHV-7 gB polypeptide in an amount sufficient to produce anti-HHV-6 or anti-HHV-7 high titer immunoglobulin; and / or (ii) collecting anti-HHV-6 or anti-HHV-7 high titer immunoglobulin from the one or more human donor subjects. In certain embodiments, the HHV-6 or HHV-7 gB polypeptide is a monomer, dimer, or trimer, and the gH / gL heterodimer is a monomer, dimer, trimer, or tetramer.

[0046] Also disclosed are methods for passively transferring immunity against Kaposi's Sarcoma Herpesvirus (KSHV). These methods accomplish passive transfer by administering immune cells or high titer anti-KSHV immunoglobulins to a subject in need thereof, the immune cells or high titer anti-KSHV immunoglobulins being obtained from one or more blood, plasma or serum samples, optionally human blood, plasma or serum samples, selected for high titer anti-KSHV immunoglobulins. Optionally, the blood, plasma or serum samples are obtained from donors immunized with two or more KSHV fusion and host cell entry proteins. Also, the blood, plasma or serum samples can be obtained from donors immunized with a single multimeric KSHV protein involved in mediating KSHV binding, fusion and entry into host cells, including but not limited to trimeric gH / gL or trimeric gB. In another embodiment of this method, the subject is at risk of developing KSHV-associated Kaposi's sarcoma, primary effusion lymphoma, multicentric Castleman's disease, KSHV-associated inflammatory cytokine syndrome, or KSHV immune reconstitution inflammatory syndrome.

[0047] In another embodiment of the method for passive transfer of KSHV, the method includes one or more of the following steps prior to the step of administering high titer anti-KSHV immunoglobulin to the subject: (i) administering to one or more human donor subjects at least two of a KSHV gH / gL heterodimer comprising a KSHV gH polypeptide and a KSHV gL polypeptide, a KSHV gB polypeptide, a KSHV gM polypeptide, a KSHV gN polypeptide, a KSHV ORF68 polypeptide, and a KSHV K8.1 polypeptide in an amount sufficient to generate high titer anti-KSHV immunoglobulin, and / or (ii) collecting the high titer anti-KSHV immunoglobulin from the one or more human donor subjects. In certain embodiments, the KSHV gB polypeptide is a monomer, dimer, or trimer, and the gH / gL heterodimer is a monomer, dimer, trimer, or tetramer.

[0048] The accompanying drawings, which are incorporated herein by reference, illustrate several embodiments and, together with the description, serve to explain several principles of the constructs and methods disclosed herein. [Brief description of the drawings]

[0049] [Figure 1] FIG. 1 is a schematic diagram of recombinant constructs for expressing non-limiting embodiments of multimeric EBV gp350, gH / gL and gB. FIG. 1 discloses "(Gly4Ser1)3" as SEQ ID NO:3, "His6" as SEQ ID NO:49, and "RRRRRD" as SEQ ID NO:55. [Figure 2A] FIG. 1 shows Western blot images of monomeric and multimeric EBV gH / gL polypeptides. [Figure 2B] FIG. 1 shows Western blot images of monomeric and multimeric EBV gB polypeptides. [Figure 2C] FIG. 1 shows Western blot images of monomeric and multimeric EBV gp350 polypeptides. [Diagram 3] FIG. 1 shows EBV in vitro neutralization analysis of sera from rabbits immunized with gp350 monomer (left panel, open circles), gp350 tetramer (left panel, filled circles), gB trimer (right panel), gH / gL monomer (middle panel, open circles), and gH / gL trimer (middle panel, filled circles). [Figure 4A] FIG. 1 shows neutralization titers of sera from rabbits immunized with monomeric or tetrameric EBV gp350, monomeric or trimeric EBV gH / gL, or trimeric EBV gB in alum + CpG-ODN adjuvant in Raji cells. [Figure 4B] FIG. 1 shows neutralization titers of sera from rabbits immunized with monomeric or tetrameric EBV gp350, monomeric or trimeric EBV gH / gL, or trimeric EBV gB in alum + CpG-ODN adjuvant in naive peripheral blood human B cells. [Diagram 5]FIG. 1 shows the EBV neutralizing activity of immune sera from rabbits immunized with trimeric EBV gB or monomeric EBV gH / gL, or a synergistic combination of trimeric EBV gB and monomeric EBV gH / gL. [Figure 6A] FIG. 1 shows the EBV neutralizing activity of pooled immune sera from rabbits (n=5) immunized with tetrameric EBV gp350, trimeric EBV gB, trimeric EBV gH / gL or synergistic combinations thereof. [Figure 6B] FIG. 1 shows the EBV neutralizing activity of pooled immune sera from rabbits immunized (n=5) with monomeric EBV gp350, trimeric EBV gB, monomeric EBV gH / gL or their synergistic combinations. [Figure 7A] Passive transfer of immune rabbit serum prior to EBV infection of humanized mice reduced EBV DNA load and increased survival of challenged mice, Figure 7A shows survival of mice exposed to infection with high doses of live EBV after passive transfer of serum from rabbits immunized with tetrameric EBV gp350, trimeric EBV gH / gL, trimeric EBV gB, or adjuvant alone (control). [Figure 7B] Figure 1 shows that passive transfer of immune rabbit serum prior to EBV infection of humanized mice reduced EBV DNA load and increased survival of challenged mice.Figure 2 shows that pooled immune serum from rabbits immunized with tetrameric EBV gp350 or trimeric EBV gH / gL reduced EBV DNA copy number (geometric mean) in multiple organs of three humanized mice. [Figure 7C] Figure 1 shows that passive transfer of immune rabbit serum prior to EBV infection of humanized mice reduced EBV DNA load and increased survival of challenged mice.Figure 2 shows that pooled immune serum from rabbits immunized with tetrameric EBV gp350, trimeric EBV gH / gL or trimeric EBV gB significantly reduced EBV viral load in peripheral blood (geometric mean of 3 mice) compared to controls. [Figure 8]8 shows an outline of recombinant constructs for expressing non-limiting embodiments of wild-type and trimeric HCMV gB polypeptides. FIG. 8 discloses "GGGGSGGGGSGGGGS" as SEQ ID NO:3, "His6" as SEQ ID NO:49, and "RTKRS" as SEQ ID NO:53. [Figure 9A] FIG. 1 shows an image of a Western blot of monomeric HCMV gB. [Figure 9B] FIG. 1 shows an image of a Western blot of trimeric HCMV gB. [Figure 9C] FIG. 1 shows an image of a Western blot of monomeric HCMV gH / gL. [Figure 9D] FIG. 1 shows an image of a Western blot of trimeric HCMV gH / gL. [Figure 9E] FIG. 1 shows an image of a Western blot of monomeric HCMV UL128 / 130 / 131A. [Figure 10] Figure 10 shows an outline of a non-limiting cloning strategy for expressing recombinant trimeric UL128 / 130 / 131A. Figure 10 discloses "(Gly4Ser)3" as SEQ ID NO:3 and "His6" as SEQ ID NO:49. [Figure 11] FIG. 1 shows serum IgG titers of anti-gH / gL antibodies (left panel) and anti-gB antibodies (right panel) after immunization of rabbits with complexes of monomeric HCMV gH / gL, trimeric HCMV gB, trimeric HCMV gB + monomeric HCMV gH / gL, or trimeric HCMV gB + monomeric HCMV gH / gL. [Figure 12A] FIG. 1 shows the in vitro HCMV neutralization titers (IC50) of non-heat-inactivated sera from rabbits immunized with monomeric HCMV gH / gL, HCMV UL128 / UL130 / UL131A, monomeric HCMV gB (Sino gB), trimeric gB, or certain synergistic combinations thereof, using the ARPE19 epithelial cell line. [Figure 12B]FIG. 1 shows the in vitro HCMV neutralization titers (IC50) using the MRC-5 fibroblast cell line of heat-inactivated sera from rabbits immunized with monomeric HCMV gB (Sino gB), trimeric HCMV gB, monomeric HCMV gH / gL, or a synergistic combination of trimeric HCMV gB and monomeric HCMV gH / gL. [Figure 13] FIG. 1 shows a schematic diagram of a non-limiting DNA construct for expression of the pentameric complex gH / gL / UL128 / UL130 / UL131A. [Figure 14] FIG. 1 shows a schematic diagram of a non-limiting DNA construct for expression of the gH / gL / gO complex. [Figure 15] FIG. 1 shows the in vitro HCMV neutralizing activity of pooled immune sera from rabbits immunized with monomeric HCMV gB. [Figure 16] FIG. 1 shows the in vitro HCMV neutralizing activity of pooled immune sera from rabbits immunized with trimeric HCMV gB. [Figure 17] FIG. 1 shows the in vitro HCMV neutralizing activity of pooled immune sera from rabbits immunized with monomeric HCMV gH / gL. [Figure 18] FIG. 1 shows the in vitro HCMV neutralizing activity of in vitro combined immune sera from rabbits immunized with monomeric HCMV gB and monomeric HMCV gH / gL. [Figure 19] FIG. 1 shows the in vitro HCMV neutralizing activity of in vitro combined immune sera from rabbits immunized with trimeric HCMV gB and monomeric HMCV gH / gL. [Figure 20]FIG. 1 compares the in vitro HCMV neutralizing activity of pooled immune sera from rabbits immunized with individual HCMV proteins (monomeric gB, trimeric gB and monomeric gH / gL) or in vitro combinations of sera from rabbits immunized with HCMV proteins (monomeric gB and monomeric gH / gL, or trimeric gB and monomeric gH / gL) and shows that combinations of HCMV proteins exhibit synergistic activity. [Figure 21A] FIG. 1 shows mouse serum titers of gB-specific IgG from mice immunized with different amounts of HCMV trimeric gB or HCMV monomeric gB. [Figure 21B] FIG. 1 shows neutralization titers (IC50) of heat-inactivated sera from mice immunized with various amounts (1 μg, 5 μg, and 25 μg) of monomeric or trimeric HCMV gB, or with 10 mg / mL CytoGam™ IVIg (CSL Behring, King of Prussia, PA, USA) as a control. [Figure 21C] FIG. 1 shows neutralization titers (IC50) of non-heat inactivated sera from mice immunized with various amounts (1 μg, 5 μg, and 25 μg) of monomeric or trimeric HCMV gB, or with 10 mg / mL CytoGam™ IVIg (CSL Behring, King of Prussia, PA, USA) as a control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] It is to be understood that the following detailed description is presented to provide the reader with a more complete understanding of the details of certain embodiments, features and aspects of the present invention, and should not be construed as limiting the scope of the invention.

[0051] definition In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the Detailed Description.

[0052] The term "antibody" as used in this disclosure refers to an immunoglobulin or an antigen-binding fragment thereof. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies. Antibodies may include constant regions, such as kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, or portions thereof. Heavy chain constant regions of various isotypes can be used, including, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. By way of example, the light chain constant region may be kappa or lambda.

[0053] The terms "antigen-binding domain" and "antigen-binding fragment" refer to a portion of an antibody molecule that contains the amino acids responsible for the specific binding between the antibody and the antigen. For some antigens, the antigen-binding domain or antigen-binding fragment may only bind to a portion of the antigen. The part of the antigen that is specifically recognized and bound by an antibody is called an "epitope" or "antigenic determinant". Antigen-binding domains and antigen-binding fragments include Fab (Fragment antigen-binding), F(ab')2 fragments, a bivalent fragment having two Fab fragments linked at the hinge region by a disulfide bridge, Fv fragments, single chain Fv fragments (scFv) (see, e.g., Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883), two VF fragments, and the like. H Domain and C H Fd fragment having 1 domain, dA b (Ward et al. (1989) Nature 341:544-546), and other antibody fragments that retain antigen-binding function. The Fab fragment contains V covalently linked by disulfide bonds between constant regions. H -C H 1 Domain and V L -C L F VThe fragments are smaller and not covalently bound V H Domain and V L To overcome the tendency of non-covalently linked domains to dissociate, the scF V It is possible to construct scF V (1) V H The C-terminus of V L or (2) V L The C-terminus of V H The antibody fragments include a flexible polypeptide linked to the N-terminus of the 15-mer (Gly4Ser)3 peptide (SEQ ID NO:3) can be used as a linker, although other linkers are known in the art. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are evaluated for function in the same manner as intact antibodies.

[0054] As used herein, "antigen" refers to a protein or fragment thereof, or a polysaccharide linked to a protein carrier, that is capable of eliciting an immune response when expressed in an animal or human cell or tissue. The protein or fragment thereof may be glycosylated. Or it may be unglycosylated.

[0055] The term "extracellular domain" refers to the portion of a full-length polypeptide that extends beyond the cell membrane into the medium in which the cell inside the polypeptide resides. Polypeptides are generally known to include an intracellular domain, a transmembrane domain, and the remainder is the extracellular domain ("ECD"). As used herein, the term "extracellular domain" or "ECD" refers to the amino acids of a polypeptide that in its wild-type form extend beyond the cell membrane, or any portion thereof that is recognizable by an antibody. Thus, an extracellular domain includes the entire domain, or any number of residues that can be included in recombinant expression and antigenic compositions, including polypeptides that represent 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the entire wild-type extracellular domain of a polypeptide. That is, the extracellular domain may be shortened or truncated by methods known in the art to remove extraneous domains at either the carboxy or amino terminus of the polypeptide, or both, as needed to obtain more efficient and robust expression of the extracellular domain of a polypeptide.

[0056] The term "full length" for a given polypeptide refers to the form of the polypeptide naturally translated from a coding DNA sequence, beginning with the ATG start codon, which codes for the first methionine in the amino acid sequence, and ending with a TGA, TAG, or TTA stop codon, or any stop codon utilized by the organism.

[0057] The term "fusion protein" refers to a protein translated from a nucleic acid transcript produced by combining a first nucleic acid sequence encoding a first protein with at least a second nucleic acid encoding a second protein, where the fusion protein is not a naturally occurring protein. A nucleic acid construct may encode two or more proteins that are joined into a fusion protein to create a single polypeptide chain. The two or more nucleic acid sequences are optionally operably linked to a single promoter or to two or more separate promoters.

[0058] The term "glycoprotein" refers to a polypeptide having one or more sugar moieties or oligosaccharide chains covalently attached to it. Sugar moieties are usually attached to the glycoprotein cotranslationally or as a post-translational modification.

[0059] The term "isolated," when used in reference to a polypeptide or nucleic acid, refers to a polypeptide or nucleic acid that is substantially free of its natural environment and thus distinguishable from a naturally occurring polypeptide or nucleic acid. For example, an isolated polypeptide or nucleic acid is substantially free of cellular material or other polypeptides or nucleic acids from the cell or tissue source from which it is derived. The term also refers to preparations in which the isolated polypeptide or nucleic acid is sufficiently pure for a pharmaceutical composition, or at least 70% (w / w) to 80% (w / w), or at least 80% (w / w) to 90% (w / w), or at least 90% (w / w) to 95% (w / w), or at least 95% (w / w), 96% (w / w), 97% (w / w), 98% (w / w), 99% (w / w), or 100% (w / w) pure.

[0060] The term "leader sequence" refers to a short peptide sequence at the N-terminus of a recombinant protein that directs the recombinant protein to be secreted from a host cell.

[0061] The term "HHV fusion and host cell entry protein" refers to a human herpesvirus gB polypeptide, a gH polypeptide, a gL polypeptide, a gH / gL heterodimer, or Refers to the gp350 polypeptide.

[0062] The term "HHV accessory protein" refers to a human herpesvirus polypeptide other than gB, gH, gL, gH / gL or gp350 that is involved in mediating viral binding, fusion and host cell entry, including, but not limited to, gp42, gM, gN, gI, gC, gD, ORF68, BMRF-2, BDLF2, UL128, UL130, UL131A and gpK8.1.

[0063] The term "immune cell" refers to any cell of the hematopoietic lineage that is involved in regulating the immune response to an antigen (e.g., a self-antigen). In an exemplary embodiment, the immune cell is a leukocyte, such as a white blood cell. Immune cells include neutrophils, leukocytes, and the like. The immune cells may be eosinophils, basophils, lymphocytes and / or monocytes. Lymphocytes include T lymphocytes and B lymphocytes. The immune cells may also be dendritic cells, natural killer (NK) cells and / or mast cells.

[0064] The term "intracellular domain" refers to that portion of a polypeptide that is present in the cytoplasm of a host cell. The intracellular domain includes that portion of a polypeptide that is not the transmembrane domain and that is not the extracellular domain.

[0065] The term "gH / gL heterodimer" refers to a polypeptide or a complex of polypeptides comprising an HHV gH polypeptide and an HHV gL polypeptide. For example, the heterodimer may be a complex non-covalently associated between an HHV gH polypeptide and an HHV gL polypeptide. Alternatively, the heterodimer may be a recombinant fusion protein comprising an HHV gH protein linked to an HHV gL protein. The HHV gH protein may be linked to the HHV gL protein by a peptide linker.

[0066] As used herein, the term "modified gB polypeptide" refers to an HHV gB polypeptide in which the furin cleavage site in the extracellular domain of the gB polypeptide has been replaced by a linker sequence, as described in Patent Document 2.

[0067] The term "operably linked" means that a promoter or similar regulatory element is positioned next to an expressible nucleotide sequence, or coding region, such that transcription of the coding region is controlled and regulated by the promoter.

[0068] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids.

[0069] The term "peptide linker" refers to a short, non-native peptide sequence that links two proteins or fragments of proteins.

[0070] The term "recombinant," when used in reference to nucleic acids, refers to a nucleic acid having nucleotide sequences that are not naturally linked, and may be made by artificially combining two segments of otherwise isolated sequences. This artificial combination is often accomplished by chemical synthesis, or, more commonly, by the artificial manipulation of isolated segments of nucleic acid, e.g., by genetic engineering techniques. Recombinant nucleic acids include nucleic acid vectors that contain amplified or associated nucleic acids that can be used to transform or transfect suitable host cells. A host cell that contains a recombinant nucleic acid is called a "recombinant host cell." Genes are then expressed in the recombinant host cell to produce a "recombinant polypeptide." Recombinant nucleic acids may also include those that contain non-coding functions (e.g., promoters, origins of replication, ribosomal proteins, and / or polypeptides). These may act as ligands (e.g., phospholipid binding sites).

[0071] The term "transmembrane domain" (or "TM") refers to a portion of a polypeptide that naturally passes completely through the cellular membrane, the hydrophobic phospholipid bilayer that separates the cytoplasm from the external medium in which the host cell resides. Transmembrane domains are typically about 20 amino acids to about 25 amino acids in length, depending on the polypeptide. Transmembrane forms are typically not included in the antigenic compositions disclosed herein because they are typically lipophilic and therefore difficult to express, purify and solubilize.

[0072] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" means solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art. In certain embodiments, the pharmaceutically acceptable carrier or excipient is not naturally occurring.

[0073] The terms "prevent, preventing" when used in reference to a disease or disease state, refer to the prophylactic administration of a composition that arrests or delays the onset of a pathological feature or symptom of the disease or disorder.

[0074] The term "treat, treating" when used in reference to a disease or disease state can mean to ameliorate, ameliorate or cure the disease, disorder, or symptoms of the disease or a condition associated with the disease, or can mean to completely or partially arrest the biochemical basis of the disease at the molecular level, such as stopping the replication of a virus.

[0075] The term "therapeutically effective amount" when used in reference to an amount of an active agent means an amount that results in an improvement or remediation of a disease, disorder, or symptoms of a disease or condition.

[0076] The term "passive transfer" or "passive immunotherapy" or "passive immunity" refers to obtaining antibodies and / or immune cells from a subject exposed to an antigen and administering those antibodies and / or immune cells to a second subject, thereby providing the second subject with immune protection against antigen challenge. The antibodies or immune cells can be transferred in the form of blood, plasma, purified antibodies or immune cells, serum, etc. The second subject may be immunocompromised and / or naive (never exposed to an antigen) (Keller et al., 2002). See Clin. Microbiol. Rev., 13(4):602-614, 2000).

[0077] Human Herpesviruses. The Herpesviridae family is subdivided into three subfamilies, Alphaherpesviruses, Betaherpesviruses, and Gammaherpesviruses, based on biological characteristics and DNA genome similarities (Davison et al., Antiviral Res., 56:1-11, 2002; MacDonald et al., Am. J. Cardiol., 64:359-362, 1989) (Table 1; see Willis et al., Br. Med. Bull., 62(1):125-138, 2002). The Alphaherpesviruses include H These include HV-1, HHV-2, VZV and pseudorabies virus (PRV) and are neurotropic, i.e., they tend to primarily infect or attack the nervous system of the host. The Alphaherpesviridae family has the broadest host range and spreads rapidly in cell culture. Latent Alphaherpesvirus infection is usually established in sensory neurons, and lytic infection occurs in epidermal cells (Roizman B, Sears AE. Herpes simplex viruses and their replication. In: Fields BN, Knipe DM, Howley PM, eds. Fields virology. Philadelphia: Lippincott-Raven, 1996:2231-95).

[0078] [Table 1]

[0079] The Betaherpesvirinae subfamily consists of all cytomegaloviruses, including human cytomegalovirus (HCMV, HHV-8), HHV-6 and HHV-7, commonly referred to as roseoloviruses. The Betaherpesviridae family has a restricted host range and a long infectious cycle. Viral latency of Betaherpesviruses is maintained in secretory glands, kidneys and other tissues (Hendrix et al., Expert Rev. Anti Infect. Ther., 5:427-439, 2007).

[0080] The Gammaherpesvirinae subfamily is divided into EBV, Rhadinovirus, and Lymphocryptovirus, which includes HHV-8 (KSHV). Gammaherpesviruses have a very narrow host range, and viral replication typically occurs in lymphoblastoid cells, but they can also lytically infect epithelial and fibroblastic cells. Latent forms of Gammaherpesvirus infection are primarily observed in B and T lymphocytes (Ackerman, Vet. Microbiol., 113:211-222, 2006).

[0081] Gammaherpesviruses: Epstein-Barr virus (EBV, HHV-4) and Kaposi's sarcoma virus-associated herpes (KSHV, HHV-8) Epstein-Barr Virus (EBV, HHV-4). Epstein-Barr Virus (EBV) was the first discovered human cancer virus and is strongly implicated in the pathogenesis of post-transplant lymphoproliferative disorder (PTLD) and undifferentiated nasopharyngeal carcinoma (NPC). In both instances, disease onset and severity positively correlated with the level of EBVemia, strongly suggesting a role for lytic EBV reactivation in the perpetuation of disease. Epstein-Barr virus (EBV), also known as human herpesvirus 4 (HHV-4), is responsible for pathological entities such as Burkitt's lymphoma, nasopharyngeal carcinoma, infectious mononucleosis, a subset of Hodgkin's disease, and lymphoproliferative syndromes in immunosuppressed patients and is a major cause of morbidity and mortality worldwide (Cohen JI, Curr. Opin. Immunol., 1999 Aug; 11(4):365-70; Thorley-Lawson DA, J., Allergy Clin. Immunol., 2005 Aug; 116(2):251-61; quiz 62; and Vetsika EK, Callan M., Expert Rev. Mol. Med., 2004 Nov 5;6(23):1-16). EBV has a double-stranded linear DNA genome. The nucleotide sequence of the EBV genome, and the amino acid sequences of the viral proteins encoded thereby, are known and are set forth in NCBI Reference No. NC_009334, Version NC_009334.1, GI:139424470, which sequences are incorporated herein by reference.

[0082] EBV is a member of the Gammaherpesvirinae subfamily, which is further divided into the Lymphocryptoviruses, of which KSHV (HHV-8) is also a member. Replication for these family members typically occurs in lymphoblastoid cells, but they can also infect epithelial cells (e.g., nasopharyngeal epithelial cells) and fibroblasts. Latent infection is observed primarily in B and T lymphocytes (Ackerman, Vet. Microbiol., 113:211-222, 2006).

[0083] Post-transplant lymphoproliferative disorder (PLTD). Patients undergoing solid organ or stem cell transplantation are at risk for developing post-transplant lymphoproliferative disorder (PTLD), characterized by uncontrolled EBV-driven B-cell proliferation that can progress to non-Hodgkin's lymphoma (LaCasce, Oncologist, 11:674-80, 2006). PTLD can result from EBV reactivation in seropositive recipients or primary EBV infection from the donor allograft, posing an even greater risk (Dharnidharka et al., Am. J. Transplant, 12:976-83, 2012). Similar phenomena have been reported in patients with EBV-associated B-cell grafts. This also occurs in people with AIDS.

[0084] Most cases of PTLD involve an EBV-driven proliferation of excessive B cells, with a minority (10%-15%) of cases being of NK / T cell type (Petrara et al., Cancer Lett., 369(1):37-44, 2015, and Starzl et al., Lancet, 1:583-7, 1984). The frequency of PTLD ranges from 1% to 20%, depending on the type of transplant, the age of the recipient, and the duration and type of immunosuppressive treatment (Ibrahim et al., Adv Hematol., 2012:230173, 2012, and Smets et al., Recent Results Cancer Res., 193:173-90, 2014). EBV-seronegative, more Younger patients are at highest risk of developing PTLD after hematopoietic stem cell or solid organ transplantation due to the lack of prior immunity. Patients with primary immunodeficiency syndromes are also at high risk of developing EBV-driven B-cell lymphoproliferation and lymphoma (Rickinson et al., Trends Immunol., 35:159-69, 2014). The WHO defines three main histological types of PTLD in ascending order of severity: primary lesion, polymorphic (P-PTLD), and monomorphic (M-PTLD) (Harris M-PTLD is typically characterized by a non-Hodgkin's syndrome. It manifests as kin lymphoma.

[0085] The initial management of PTLD is reduction of immunosuppression. Additional treatment options include B cell-depleting anti-CD20 mAb therapy, antiviral therapy, intravenous immunoglobulin (IVIg), and interferon (IFN)-γ (LaCasce AS, Oncologist, 11:674-80, 2006). In particular, IVIg has been used empirically in combination with other therapies to treat PTLD, but there have been no studies evaluating its possible clinical benefits.

[0086] Nasopharyngeal carcinoma and EBV. Nonkeratinizing variants of squamous cell carcinoma (NPC) of the nasopharyngeal tract are endemic in East and South-East Asia, as well as parts of North and East Africa, and accounted for 86,500 cancer cases worldwide in 2012 (Chua et al., Lancet, 387(10022):1012-1024, 2016). NPC is associated with frequent metastases to cervical lymph nodes, leading to epistaxis, unilateral nasal congestion, and nasopharyngeal congestion. It manifests clinically as blockage, auditory complaints and cranial nerve palsies. Radiotherapy is the primary treatment for NPC, with additional chemotherapy utilized in more advanced cases. (Ibid.) Five-year survival is 70%-98%, depending on stage, but NPC is prone to recurrence.

[0087] Undifferentiated NPCs are invariably associated with EBV, which appears to play a pathogenic role in tumor initiation and progression (Tsang et al., Virol. Sin., 30:107-21, 2015). Establishment of latent EBV infection in premalignant nasopharyngeal epithelial cells appears to further drive malignant transformation. Elevated serum IgA levels specific for EBV lytic antigens, such as viral capsid and early antigens, correlate with progression to NPC (Ji et al., Br. J. Cancer, 96:623-30, 2007). Plasma EBV V DNA levels directly correlate with NPC tumor burden (To et al., Clin. Cancer Res., 9:3254-9, 2003). Thus, reactivation of latent EBV is a key feature of NPC formation and progression, suggesting a possible role for antibody-based immunotherapy. Although multiple strains of EBV can be isolated from the blood and saliva of healthy seropositive individuals, typically only a single strain of EBV is isolated from NPC cells, consistent with its pathogenic role (Tsang et al., Virol. Sin., 30:107-21, 2015). Although strain variations in the sequences of EBNA2, 3A, 3B and 3C have been described, the envelope proteins gp350, gH / gL and gB are highly conserved, making these latter proteins ideal vaccine candidates for cross-strain protection (Sample et al., J. Virol., 64:4084-92, 1990, and Rowe et al., J. Virol., 63:1031-9, 1989).

[0088] Circulating EBV DNA copy number positively correlates with the impending development and clinical severity of EBV-associated malignancies. EBV qPCR assays are routinely used after transplantation (Meerbach et al., J. Med. Virol., 80:441-54, 2008; Tsai et al., Am. J. Transplant, 8:1016-24, 2008; Wagner et al., Transplantation, 74:656-64, 2002; and van Esser et al., Blood 98:972-8, 2001). Elevated levels of EBV DNA in the blood are associated with an increased risk for PTLD, whereas reductions correlate with treatment success (Baldanti et al., J. Clin. Microbiol., 38:613-9, 2000; Hakim et al., J. Clin. Microbiol., 45:2151-5, 2007; Wagner et al., Transplantation, 72:1012-9, 2001; and Clave et al., Transplantation, 77:76-84, 2004). Circulating EBV DNA has also been implicated in the development of Hodgkin's lymphoma (Kanakry et al., J. Clin. Microbiol., 38:613-9, 2000). al., Blood, 121:3547-53, 2013) and extranodal NK / T cell lymphoma, which is also pathogenically associated with EBV (Wang et al., Oncotarget., 6(30):30317-30326, 2015), and positively correlates with adverse survival outcomes in NPC (Jin et al., Eur. J. Cancer, 48:882-8, 2012; Hsu et al., Head Neck, 34:1064-70, 2012; and Hsu et al., Oral Oncol., 49:620-5, 2013).

[0089] In the developing world, EBV seroconversion typically occurs during early childhood, whereas in developed countries, the disease tends to affect adolescents more frequently. Infectious mononucleosis typically occurs only in this latter group (Vetsika et al., Expert Rev. Mol. Med., 2004 Nov 5; 6(23):1-16). The main human reservoir for latent EBV and EBV transmission are resting memory B lymphocytes (Babcock et al., Immunity, 1998 Sep; 9(3):395-404). EBV depends on the gp350-CD21 binding event for viral entry into B cells (Tanner et al., Cell, 1987 Jul 17; 50(2):203-13, and Tanner et al., J. Virology, 1988; 62(12):4452-64), which is a critical event for infectivity and B cell neoplastic transformation (Thorley-Lawson DA, J. Allergy Clin. Immunol., 2005 Aug; 116(2):251-61; quiz 62). gp350 is the major EBV outer membrane glycoprotein, while CD21, also known as the type 2 complement receptor (CR2), is a receptor on the B cell surface that binds to the iC3b complement protein. Sera from patients with active EBV infection contain antibodies that block EBV entry into B cells ("neutralizing" antibodies). Adsorption of these sera with gp350 eliminates most of this neutralizing activity (Thorley-Lawson et al., J. Virology, 1982 Aug; 43(2):730-6), suggesting that gp350 is a protective These results indicate that EBV plays a role as the major EBV antigen against which the target humoral immune response is directed.

[0090] Many studies have demonstrated that immunization of non-human primates with subunit gp350 vaccines in adjuvant protects against experimental EBV-induced lymphoma or EBV replication. Thus, purified native gp35 vaccines injected into cottontop marmosets (CTMs) with liposomes, ISCOMs or muramyl dipeptide were used to protect against EBV-induced lymphoma and EBV replication. 0 protected against EBV-induced lymphomas (Morgan et al., J. Med. Virol., 1984; 13(3):281-92, and Morgan et al., J. Med. Virol., 1989 Sep; 29(1):74-8). Recombinant gp350 in alum or muramyl dipeptide was similarly protective (Finerty et al., J. Gen. Virol., 1992 Feb; 73 (Pt 2):449-53, and Finerty et al., Vaccine, 1994 Oct; 12(13):1180-4). Common marmosets have also been shown to be protected from EBV-induced lymphomas by recombinant gp350 in alum. Immunization with p350 has been shown to reduce viral replication after EBV challenge (Cox et al., J. Med. Virol., 1998 Aug; 55(4):255-61). Non-human primate studies using gp350 expressed by adenovirus or vaccinia virus vectors have similarly shown protection against experimental EBV-induced lymphomas or EBV replication in CTM or common marmosets (Mackett et al., J. Med. Virol., 1996 Nov; 50(3):263-71; Ragot et al., J. Gen. Virol., 1993 Mar; 74 (Pt 3):501-7; and Morgan et al., J. Med. Virol., 1988 Jun; 25(2):189-95).

[0091] Also, pilot studies in humans suggested a possible role for gp350 vaccination in host protection against EBV. In a study by Gu et al. (Dev. Biol. Stand., 1995; 84:171-7), a single dose of gp350 / 220 expressed by vaccinia virus (VV) was given by scarification to 1- to 3-year-old children who were EBV-seronegative and VV-seronegative. These children generated neutralizing antibodies against EBV (1:40-1:160). Ten out of ten unvaccinated controls were infected at 16 months of follow-up, whereas only three out of nine vaccinated children were infected during this time period. More recently, a phase I / II study was performed in which healthy EBV-seronegative adults were immunized with recombinant monomeric gp350 protein in alum + / - monophosphoryl lipid A (Non-Patent Document 6, and Non-Patent Document 7). After three doses, up to 82% of subjects had detectable neutralizing serum anti-gp350 antibody titers. The vaccine demonstrated 78.0% efficacy in preventing the development of infectious mononucleosis but not in preventing asymptomatic EBV infection. Finally, an additional phase I trial of recombinant monomeric gp350 protein in alum given to children with chronic kidney disease demonstrated that only a minority of subjects demonstrated detectable neutralizing serum anti-gp350 titers (8).

[0092] Currently, there is no effective immunotherapy for EBV-associated disease and no clinically licensed prophylactic EBV vaccine. EBV gp350, gH / gL complex, and gB are three envelope proteins that are potential vaccine target antigens against EBV. EBV gp350 mediates EBV attachment to B cells through its binding to CD21. EBV gH / gL and gB are involved in mediating EBV fusion and entry into both B cells and epithelial cells.

[0093] EBV gp350 / gp220. The EBV glycoprotein gp350 and the related splice variant gp220 are responsible for the attachment of EBV with high affinity to CR2 on B cells. Antibodies against gp350 or gp220 that block EBV binding neutralize B cell infection. gp350 and gp220 are highly glycosylated single-pass transmembrane proteins, respectively. As a result of alternative splicing, the viral glycoprotein appears in two forms, with masses of approximately 350 kDa and 220 kDa. The 200 kDa splice form lacks residues 500-757 of the full-length gp350. Both gp350 and gp220 retain the CR2-binding domain at the amino terminus. Truncated forms of gp350 or gp220 having amino acids 1-470 of gp350 retain the ability to bind CR2 and can inhibit EBV binding to CR2 and can be substituted for full-length gp350 or gp200, or the extracellular domain form of gp350 in the compositions described herein (Sarrias et al., J. Immunol., 2001 Aug 1;167(3):1490-9). Additionally, the gp350 sequence The portion of gp220 between amino acids 21-26 and amino acids 372-378 of the gp350 sequence is involved in CR2 binding (Tanner et al., Cell, 203-213 (1987), and Nemerow et al., Cell, 61:1416-20, 1987). The term protein or gp350 antigen (or gp220 protein or antigen) refers not only to full-length gp350 or gp220 proteins, but also to fragments or modified forms thereof that retain the ability to bind CR2.

[0094] The amino acid and nucleic acid sequences of gp350 published in GenBank under Accession No. M10593, Version M10593.1, GI 330360, are incorporated herein by reference. The amino acid sequence of gp350 is as follows (SEQ ID NO:1): MEAALLVCQY TIQSLIHLTG EDPGFFNVEI PEFPFYPTCN VCTADVNVTI 50 NFDVGGKKHQ LDLDFGQLTP HTKAVYQPRG AFGGSENATN LFLLELLGAG 100 ELALTMRSKK LPINVTTGEE QQVSLESVDV YFQDVFGTMW CHHAEMQNPV 150 YLIPETVPYI KWDNCNSTNI TAVVRAQGLD VTLPLSLPTS AQDSNFSVKT 200 EMLGNEIDIE CIMEDGEISQ VLPGDNKFNI TCSGYESHVP SGGILTSTSP 250 VATPIPGTGY AYSLRLTPRP VSRFLGNNSI LYVFYSGNGP KASGGDYCIQ 300 SNIVFSDEIP ASQDMPTNTT DITYVGDNAT YSVPMVTSED ANSPNVTVTA 350 FWAWPNNTET DFKCKWTLTS GTPSGCENIS GAFASNRTFD ITVSGLGTAP 400 KTLIITRTAT NATTTTHKVI FSKAPESTTT SPTLNTTGFA DPNTTTGLPS 450 STHVPTNLTA PASTGPTVST ADVTSPTPAG TTSGASPVTP SPSPWDNGTE 500 SKAPDMTSST SPVTTPTPNA TSPTPAVTTP TPNATSPTPA VTTPTPNATS 550 PTLGKTSPTS AVTTPTPNAT SPTLGKTSPT SAVTTPTPNA TSPTLGKTSP 600 TSAVTTPTPN ATGPTVGETS PQANATNHTL GGTSPTPVVT SQPKNATSAV 650 TTGQHNITSS STSSMSLRPS SNPETLSPST SDNSTSHMPL LTSAHPTGGE 700 NITQVTPASI STHHVSTSSP EPRPGTTSQA SGPGNSSTST KPGEVNVTKG 750 TPPQNATSPQ APSGQKTAVP TVTSTGGKAN STTGKHTTG HGARTSTEPT 800 TDYGGDSTTP RPRYNATTYL PPSTSSKLRP RWTFTSPPVT TAQATVPVPP 850 TSQPRFSNLS MLVLQWASLA VLTLLLLLVM ADCAFRRNLS TSHTYTTPPY 900 DDAETYV 907

[0095] The sequence of gp220 amino acids, as published in GenBank under Accession No. M10593, Version M10593.1, GI 330360, and incorporated herein by reference, is as follows (SEQ ID NO:2): MEAALLVCQY TIQSLIHLTG EDPGFFNVEI PEFPFYPTCN VCTADVNVTI 50 NFDVGGKKHQ LDLDFGQLTP HTKAVYQPRG AFGGSENATN LFLLELLGAG 100 ELALTMRSKK LPINVTTGEE QQVSLESVDV YFQDVFGTMW CHHAEMQNPV 150 YLIPETVPYI KWDNCNSTNI TAVVRAQGLD VTLPLSLPTS AQDSNFSVKT 200 EMLGNEIDIE CIMEDGEISQ VLPGDNKFNI TCSGYESHVP SGGILTSTSP 250 VATPIPGTGY AYSLRLTPRP VSRFLGNNSI LYVFYSGNGP KASGGDYCIQ 300 SNIVFSDEIP ASQDMPTNTT DITYVGDNAT YSVPMVTSED ANSPNVTVTA 350 FWAWPNNTET DFKCKWTLTS GTPSGCENIS GAFASNRTFD ITVSGLGTAP 400 KTLIITRTAT NATTTTHKVI FSKAPESTTT SPTLNTTGFA DPNTTTGLPS 450 STHVPTNLTA PASTGPTVST ADVTSPTPAG TTSGASPVTP SPSPWDNGTE 500 STPPQNATSP QAPSGQKTAV PTVTSTGGKA NSTTGGKHTT GHGARTSTEP 550 TTDYGGDSTT PRPRYNATTY LPPSTSSKLR PRWTFTSPPV TTAQATVPVP 600 PTSQPRFSNL SMLVLQWASL AVLTLLLLLV MADCAFRRNL STSHTYTTPP 650 YDDAETYV 658

[0096] EBV gH, gL, gB and gp42. The minimum requirements for viral fusion with B cells include the EBV glycoproteins gH, gL, gB and gp42. For infection of B cells, gp42 binds to host cell MHC class II molecules and initiates viral-cell membrane fusion. On the other hand, for infection of epithelial cells, gp42 is not required. Rather, EBV gH, gL and gB proteins are sufficient for viral fusion with epithelial cells. EBV gH / gL exists in certain environments as a non-covalently associated complex.

[0097] The amino acid sequence of EBV gH is as follows (SEQ ID NO:4): MQLLCVFCLV LLWEVGAASL SEVKLHLDIE GHASHYTIPW TELMAKVPGL 50 SPEALWREAN VTEDLASMLN RYKLIYKTSG TLGIALAEPV DIPAVSEGSM 100 QVDASKVHPG VISGLNSPAC MLSAPLEKQL FYYIGTMLPN TRPHSYVFYQ 150 LRCHLSYVAL SINGDKFQYT GAMTSKFLMG TYKRVTEKGD EHVLSLIFGK 200 TKDLPDLRGP FSYPSLTSAQ SGDYSLVIVT TFVHYANFHN YFVPNLKDMF 250 SRAVTMTAAS YARYVLQKLV LLEMKGGCRE PELDTETLTT MFEVSVAFFK 300 VGHAVGETGN GCVDLRWLAK SFFELTVLKD IIGICYGATV KGMQSYGLER 350 LAAVLMATVK MEELGHLTTE KQEYALRLAT VGYPKAGVYS GLIGGATSVL 400 LSAYNRHPLF QPLHTVMRET LFIGSHVVLR ELRLNVTTQG PNLALYQLLS 450 TALCSALEIG EVLRGLALGT ESGLFSPCYL SLRFDLTRDK LLSMAPQEAM 500 LDQAAVSNAV DGFLGRLSLE REDRDAWHLP AYKCVDRLDK VLMIIPLINV 550 TFIISSDREV RGSALYEAST TYLSSSLFLS PVIMNKCSQG AVAGEPRQIP 600 KIQNFTRTQK SCIFCGFALL SYDEKEGLET TTYITSQEVQ NSILSSNYFD 650 FDNLHVHYLL LTTNGTVMEI AGLYEERAHV VLAIILYFIA FALGIFLVHK 700 IVMFFL 706

[0098] The amino acid sequence of EBV gL is as follows (SEQ ID NO:5): MRTVGVFLAT CLVTIFVLPT WGNWAYPCCH VTQLRAQHLL ALENISDIYL 50 VSNQTCDGFS LASLNSPKNG SNQLVISRCA NGLNVVSFFI SILKRSSSAL 100 TGHLRELLTT LETLYGSFSV EDLFGANLNR YAWHRGG 137

[0099] The amino acid sequence of EBV gB is as follows (SEQ ID NO:6): MTRRRVLSVV VLLAALACRL GAQTPEQPAP PATTVQPTAT RQQTSFPFRV 50 CELSSHGDLF RFSSDIQCPS FGTRENHTEG LLMVFKDNII PYSFKVRSYT 100 KIVTNILIYN GWYADSVTNR HEEKFSVDSY ETDQMDTIYQ CYNAVKMTKD 150 GLTRVYVDRD GVNITVNLKP TGGLANGVRR YASQTELYDA PGWLIWTYRT 200 RTTVNCLITD MMAKSNSPFD FFVTTTGQTV EMSPFYDGKN KETFHERADS 250 FHVRTNYKIV DYDNRGTNPQ GERRAFLDKG TYTLSWKLEN RTAYCPLQHW 300 QTFDSTIATE TGKSIHFVTD EGTSSFVTNT TVGIELPDAF KCIEEQVNKT 350 MHEKYEAVQD RYTKGQEAIT YFITSGGLLL AWLPLTPRSL ATVKNLTELT 400 TPTSSPPSSP SPPAPPAARG STSAAVLRRR RRDAGNATTP VPPAAPGKSL 450 GTLNNPATVQ IQFAYDSLRR QINRMLGDLA RAWCLEQKRQ NMVLRELTKI 500 NPTTVMSSIY GKAVAAKRLG DVISVSQCVP VNQATVTLRK SMRVPGSETM 550 CYSRPLVSFS FINDTKTYEG QLGTDNEIFL TKKMTEVCQA TSQYYFQSGN 600 EIHVYNDYHH FKTIELDGIA TLQTFISLNT SLIENIDFAS LELYSRDEQR 650 ASNVFDLEGI FREYNFQAQN IAGLRKDLDN AVSNGRNQFV DGLGELMDSL 700 GSVGQSITNL VSTVGGLFSS LVSGFISFFK NPFGGMLILV LVAGVVILVI 750 SLTRRTRQMS QQPVQMLYPG IDELAQQHAS GEGPGINPIS KTELQAIMLA 800 LHEQNQEQKR AAQRAAGPSV ASRALQAARD RFPGLRRRRY HDPETAAALL 850 GEAETEF 857

[0100] The amino acid sequence of EBV gp42 is as follows (SEQ ID NO:7): MVSFKQVRVP LFTAIALVIV LLLAYFLPPR VRGGGRVSAA AITWVPKPNV 50 EVWPVDPPPP VNFNKTAEQE YGDKEIKLPH WTPTLHTFQV PKNYTKANCT 100 YCNTREYTFS YKERCFYFTK KKHTWNGCFQ ACAELYPCTY FYGPTPDILP 150 VVTRNLNAIE SLWVGVYRVG EGNWTSLDGG TFKVYQIFGS HCTYVSKFST 200 VPVSHHECSF LKPCLCVSQR SNS 223

[0101] The amino acid sequence of EBV BMRF-2 is as follows (SEQ ID NO:8): MFSCKQHLSL GACVFCLGLL ASTPFIWCFV FANLLSLEIF SPWQTHVYRL 50 GFPTACLMAV LWTLVPAKHA VRAVTPAIML NIASALIFFS LRVYSTSTWV 100 SAPCLFLANL PLLCLWPRLA IEIVYICPAI HQRFFELGLL LACTIFALSV 150 VSRALEVSAV FMSPFFIFLA LGSGSLAGAR RNQIYTSGLE RRRSIFCARG 200 DHSVASLKET LHKCPWDLLA ISALTVLVVC VMIVLHVHAE VFFGLSRYLP 250 LFLCGAMASG GLYLGHSSII ACVMATLCTL TSVVVYFLHE TLGPLGKTVL 300 FISIFVYYFS GVAALSAAMR YKLKKFVNGP LVHLRVVYMC CFVFTFCEYL 350 LVTFIKS

[0102] The amino acid sequence of EBV BDLF2 is as follows (SEQ ID NO:9): MVDEQVAVEH GTVSHTISRE EDGVVHERRV LASGERVEVF YKAPAPRPRE 50 GRASTFHDFT VPAAAAVPGP EPEPEPHPPM PIHANGGGET KTNTQDQNQN 100 QTTRTRTNAK AEERTAEMDD TMASSGGQRG APISADLLSL SSLTGRMAAM 150 APSWMKSEVC GERMRFKEDV YDGEAETLAE PPRCFMLSFV FIYYCCYLAF 200 LALLAFGFNP LFLPSFMPVG AKVLRGKGRD FGVPLSYGCP TNPFCKVYTL 250 IPAVVINNVT YYPNNTDSHG GHGGFEAAAL HVAALFESGC PNLQAVTNRN 300 RTFNVTRASG RVERRLVQDM QRVLASAVVV MHHHCHYETY YVFDGVGPEF 350 GTIPTPCFKD VLAFRPSLVT NCTAPLKTSV KGPNWSGAAG GMKRKQCRVD 400 RLTDRSFPAY LEEVMYVMVQ

[0103] The antigenic compositions and methods of the present application typically include two or more HHV proteins involved in mediating HHV binding, fusion and entry into host cells. In certain embodiments, two or more EBV proteins disclosed herein are combined in an antigenic composition. Two or more EBV proteins can be administered simultaneously or separately to induce an immune response or treat or prevent EBV infection in a subject. In certain embodiments, the antigenic composition (or administration method) includes two or more of the following EBV polypeptides (or nucleic acids encoding same): gB, gH, gL and gp350. In some embodiments, the gB polypeptide is a monomer, dimer, or trimer. In some embodiments, the gH and gL polypeptides are a monomer, dimer, trimer, or tetramer. Typically, gH and gL form a gH / gL heterodimer. In some embodiments, the gp350 polypeptide is a monomer, dimer, trimer, or tetramer.

[0104] In certain embodiments, the two or more EBV proteins (or nucleic acids encoding same) include monomeric or multimeric gp350 and monomeric or multimeric gB. In certain embodiments, gp350 is monomeric or tetrameric and gB is monomeric or trimer. In certain embodiments, gp350 is monomeric and gB is trimer. In certain embodiments, gp350 is tetrameric and gB is trimer.

[0105] In certain embodiments, the two or more EBV proteins (or nucleic acids encoding same) include a monomeric or multimeric gp350 and a monomeric or multimeric gH / gL heterodimer. In certain embodiments, gp350 is a monomer or a tetramer and the gH / gL heterodimer is a monomer or a trimer. In certain embodiments, gp350 is a monomer and the gH / gL heterodimer is a monomer. In certain embodiments, gp350 is a tetramer and the gH / gL heterodimer is a trimer.

[0106] In certain embodiments, the two or more EBV proteins (or nucleic acids encoding same) include a monomeric or multimeric gB and a monomeric or multimeric gH / gL heterodimer. In certain embodiments, gB is a monomer, a dimer, or a trimer, and the gH / gL heterodimer is a monomer or a trimer. In certain embodiments, gB is a monomer and the gH / gL heterodimer is a monomer or a trimer. In certain embodiments, gB is a trimer and the gH / gL heterodimer is a monomer. In certain embodiments, gB is a trimer. and the gH / gL heterodimer is a trimer. In certain embodiments, the EBV gB, gH and gL polypeptides form a protein complex when mixed together. In certain embodiments, the EBV gB, gH and gL polypeptides are not administered as a protein complex comprising gB, gH and gL polypeptides. For example, gB can be administered separately from gH and / or gL, or can be administered with gH and gL but not as a protein complex.

[0107] In certain embodiments, the two or more EBV proteins (or nucleic acids encoding same) include monomeric or multimeric gp350, monomeric or multimeric gB, and monomeric or multimeric gH / gL heterodimer. In certain embodiments, gp350 is monomeric or tetrameric, gB is monomeric or trimer, and gH / gL heterodimer is monomeric or trimer. In certain embodiments, gp350 is monomeric, gB is trimer, and gH / gL heterodimer is monomeric. In certain embodiments, gp350 is tetrameric, gB is trimer, and gH / gL heterodimer is trimer.

[0108] In some embodiments, the two or more EBV proteins further include one or more of a BMRF-2 polypeptide, a BDLF2 polypeptide, and / or a gp42 polypeptide, which may be monomeric or multimeric (e.g., dimer, trimer, or tetramer).

[0109] Kaposi's Sarcoma Virus-Associated Herpes (KSHV, HHV-8). Two human gammaherpesviruses, Epstein-Barr virus (EBV), a gamma-1 lymphocryptovirus, and Kaposi's Sarcoma-Associated Virus (KSHV), a gamma-2 rhadinovirus, have many common features. They share a structure unique to all members of the Herpesviridae family, they share the ability to establish latency in lymphocytes, and they are both initiators or potentiators of human tumors (Chandran et al., Human Herpesviruses: Biology, Therapy, and Imunoprophylaxis, Eds. Arvin, A., Campadelli-Fiume, G., and Mocarski E., et al., Cambridge University Press, 2007, Ch. 23). KSHV is a virulent pathogen that induces B-cell, primary effusion lymphoma (PEL) from peripheral blood. KSHV broadly infects many types of host cells, including B cells in EBV (PEL) or body-cavity based B-cell lymphomas (BCBL) and multicentric Castleman disease (MCD), squamous endothelial cells adhering to the vascular lumen of Kaposi's sarcoma (KS) lesions, typical KS spindle cells, CD45+ / CD68+ monocytes, keratinocytes, and epithelial cells in KS lesions (Id.). Furthermore, KSHV infection has been associated with multiple myeloma (Rettig et al., Science, 276:1851-4, 1997). Like EBV, KSHV also expresses gB, gH, and gL, which mediate cell fusion and entry. KSHV also expresses conserved glycoproteins gM and gN, which mediate similar, if not identical, roles compared to their EBV counterparts (Id.).

[0110] However, the gp350 glycoprotein of EBV is replaced in KSHV by a polypeptide designated K8.1. The K8.1 gene encodes 197 amino acids with a predicted molecular weight of approximately 22 kDa and has no sequence corresponding to a TM domain. Similar to EBV gp350 / 220, the KSHV K8.1 gene encodes two ORFs, designated gpK8.1A and gpK8.1B, from the spliced ​​message. The larger cDNA is 752 bp long (from 76214 bp to 76941 bp) and utilizes a polyadenylation signal sequence (AATAAA) at position 77013 bp. The encoded protein, 228 amino acids long, is designated gpK8.1A and contains a signal sequence, a transmembrane domain, and four N-glycosylation sites. Alternatively, the KSHV gpK18.1 polypeptide may serve a similar function as reported for EBV gp350, forming a complex with gB to bind to cell surface heparin sulfate molecules on host cells.

[0111] KSHV ORF68 is a late lytic infection, immediate early structural and assembly gene that encodes a transmembrane glycoprotein that is a component of the KSHV envelope (Nakamura et al., J. Virol., 77(7):4205-20, 2003, and Jha et al., mBio, 5(6):e02261-14, 2014, and (Stuerzl et al., Thromb. Haemost., 102:1117-34, 2009). ORF68 is expressed in the host cell It is known that it inhibits protein degradation by interacting with and inhibiting the ubiquitin proteasome pathway (Gardner, M., 8 th Annual CEND Symposium, 22 March 2016). ORF68 is essential for viral genome replication in KSHV. It is hypothesized that KSHV ORF68 encodes a protein that suppresses proteasome-mediated degradation of proteins in the cytoplasm of host cells that are essential for KSHV DNA replication (Id.).

[0112] The antigenic compositions and methods of the present application typically include two or more HHV proteins involved in mediating HHV binding, fusion and entry into host cells. In certain embodiments, two or more KSHV proteins disclosed herein are combined in an antigenic composition. Two or more KSHV proteins can be administered simultaneously or separately to induce an immune response or treat or prevent KSHV infection in a subject. In certain embodiments, the antigenic composition (or administration method) includes two or more of the following KSHV polypeptides (or nucleic acids encoding same): gB, gH, and gL. In some embodiments, the gB polypeptide is a monomer, dimer, or trimer. In some embodiments, the gH and gL polypeptides are a monomer, dimer, trimer, or tetramer. Typically, gH and gL form a gH / gL heterodimer.

[0113] In certain embodiments, the two or more KSHV proteins (or nucleic acids encoding same) include a monomeric or multimeric gB and a monomeric or multimeric gH / gL heterodimer. In certain embodiments, gB is a monomer, dimer, or trimer, and the gH / gL heterodimer is a monomer or trimer. In certain embodiments, gB is a monomer and the gH / gL heterodimer is a monomer or trimer. In certain embodiments, gB is a trimer and the gH / gL heterodimer is a monomer. In certain embodiments, gB is a trimer and the gH / gL heterodimer is a trimer. In certain embodiments, when mixed together, the KSHV gB, gH, and gL polypeptides form a protein complex. In certain embodiments, the KSHV gB, gH, and gL polypeptides are not administered as a protein complex comprising gB, gH, and gL polypeptides. For example, gB can be administered separately from gH and / or gL, or can be administered together with gH and gL rather than as a protein complex.

[0114] In certain embodiments, the two or more KSHV proteins further include one or more of a gN polypeptide, a gM polypeptide, an ORF68 polypeptide, and / or a gpK8.1 polypeptide, which may be monomeric or multimeric (e.g., dimer, trimer, or tetramer).

[0115] The amino acid and nucleic acid sequences of KSHV gpK8.1A, published in GenBank under accession numbers AAC63270.1, GI3414867, are incorporated herein by reference. The amino acid sequence of gpK8.1 is as follows (SEQ ID NO:10): 1 msstqirtei pvallilclc lvachancpt yrshlgfwqe gwsgqvyqdw lgrmncsyen 61 mtaleavsln gtrlaagsps seypnvsvsv edtsasgsge daidesgsge eerpvtshvt 121 fmtqsvqatt eltdalisaf sgsyssgeps rttrirvspv aengrnsgas nrvpfsattt 181 ttrgrdahyn aeirthlyil wavglllglv lilylcvprc rrkkpyiv

[0116] GenBa under accession numbers AJE29698.1 and GI748016404. The amino acid and nucleic acid sequences of KSHV gpK8.1B are disclosed in US Pat. No. 6,399,341 and incorporated herein by reference. The amino acid sequence of gpK8.1B is as follows (SEQ ID NO:11): 1 MSSTQIRTEI PVALLILCLC LVACHANCPT YRSHLGFWQE GWSGQVYQDW LGRMNCSYEN 61 MTALEAVSLN GTRLAAGSPS RSYSSGEPSR TTRIRVSPVA ENGRNSGASN RVPFSATTTT 121 TRGRDAHYNA EIRTHLYILW AVGLLLGLVL ILYLCVPRCR RKKPYIV

[0117] The amino acid sequence of KSHV gH is as follows (SEQ ID NO:12): MQGLAFLAAL ACWRCISLTC GATGALPTTA TTITRSATQL INGRTNLSIE 50 LEFNGTSFFL NWQNLLNVIT EPALTELWTS AEVAEDLRVT LKKRQSLFFP 100 NKTVVISGDG HRYTCEVPTS SQTYNITKGF NYSALPGHLG GFGINARLVL 150 GDIFASKWSL FARDTPEYRV FYPMNVMAVK FSISIGNNES GVALYGVVSE 200 DFVVVTLHNR SKEANETASH LLFGLPDSLP SLKGHATYDE LTFARNAKYA 250 LVAILPKDSY QTLLTENYTR IFLNMTESTP LEFTRTIQTR IVSIEARRAC 300 AAQEAAPDIF LVLFQMLVAH FLVARGIAEH RFVEVDCVCR QYAELYFLRR 350 ISRLCMPTFT TVGYNHTTLG AVAATQIARV SATKLASLPR SSQETVLAMV 400 QLGARDGAVP SSILEGIAMV VEHMYTAYTY VYTLGDTERK LMLDIHTVLT 450 DSCPPKDSGV SEKLLRTYLM FTSMCTNIEL GEMIARFSKP DSLNIYRAFS 500 PCFLGLRYDL HPAKLRAEAP QSSALTRTAV ARGTSGFAEL LHALHLDSLN 550 LIPAINCSKI TADKIIATVP LPHVTYIISS EALSNAVVYE VSEIFLKSAM 600 FISAIKPDCS GFNFSQIDRH IPIVYNISTP RRGCPLCDSV IMSYDESDGL 650 QSLMYVTNER VQTNLFLDKS PFFDNNNLHI HYLWLRDNGT VVEIRGMYRR 700 RAASALFLIL SFIGFSGVIY FLYRLFSILY

[0118] The amino acid sequence of KSHV gL is as follows (SEQ ID NO:13): MGIFALFAVL WTTLLVTSHA YVALPCCAIQ ASAASTLPLF FAVHSIHFAD 50 PNHCNGVCIA KLRSKTGDIT VETCVNGFNL RSFLVAVVRR LGSWASQENL 100 RLLWYLQRSL TAYTVGFNAT TADSSIHNVN IIIISVGKAM NRTGSVSGSQ 150 TRAKSSSRRA HAGQKGK

[0119] The amino acid sequence of KSHV gB is as follows (SEQ ID NO:12): MQGLAFLAAL ACWRCISLTC GATGALPTTA TTITRSATQL INGRTNLSIE 50 LEFNGTSFFL NWQNLLNVIT EPALTELWTS AEVAEDLRVT LKKRQSLFFP 100 NKTVVISGDG HRYTCEVPTS SQTYNITKGF NYSALPGHLG GFGINARLVL 150 GDIFASKWSL FARDTPEYRV FYPMNVMAVK FSISIGNNES GVALYGVVSE 200 DFVVVTLHNR SKEANETASH LLFGLPDSLP SLKGHATYDE LTFARNAKYA 250 LVAILPKDSY QTLLTENYTR IFLNMTESTP LEFTRTIQTR IVSIEARRAC 300 AAQEAAPDIF LVLFQMLVAH FLVARGIAEH RFVEVDCVCR QYAELYFLRR 350 ISRLCMPTFT TVGYNHTTLG AVAATQIARV SATKLASLPR SSQETVLAMV 400 QLGARDGAVP SSILEGIAMV VEHMYTAYTY VYTLGDTERK LMLDIHTVLT 450 DSCPPKDSGV SEKLLRTYLM FTSMCTNIEL GEMIARFSKP DSLNIYRAFS 500 PCFLGLRYDL HPAKLRAEAP QSSALTRTAV ARGTSGFAEL LHALHLDSLN 550 LIPAINCSKI TADKIIATVP LPHVTYIISS EALSNAVVYE VSEIFLKSAM 600 FISAIKPDCS GFNFSQIDRH IPIVYNISTP RRGCPLCDSV IMSYDESDGL 650 QSLMYVTNER VQTNLFLDKS PFFDNNNLHI HYLWLRDNGT VVEIRGMYRR 700 RAASALFLIL SFIGFSGVIY FLYRLFSILY

[0120] The amino acid sequence of KSHV gN is as follows (SEQ ID NO:14): MTASTVALAL FVASILGHCW VTANSTGVAS STERSSPSTA GLSARPSPGP 50 TSVTTPGFYD VACSADSFSP SLSSFSSVWA LINALLVVVA TFFYLVYLCF 100 FKFVDEVVHA

[0121] The amino acid sequence of KSHV gM is as follows (SEQ ID NO:15): MRASKSDRFL MSSWVKLLFV AVIMYICSAV VPMAATYEGL GFPCYFNNLV 50 NYSALNLTVR NSAKHLTPTL FLEKPEMLVY IFWTFIVDGI AIVYYCLAAV 100 AVYRAKHVHA TTMMSMQSWI ALLGSHSVLY VAILRMWSMQ LFIHVLSYKH 150 VLMAAFVYCI HFCISFAHIQ SLITCNSAQW EIPLLEQHVP DNTMMESLLT 200 RWKPVCVNLY LSTTALEMLL FSLSTMMAVG NSFYVLVSDA IFGAVNMFLA 250 LTVVWYINTE FFLVKFMRRQ VGFYVGVFVG YLILLLPVIR YENAFVQANL 300 HYIVAINISC IPILCILAIV IRVIRSDWGL CTPSAAYMPL ATSAPTVDRT 350 PTVHQKPPPL PAKTRARAKV KDISTPAPRT QYQSDHESDS EIDETQMIFI 400

[0122] The amino acid sequence of KSHV ORF68 is as follows (SEQ ID NO:16): MFVPWQLGTI TRHRDELQKL LAASLLPEHP EESLGNPIMT QIHQSLQPSS 50 PCRVCQLLFS LVRDSSTPMG FFEDYACLCF FCLYAPHCWT STMAAAADLC 100 EIMHLHFPEE EATYGLFGPG RLMGIDLQLH FFVQKCFKTT AAEKILGISN 150 LQFLKSEFIR GMLTGTITCN FCFKTSWPRT DKEEATGPTP CCQITDTTTA 200 PASGIPELAR ATFCGASRPT KPSLLPALID IWSTSSELLD EPRPRLIASD 250 MSELKSVVAS HDPFFSPPLQ ADTSQGPCLM HPTLGLRYKN GTASVCLLCE 300 CLAAHPEAPK ALQTLQCEVM GHIENNVKLV DRIAFVLDNP FAMPYVSDPL 350 LRELIRGCTP QEIHKHLFCD PLCALNAKVV SEDVLFRLPR EQEYKKLRAS 400 AAAGQLLDAN TLFDCEVVQT LVFLFKGLQN ARVGKTTSLD IIRELTAQLK 450 RHRLDLAHPS QTSHLYA

[0123] Betaherpesviruses: Human cytomegalovirus (HCMV, HHV-5), human herpesvirus 6 (HHV-6), and human herpesvirus 7 (HHV-7) Human cytomegalovirus (HCMV, HHV-5). Human cytomegalovirus (HCMV) is an enveloped, double-stranded DNA beta-herpesvirus in the family Herpesviridae. HCMV belongs to the subfamily Betaherpesvirinae, of which HHV-6 and HHV-7 are also members. Cells infected by viruses of this family often become enlarged (giant cell tumors). HCMV is the leading non-genetic cause of childhood hearing loss and a significant cause of neurodevelopmental delay, including mental retardation (Demmler-Harrison GJ, J. Clin. Virol., 46 Suppl 4, 2009: S1-5; Jeon et al., Infect. Dis. Obstet. Gynecol., 2006:80383, 2006; and Morton et al., N. Engl. J. Med., 354:2151-64, 2006). In the United States, 20,000-40,000 infants are born with HCMV infections per year, accounting for 8,000 permanent disabilities and $1.86 billion in medical costs per year. HCMV also causes significant clinical disease in immunosuppressed individuals, including transplant recipients and patients with AIDS (Bonaros et al., Clin. Transplant., 22:89-97, 2008, and Steininger et al., J. Clin. Virol., 37:1-9, 2006). Infection is generally asymptomatic, but may result in mononucleosis syndrome in 10% of primary infections in older children and adults (Horwitz et al., Medicine (Baltimore), 65:124-34, 1986). In 2001, the Institute of Medicine of the National Academy of Sciences published a paper entitled "Congenital HCMV Infection." He stated that a vaccine to prevent infection is a top priority in the United States in particular (Stratton et al., "Vaccines for the 21st Century: A tool for decisionmaking," Washington, DC, National Academy Press, 2001).

[0124] HCMV is spread primarily through saliva and urine, and via transplacental infection to the fetus (Krause et al., Vaccine, 32:4-10, 2014). HCMV can also be transmitted to infants via breast milk (Maschmann et al., Clin. Infect. Dis., 33:1998-2003, 2001) and through sexual activity to solid organs or hematopoiesis. It can be transmitted through stem cell transplants and, rarely, by infusion of blood products. HCMV primarily infects fibroblasts, epithelial cells, endothelial cells, monocytes / macrophages, hepatocytes, and neurons. The mechanism of fusion and entry of HCMV into mammalian cells is similar to that used by other members of the Herpesviridae family (Heldwein et al., Cell. Mol. Life Sci., 65:1653-68, 2008, and White et al., Crit. Rev. Biochem. Mol. Biol., 43:189-219, 2008). HCMV invades cells by fusing its envelope with either the plasma membrane (fibroblasts) (Compton et al., Virology, 191:387-95, 1992) or endosomal membrane (epithelial and endothelial cells) (Ryckman et al., J. Virol., 80:710-22, 2006). Enter.

[0125] HCMV gB, gH, gL, gO (UL74), gM, gN (gpUL73) and UL128 / 130 / 131A. Nine glycoproteins, gB, gH, gL, gO (UL74), gM, gN (gpUL73), and UL128 / 130 / 131A, have been collectively identified as envelope glycoproteins that play an important role in HCMV fusion and entry into host cells (Hahn et al., J. Virol., 78:10023-33, 2004; Ryckman et al., J. Virol., 82:60-70, 2008; Wang et al., Proc. Natl. Acad. Sci. USA, 102:18153-8, 2005, and Wille et al., J. Virol., 84:2585-96, 2010). Similarly, the HCMV gH / gL and gB proteins play important roles in HCMV fusion and entry into host cells. The gB protein is the direct mediator of HCMV fusion with all host cell membranes. Activation of HCMV gB and its fusion activity requires the association with gH / gL and gO, which together form the gH / gL / gO heterotrimeric protein complex. However, the gH / gL / UL128 / 130 / 131A (pentameric complex) proteins are also important for efficient targeting of HCMV to epithelial and endothelial cells, as the UL128 / 130 / 131A mutant failed to infect these cells (Ryckman et al., J. Virol., 80:710-22, 2006; Hahn et al., J. Virol., 78:10023-33, 2004; Adler et al., J. Gen. Virol., 87: 2451-60, 2006, and Wang et al., J. Virol., 79:10330-8, 2005). In contrast, gO appears to be involved in HCMV fusion with all HCMV host cells, as gO-null HCMV was unable to infect all cell types tested, including fibroblasts, epithelial and endothelial cells, and infection of both fibroblasts and epithelial cells generally correlated with the abundance of the gH / gL / gO complex but not the pentameric complex gH / gL / UL128 / UL130 / UL131A (Wille et al., J. Virol., 84:2585-96, 2010; Jiang et al., J. Virol., 82:2802-12, 2008; and Zhou et al., J. Virol., 89(17):8999-9009, 2015). All three UL128-131 genes share a common structure that includes an amino-terminal signal peptide, a central chemokine-like domain, and a carboxy-terminal domain that has no homology to any known class of proteins (Patrone et al., J. Virol., 79(13):8361-8373, 2005). CMV gB or gH / gL proteins have been shown to elicit serum HCMV neutralizing antibodies against both fibroblasts and epithelial cells. However, the pentameric complex induces the highest serum neutralizing titers against epithelial and endothelial cells, but does not improve further against fibroblasts (Wen et al., Vaccine, 32:3796-804, 2014; Freed et al., Proc. Natl. Acad. Sci. USA, 110:E4997-5005, 2013; and Schuessler et al., J. Virol., 86:504-12, 2012). HCMV gH / gL / gO complex induces serum neutralizing antibodies against mammalian cells (HE). K-239) (Kinzler et al., J. Clin. Virol., 25 Suppl 2:S87-95, 2002), but there have been no reports on its ability to induce HCMV neutralizing antibodies.

[0126] The glycoproteins M and N polypeptides are glycoprotein complex II (GCII) antigens. Glycoprotein N is an envelope component of mature virus particles with a portion exposed on the viral surface and a portion that extends to the interior of the envelope. Glycoprotein N is present in the matrix and "block holes" of defense bodies (Pignatelli, et al., Arch. Virol., 147:1247, 2002). The HCMV gM polypeptide is 372 amino acids long and has a molecular weight of approximately 42 kDa. and has seven TM domains. HCMV gN is 129 amino acids long and has a predicted molecular weight of approximately 15 kDa, but due to heavy glycosylation tends to appear as a 40 kDa-50 kDa protein. Glycoprotein M (gM, UL100) and glycoprotein N (gN, UL73) form the gM / gN protein complex, which is the most abundant protein component of the HCMV envelope. Recent studies have shown that deletion of the viral genes encoding either gM or gN is lethal for HCMV but not for other HHVs (Baines et al., J. Virol., 67:1441-1452, 1993; Fuchs et al., Virus Res., 112:108-114, 2005; Hobom et al., J. Virol., 74:7720-7729, 2000; Mach et al., J. Virol., 81:5212-5224, 2007; and MacLean et al., J. Gen. Virol., 74(pt. 6):975-983, 1993).

[0127] The antigenic compositions and methods of the present application typically include two or more HHV proteins involved in mediating HHV binding, fusion and entry into host cells. In certain embodiments, two or more HCMV proteins disclosed herein are combined in an antigenic composition. Two or more HCMV proteins can be administered simultaneously or separately to induce an immune response or treat or prevent HCMV infection in a subject. In certain embodiments, the antigenic composition (or administration method) includes two or more of the following HCMV polypeptides (or nucleic acids encoding same): gB, gH and gL. In some embodiments, the gB polypeptide is a monomer, dimer or trimer. In some embodiments, the gH and gL polypeptides are a monomer, dimer, trimer or tetramer. Typically, gH and gL form a gH / gL heterodimer.

[0128] In certain embodiments, the two or more HMCV proteins (or nucleic acids encoding same) include a monomeric or multimeric gB and a monomeric or multimeric gH / gL heterodimer. In certain embodiments, gB is a monomer, dimer, or trimer, and the gH / gL heterodimer is a monomer or trimer. In certain embodiments, gB is a monomer and the gH / gL heterodimer is a monomer or trimer. In certain embodiments, gB is a trimer and the gH / gL heterodimer is a monomer. In certain embodiments, gB is a trimer and the gH / gL heterodimer is a trimer. In certain embodiments, when mixed together, the HMCV gB, gH, and gL polypeptides form a protein complex. In certain embodiments, the HMCV gB, gH, and gL polypeptides are not administered as a protein complex comprising gB, gH, and gL polypeptides. For example, gB can be administered separately from gH and / or gL, or can be administered together with gH and gL rather than as a protein complex.

[0129] In some embodiments, the two or more HCMV proteins (or nucleic acids encoding same) further comprise a gO polypeptide, which is optionally multimeric (e.g., dimeric, trimeric, or tetrameric). In other embodiments, the two or more HCMV proteins (or nucleic acids encoding same) further comprise a gN and / or gM polypeptide, which may be monomeric or multimeric (e.g., dimeric, trimeric, or tetrameric). In yet other embodiments, the two or more HCMV proteins (or nucleic acids encoding same) comprise a gB polypeptide, a gH polypeptide, and a gL polypeptide, and a UL128 polypeptide, a UL130 polypeptide, and a UL131A polypeptide. In certain embodiments, the two or more HCMV proteins (or nucleic acids encoding same) comprise a trimeric gB, a monomeric gH / gL, and UL128, UL130, and UL131A, with UL128, UL130, and UL131A preferably combined as a fusion protein. In certain embodiments, the five HCMV polypeptides are present in the composition as a pentameric protein complex, hi certain embodiments, the polypeptides are present in the composition as a fusion protein.

[0130] Also includes HHV polypeptides gH, gL, UL128, UL130, and UL131A. The present invention discloses recombinant nucleic acids encoding protein complexes or fusion proteins comprising the HHV polypeptides. The sequences of these HHV polypeptides constituting the pentameric complex may be derived from any member of the beta-herpesvirinae subfamily, including, for example, HCMV. An embodiment of a nucleic acid construct encoding all five HCMV polypeptides of the pentameric complex, including, for example, an operably linked promoter sequence, is shown in FIG. 13. Additional nucleic acid sequences, such as his-tag sequences known in the art as protein purification tags, or immunoglobulin kappa sequences, may be included in such nucleic acid sequences to aid in purification. In another embodiment, the nucleic acid construct may include sequences encoding the HHV polypeptides gH, gL, and gB. These highly conserved polypeptides are found in all HHV genomes and may correspond to any known HHV gB, gH, and / or gL sequences.

[0131] The amino acid sequence of HCMV gH is as follows (SEQ ID NO:17): MRPGLPPYLT VFTVYLLSHL PSQRYGADAA SEALDPHAFH LLLNTYGRPI 50 RFLRENTTQC TYNSSLRNST VVRENAISFN FFQSYNQYYV FHMPRCLFAG 100 PLAEQFLNQV DLTETLERYQ QRLNTYALVS KDLASYRSFS QQLKAQDSLG 150 QQPTTVPPPI DLSIPHVWMP PQTTPHDWKG SHTTSGLHRP HFNQTCILFD 200 GHDLLFSTVT PCLHQGFYLM DELRYVKITL TEDFFVVTVS IDDDTPMLLI 250 FGHLPRVLFK APYQRDNFIL RQTEKHELLV LVKKAQLNRH SYLKDSDFLD 300 AALDFNYLDL SALLRNSFHR YAVDVLKSGR CQMLDRRTVE MAFAYALALF 350 AAARQEEAGT EISIPRALDR QAALLQIQEF MITCLSQTPP RTTLLLYPTA 400 VDLAKRALWT PDQITDITSL VRLVYILSKQ NQQHLIPQWA LRQIADFALQ 450 LHKTHLASFL SAFARQELYL MGSLVHSMLV HTTERREIFI VETGLCSLAE 500 LSHFTQLLAH PHHEYLSDLY TPCSSSGRRD HSLERLTRLF PDATVPATVP 550 AALSILSTMQ PSTLETFPDL FCLPLGESFS ALTVSEHVSY VVTNQYLIKG 600 ISYPVSTTVV GQSLIITQTD SQTKCELTRN MHTTHSITAA LNISLENCAF 650 CQSALLEYDD TQGVINIMYM HDSDDVLFAL DPYNEVVVSS PRTHYLMLLK 700 NGTVLEVTDV VVDATDSRLL MMSVYALSAI IGIYLLYRML KTC

[0132] The amino acid sequence of HCMV gL is as follows (SEQ ID NO:18): MCRRPDCGFS FSPGPVILLW CCLLLPIVSS AAVSVAPTAA EKVPAECPEL 50 TRRCLLGEVF EGDKYESWLR PLVNVTGRDG PLSQLIRYRP VTPEAANSVL 100 LDEAFLDTLA LLYNNPDQLR ALLTLLSSDT APRWMTVMRG YSECGDGSPA 150 VYTCVDDLCR GYDLTRLSYG RSIFTEHVLG FELVPPSLFN VVVAIRNEAT 200 RTNRAVRLPV STAAAPEGIT LFYGLYNAVK EFCLRHQLDP PLLRHLDKYY 250 AGLPPELKQT RVNLPAHSRY GPQAVDAR

[0133] The amino acid sequence of HCMV gB is as follows (SEQ ID NO:19): MESRIWCLVV CVNLCIVCLG AAVSSSSSTSH ATSSTHNGSH TSRTTSAQTR 50 SVYSQHVTSS EAVSHRANET IYNTTLKYGD VVGVNTTKYP YRVCSMAQGT 100 DLIRFERNII CTSMKPINED LDEGIMVVYK RNIVAHTFKV RVYQKVLTFR 150 RSYAYIYTTY LLGSNTEYVA PPMWEIHHIN KFAQCYSSYS RVIGGTVFVA 200 <h2 style=";text-align:left;direction:ltr">YHRDSYENKT MQLIPDDYSN THSTRYVTVK DQWHSRGSTW LYRETCNLNC 250<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> MLITTARSK YPYHFFATST GDVVYISPFY NGTNRNASYF GENADKFFIF 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> PNYTIVSDFG RPNAAPETHR LVAFLERADS VISWDIQDEK NVTCQLTFWE 350<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ASERTIRSEA EDSYHFSSAK MTATFLSKKQ EVNMSDSALD CVRDEAINKL 400<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> QQIFNTSYNQ TYEKYGNVSV FETSGGLVVF WQGIKQKSLV ELERLANRSS 450<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LNITHRTRRS TSDNNTTHLS SMESVHNLVY AQLQFTYDTL RGYINRALAQ 500<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> IAEAWCVDQR RTLEVFKELS KINPSAILSA IYNKPIAARF MGDVLGLASC 550<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VTINQTSVKV LRDMNVKESP GRCYSRPVVI FNFANSSYVQ YGQLGEDNEI 600<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LLGNHRTEEC QLPSLKIFIA GNSAYEYVDY LFKRMIDLSS ISTVDSMIAL 650<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> DIDPLENTDF RVLELYSQKE LRSSNVFDLE EIMREFNSYK QRVKYVEDKV 700<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VDPLPPYLKG LDDLMSGLGA AGKAVGVAIG AVGGAVASVV EGVATFLKNP 750<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> FGAFTIILVA IAVVITYLI YTRQRRLCTQ PLQNLFPYLV SADGTTVTSG 800<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> STKDTSLQAP PSYEESVYNS GRKGPGPPSS DASTAAPPYT NEQAYQMLLA 850<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LARLDAEQRA QQNGTDSLDG QTGTQDKGQK PNLLDRLRHR KNGYRHLKDS 900<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> DEEENV

[0134] The amino acid sequence of HCMV gN is as follows (SEQ ID NO:20): MEWNTLVLGL LVLSVVAESS GNNSSTSTSA TTSKSSASVS TTKLTTVATT 50 SATTTTTTTL STTSTKLSST THDPNVMRRH ANDDFYKAHC TSHMYELSLS 100 SFAAWWTMLN ALILMGAFCI VLRHCCFQNF TATTTKGY

[0135] The amino acid sequence of HCMV gM is as follows (SEQ ID NO:21): MAPSHVDKVN TRTWSASIVF MVLTFVNVSV HLVLSNFPHL GYPCVYYHVV 50 DFERLNMSAY NVMHLHTPML FLDSVQLVCY AVFMQLVFLA VTIYYLVCWI 100 KISMRKDKGM SLNQSTRDIS YMGDSLTAFL FILSMDTFQL FTLTMSFRLP 150 SMIAFMAAVH FFCLTIFNVS MVTQYRSYKR SLFFFSRLHP KLKGTVQFRT 200 LIVNLVEVAL GFNTTVVAMA LCYGFGNNFF VRTGHMVLAV FVVYAIISII 250 YFLLIEAVFF QYVKVQFGYH LGAFFGLCGL IYPIVQYDTF LSNEYRTGIS 300 WSFGMLFFIW AMFTTCRAVR YFRGRGSGSV KYQALATASG EEVAVLSHHD 350 SLESRRLREE EDDDDDEDFE DA

[0136] The amino acid sequence of HCMV gO is as follows (SEQ ID NO:22): MGRKEMMVRD VPKMVFLISI SFLLVSFINC KVMSKALYNR PWRGLVLSKI 50 GKYKLDQLKL EILRQLETTI STKYNVSKQP VKNLTMNMTE FPQYYILAGP 100 IQNYSITYLW FDFYSTQLRK PAKYVYSQYN HTAKTITFRP PPCGTVPSMT 150 CLSEMMLNVSK RNDTGEQGCG NFTTFNPMFF NVPRWNTKLY VGPTKVNVDS 200 QTIYFLGLTA LLLRYAQRNC THSFYLVNAM SRNLFRVPKY INGTKLKNTM 250 RKLKRKQAPV KEQFEKKAKK TQSTTTPYFS YTTSAALNVT TNVTYSITTA 300 ARRVSTSTIA YRPDSSFMKS IMATQLRDLA TWVYTTLRYR QNPFCEPSRN 350 RTAVSEFMKN THVLIRNETP YTIYGTLDMS SLYYNETMFV ENKTASDSNK 400 TTPTSPSMGF QRTFIDPLWD YLDSLLFLDE IRNFSLRSPT YVNLTPPEHR 450 RAVNLSTLNS LWWWLQ

[0137] The amino acid sequence of HCMV UL128 is as follows (SEQ ID NO:23): MSPKNLTPFL TALWLLLGHS RVPRVRAEEC CEFINVNHPP ERCYDFKMCN 50 RFTVALRCPD GEVCYSPEKT AEIRGIVTTM THSLTRQVVH NKLTSCNYNP 100 LYLEADGRIR CGKVNDKAQY LLGAAGSVPY RWINLEYDKI TRIVGLDQYL 150 ESVKKHKRLD VCRAKMGYML Q

[0138] The amino acid sequence of HCMV UL130 is as follows (SEQ ID NO:24): MLRLLLRHYF HCLLLCAVWA TPCLASSWST LTANQNPSPP WSKLTYSKPH 50 DAATFYCPFL YPSPPRSPSQ FSGFQRVSTG PECRNETLYL LYNREGQTLV 100 ERSSTWVKKV IWYLSGRNQT ILQRMPRTAS KPSDGNVQIS VEDAKIFGAH 150 MVPKQTKLLR FVVNDGTRYQ MCVMKLESWA HVFRDYSVSF QVRLTFTEAN 200 NQTYTFCTHP NLIV

[0139] The amino acid sequence of HCMV UL131A is as follows (SEQ ID NO:25): MRLCRVWLSV CLCAVVLGQC QRETAEKNDY YRVPHYWDAC SRALPDQTRY 50 KYVEQLVDLT LNYHYDASHG LDNFDVLKRI NVTEVSLLIS DFRRQNRRGG 100 TNKRTTFNAA GSLAPHARSL EFSVRLFAN

[0140] Human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7). Although HHV-6 and HHV-7 are distinct from HCMV in terms of genome sequence, these human herpesviruses retained the core of 80 ORFs common to herpesviruses conserved in rodent CMV (Mocarski E., Cell. Microb., 6(8):707-717, 2004). HHV-6 was first isolated in 1986 from peripheral blood leukocytes of patients with lymphoproliferative disorders and AIDS (Flamand et al., J. Virol., 67(11):6768-6777, 1993). Within 2 years of age, It is estimated that approximately 90% of the population is infected with HHV-6 in the world today, and that the figure could reach 100% in non-industrialized countries (Salahuddin et al., Science, 234:596, 1986; Willis et al., Br. Med. Bull., 62(1):125-138, 2002). HHV-6 infection is associated with a number of inflammatory conditions, including roseola infantum (sixth disease), exanthema subitum (roseola), and erythema subitum (roseola). It causes a rash (hemophagocytic rash) and is associated with heterophile-negative infectious mononucleosis, as well as meningoencephalitis, hepatitis, fatal hemophagocytic syndrome, and interstitial pneumonia (Id.). Furthermore, the detection of HHV-6 genomic sequences in some B-cell lymphomas and the ability of HHV-6 to transform rodent cells suggest a role for HHV-6 in certain cancers. There is some evidence to suggest this (Ablashi et al., J. Virol. Methods, 21:29-48, 1988; Josephs et al., Science, 234:601-603, 1986; Razzaque, A., Oncogene, 5:1356-1370, 1990; and Torelli et al., Blood, 77:2251-2258, 1991). There are two variants of HHV-6, HHV-6A and HHV-6B, which have been identified in the wild-type human HHV-6 virus (Ablashi et al., Arch. Virol., 159(5):863-870, 2014). The genomes of the two variants are co-linear and share 90% overall sequence identity (ibid.). The highly conserved glycoproteins gH, gB, gN, and gO are identifiably and consistently different in sequence (conserved across isolates), and the two variants appear to exhibit slightly different pathogenesis and disease associations (Id.). Nevertheless, the same glycoproteins present in other HHV family members are encoded by the genomes of HHV-6 and HHV-7.

[0141] HHV-6 encodes many of the same surface glycoproteins previously mentioned for other members of the HHV family, including gB, gH, gL, and gM, for which relatively conserved homologs have been identified in all known mammalian herpesviruses (Santoro et al., J. Biol. Chem., 278:25964-25969, 2003, and Dockrell, DH, J. Med. Microbiol., 52:5-18, 2003). As with other family members, glycoproteins gH and gL are involved in the inhibition of specific antibody activity. It plays a prominent role in HHV-6 membrane fusion based on the .alpha.-antigen (Foa-Tomasi et al., J. Virol., 65:4124-4129, 1991; Gompels et al., J. Virol., 65:2393-2401, 1991; Liu et al., Virology, 197:12-22, 1993, and Qian et al., Virology, 194:380-386, 1993). As in other herpesviruses, these glycoproteins form a heterodimeric complex, and gL is required for correct folding, intracellular maturation, and surface expression of gH (Anderson et al., J. Gen. Virol., 80:1485-1494, 1999; Hutchinson et al., J. Virol., 66:2240-2250, 1992; Liu et al., J. Gen. Virol., 74:1847-1857, 1993, and Roop et al., J. Virol., 1994). 67:2285-2297, 1993). It is the most highly conserved glycoprotein among herpesviruses. The HHV-6 glycoprotein gB, which is known to be involved in the fusion / entry process, and glycoproteins gp82-gp105 (found only in HHV-6 and the related β-herpesvirus, HHV-7) are important for the fusion / entry process (Takeda et al., Virology, 222:176-183, 1996; Pfeiffer et al., J. Virol., 69:3490-3500, 1995; and Pfeiffer et al., J. Virol., 67:4611-4620, 1993).

[0142] The antigenic compositions and methods of the present application typically include two or more HHV proteins involved in mediating HHV binding, fusion and entry into host cells. In certain embodiments, two or more HHV-6 and HHV-7 proteins disclosed herein are combined in an antigenic composition. Two or more HHV-6 and HHV-7 proteins The proteins may be administered simultaneously or separately to induce an immune response or treat or prevent HHV-6 or HHV-7 infection in a subject. In certain embodiments, the antigenic composition (or method of administration) comprises two or more of the following HHV-6 and HHV-7 polypeptides (or nucleic acids encoding same): gB, gH, and gL. In certain embodiments, the gB polypeptide is a monomer, dimer, or trimer. In certain embodiments, the gH and gL polypeptides are monomers, dimers, trimers, or tetramers. Typically, gH and gL form a gH / gL heterodimer.

[0143] In certain embodiments, the two or more HHV-6 or HHV-7 proteins (or nucleic acids encoding same) comprise a monomeric or multimeric gB and a monomeric or multimeric gH / gL heterodimer. In certain embodiments, gB is a monomer, a dimer, or a trimer, and the gH / gL heterodimer is a monomer or a trimer. In certain embodiments, gB is a trimer, and the gH / gL heterodimer is a monomer. In certain embodiments, gB is a trimer, and the gH / gL heterodimer is a trimer. In certain embodiments, gB is a monomer, and the gH / gL heterodimer is a monomer or a trimer. In certain embodiments, when mixed together, the HHV-6 or HHV-7 gB, gH, and gL polypeptides form a protein complex. In certain embodiments, the HHV-6 or HHV-7 gB, gH, and gL polypeptides are not administered as a protein complex comprising gB, gH, and gL polypeptides, for example, gB can be administered separately from gH and / or gL, or can be administered together with gH and gL but not as a protein complex.

[0144] The amino acid sequence of HHV-6A gH is as follows (SEQ ID NO:26): MLLRLWVFVL LTPCYGWRPL NISNSSHCRN GNFENPIVRP GFITFNFYTK 50 NDTRIYQVPK CLLGSDITYH LFDAINTTES LTNYEKRVTR FYEPPMNDIL 100 RLSPVPSVKQ FNLDRSIQPQ VVYSLNMYPS QGIYYVRVVE VRQMQYDNVS 150 CKLPNSLKEL IFPVQVRCAK ITRYVGEDIY THFFTPDFMI LYIQNPAGDL 200 TMMYGNTTSI NFKAPYKKSS FIFKQTLTDD LLLIVEKDVI DVQYRFISDA 250 TFVDETLNDV DEVEALLLKF NNLGIQTLLR GDCKKPNYAG IPQMMFLYGI 300 VHFSYSTKNT GPMPVLRVLK THENLLSIDS FVNRCVNVSE GTLQYPKMKE 350 FLKYEPSDYS YITKNKSISV STLLTYLATA YESNVTISKY KWTDIANTLQ 400 NIYEKHMFFT NLTFSDRETL FMLAEIANII PTDERMQRHM QLLIGNLCNP 450 VEIVSWARML TADRAPNLEN IYSPCASPVR RDVTNSFLKT VLTYASLDRY 500 RSDMMEMLSV YRPPNMERVA AIQCLSPSEP AASLTLPNVT FVISPSYVIK 550 GVSLTITTTTI VATSIIITAI PLNSTCVSTN YKYAGQDLLV LRNISSQTCE 600 FCQSVVMEYD DIDGPLQYIY IKNIDELKTL TDPNNNLLVP NTRTHYLLLA 650 KNGSVFEMSE VGIDIDQVSI ILVIIYILIA IIALFGLYRL IRLC

[0145] The amino acid sequence of HHV-6B gH is as follows (SEQ ID NO:27): MLFRLWVFVL LTPCYSWRPW TISDESHCKN GNSENPIVRP GFITFNFYTK 50 NDTRIYQVPK CLLGSDITYH LFDAINTTES LTNYEKRVTR FYEPPMNDIL 100 RLSTVPAVKQ FNLDHSIQPQ IVYSLNLYPS HGIYYIRVVE VRQMQYDNVS 150 CKLPNSLNEL IFPVQVRCAK ITRYAGENIY THFFTPDFMI LYIQNPAGDL 200 TMMYGNTTDI NFKAPYRKSS FIFKQTLTDD LLLIVEKDVV DEEYRFISDA 250 TFVDETLDDV DEVEALLLKF NNLGIQTLLR GDCKKPDYAG IPQMMFLYGI 300 VHFSYSTKNT GPMPVLRVLK THENLLSIDS FVNRCVNVSE GTIQYPKMKE 350 FLKYEPSDYS YITKNKSIPV STLLTYLATA YETNVTISRY KWSDIANTLQ 400 KIYEKHMFFT NLTFSDRETL FMLAEIANFI PADERMQRHM QLLIGNLCNP 450 VEIVSWAHML TADKAPNLEN IYSPCASPVR RDVTNSFVKT VLTYASLDRY 500 RSDMMEMLSV YRPPDMARVA AIQCLSPSEP AASLPLPNVT FVISPSYVIK 550 GVSLTITTTTI VATSIIITAI PLNSTCVSTN YKYAGQDLLV LRNISSQTCE 600 FCQSVVMEYD DIDGPLQYIY IKNIDELKTL TDPNNNLLVP NTRTHYLLLA 650 KNGSVFEMSE VGIDIDQVSI ILVIIYVLIA IIALFGLYRL IRLC

[0146] The amino acid sequence of HHV-6A gL is as follows (SEQ ID NO:28): MELLLFVMSL ILLTFSKAIP LFNHNSFYFE KLDDCIAAVI NCTKSEVPLL 50 LEPIYQPPAY NEDVMSILLQ PPTKKKPFSR IMVTDEFLSD FLLLQDNPEQ 100 LRTLFALIRD PESRDNWLNF FNGFQTCSPS VGITTCIRDN CRKYSPEKIT 150 YVNNFFVDNI AGLEFNISEN TDSFYSNIGF LLYLENPAKG VTKIIRFPFN 200 SLTLFDTILN CLKYFHLKTG VELDLLKHME TYNSKLPFRS SRPTILIRNT 250

[0147] The amino acid sequence of HHV-6B gL is as follows (SEQ ID NO:29): MELLLFVMSL ILLTFSKAMP LFDHNSFYFE KLDDCIAAVI NCTRSEVPLL 50 LEPIYQPPVY NEDVMSILLK PPTKKKPFSR IMVTNEFLSD FLLLQDNPEQ 100 LRTLFALIGD PESRDNWLNF FNGFQTCSPS VGITTCISDN CRKYLPERIT 150 YVNNFFVDNI AGLEFNISEN TDSFYSNIGF LLYLENPATG ITKIIRFPFN 200 SLTLFDTILN CLKYFHLKTG VEFDLLKQME AYNSKLPFRS SRPTILIRNT 250

[0148] The amino acid sequence of HHV-6A gB is as follows (SEQ ID NO:30): MSKMAVLFLA VFLMNSVLMI YCDPDHYIRA GYNHKYPFRI CSIAKGTDLM 50 RFDRDISCSP YKSNAKMSEG FFIIYKTNIE TYTFPVRTYK KELTFQSSYR 100 DVGVVYFLDR TVMGLAMPVY EANLVNSHAQ CYSAVAMKRP DGTVFSAFHE 150 DNNKNNTLNL FPLNFKSITN KRFITTKEPY FARGPLWLYS TSTSLNCIVT 200 EATAKAKYPF SYFALTTGEI VEGSPFFNGS NGKHFAEPLE KLTILENYTM 250 IEDLMNGMNG ATTLVRKIAF LEKADTLFSW EIKEENESVC MLKHWTTVTH 300 GLRAETNETY HFISKELTAA FVAPKESLNL TDPKQTCIKN EFEKIINEVY 350 MSDYNDTYSM NGSYQIFKTT GDLILIWQPL VQKSLMFLEQ GSEKIRRRRD 400 VGDVKSRHDI LYVQLQYLYD TLKDYINDAL GNLAESWCLD QKRTITMLHE 450 LSKISPSSIV SEVYGRPISA QLHGDVLAIS KCIEVNQSSV QLHKSMRVVD 500 AKGVRSETMC YNRPLVTTFSF VNSTPEVVPG QLGLDNEILL GDHRTEECEI 550 PSTKIFLSGN HAHVYTDYTH TNSTPIEDIE VLDAFIRLKI DPLENADFKV 600 LDLYSPDELS RANVFDLENI LREYNSYKSA LYTIEAKIAT NTPSYVNGIN 650 SFLQGLGAIG TGLGSVISVT AGALGDIVGG VVSFLKNPFG GGLMLILAIV 700 VVVIIIVVFV RQRHVLSKPI DMMFPYATNP VTTVSSVTGT TVVKTPSVKD 750 VDGGTSVAVS EKEEGMADVS GQVSDDEYSQ EDALKMLKAI KSLDESYRRK 800 PSSSESHASK PSLIDRIRYR GYKSVNVEEA

[0149] The amino acid sequence of HHV-6B gB is as follows (SEQ ID NO:31): MSKMRVLFLA VFLMNSVLMI YCDSDDYIRA GYNHKYPFRI CSIAKGTDLM 50 RFDRDISCSP YKSNAKMSEG FFIIYKTNIE TYTFPVRTYK NELTFPTSYR 100 DVGVVYFLDR TVMGLAMPVY EANLVNSRAQ CYSAVAIKRP DGTVFSAYHE 150 DNNKNETLEL FPLNFKSVTN KRFITTKEPY FARGPLWLYS TSTSLNCIVT 200 EATAKAKYPF SYFALTTGEI VEGSPFFDGS NGKHFAEPLE KLTILENYTM 250 IEDLMNGMNG ATTLVRKIAF LEKGDTLFSW EIKEENESVC MLKHWTTVTH 300 GLRAETDETY HFISKELTAA FVASKESLNL TDPKQTCIKN EFEKIITDVY 350 MSDYNDAYSM NGSYQIFKTT GDLILIWQPL VQKSLMVLEQ GSVNLRRRRD 400 LVDVKSRHDI LYVQLQYLYD TLKDYINDAL GNLAESWCLD QKRTITMLHE 450 LSKISPSSIV SEVYGRPISA QLHGDVLAIS KCIEVNQSSV QLYKSMRVVD 500 AKGVRSETMC YNRPLVTFSF VNSTPEVVLG QLGLDNEILL GDHRTEECEI 550 PSTKIFLSGN HAHVYTDYTH TNSTPIEDIE VLDAFIRLKI DPLENADFKL 600 LDLYSPDELS RANVFDLENI LREYNSYKSA LYTIEAKIAT NTPSYVNGIN 650 SFLQGLGAIG TGLGSVISVT AGALGDIVGG VVSFLKNPFG GGLMLILAIV 700 VVVIIIVVFV RQKHVLSKPI DMMFPYATNP VTTVSSVTGT TVVKTPSVKD 750 ADGGTSVAVS EKEEGMADVS GQISGDEYSQ EDALKMLKAI KSLDESYRRK 800 PSSSESHASK PSLIDRIRYR GYKSVNVEEA

[0150] The amino acid sequence of HHV-7 gH is as follows (SEQ ID NO:32): MYFYINSLLL IVSINGWKHW NILNSSICVN EKTNQTIIQP GLITFNFHDY 50 NETRVYQIPK CLFGYTFVSN LFDSVNFDES FDQYKHRITR FFNPSTEKAV 100 KIYAQKFQTN IKPVSHTKTI TVSFLPLFYE KDVYFANVSE IRKLYYNQYI 150 CTLSNGLTDY LFPITERCVM RHYNYLNTVF MLALTPSFFI ISVETGMDDV 200 VFIFGNVSRI FFKAPFRKSS FIYRQTVSDD LLLITKKTTI ERFYPFLKID 250 FLDDIWKQNY DISFLIAKFN KLATVYIMEG FCGKPVNKDT FHLMFLFFGLT 300 HFLYSTRGDG LLPLLEILNT HQSIITMGRF LEKCFKMTKS HLLYPEMEKL 350 QNFQLVDYSY ITSDLTIPIS AKLAFLSLAD GRIVTVPQNK WKEIENNIET 400 LYEKHKLFTN LTQPERANLF LLSEIGNSLV FQEKIKRKIH VLLASLCNPL 450 EMYFWTHMLD NVMDIETMFS PCATATRKDL TQRVVNNILS YKNLDAYTNK 500 VMTLSVYRK KRLDMFKSIS CVSNEQAAFL TLPNITYTIS SKYILAGTSF 550 SVTSTVISTT IIITVVPLNS TCTPTNYKYS VKNIKPIYNI SSHDCVFCES 600 LVVEYDDIDG IIQFVYIMDD KQLLKLIDPD TNFIDVNPRT HYLLFLRNGS 650 VFEITALDLK SSQVSIMLVL LYLIIIIIVL FGIYHVFRLF

[0151] The amino acid sequence of HHV-7 gL is as follows (SEQ ID NO:33): MKTNIFFIFL ISILNQIYAL FNNSYYSNLE QECIKNILNC TQSKTLSLLE 50 PIDQAPIPKS DIISRLLYHT PYISRRDQVL IDEDFLETFY LLYNNPNQLH 100 TLLSLIKDSE SGHNWLGFLN NFERCLSDNT LLTCRDNVCK SYSYEKLKFT 150 GNIFVENIIG FEFNIPSNMI NFNMSILIYL ENEETRTQRI VRIDHHGINV 200 FDALLNCLRY FSRYYNFSFP LIQEMEKYNE VLPFRSEFSN LLIRTY

[0152] The amino acid sequence of HHV-7 gB is as follows (SEQ ID NO:34): MKILFLSVFI TFSLQLSLQT EADFVMTGHN QHLPFRICSI ATGTDLVRFD 50 REVSCASYGS NIKTTEGILI IYKTKIEAHT FSVRTFKKEL TFQTTYRDVG 100 TVYFLDRTVT TLPMPIEEVH MVNTEARCLS SISVKRSEEE EYVAYHKDEY 150 VNKTLDLIPL NFKSDTVRRY ITTKEPFLRN GPLWFYSTST SINCIVTDCI 200 AKTKYPFDFF ALSTGETVEG SPFYNGINSK TFNEPTEKIL FRNNYTMLKT 250 FDDGSKGNFV TLTKMAFLEK GNTIFSWEVQ NEESSICLLK HWMTIPHALR 300 AENANSFHFI AQELTASFVT GKSNYTLSDS KYNCINSNYT SILDEIYQTQ 350 YNNSHDKNGS YEIFKTEGDL ILIWQPLIQR KLTVLENFSN ASRKRRKREL 400 ETNKDIVYVQ LQYLYDTLKD YINTALGKLA EAWCLNQKRT ITVLHELSKI 450 SPSGIISAVY GKPMSAKLIG DVLAVSKCIE VNQTSVQLHK SMRLTKDSSY 500 DALRCYSRPL LTYSFANSSK ETYLGQLGLD NEILLGNHRT EECEQSNTKI 550 FLSGKFAHIF KDYTYVNSSL ITEIEALDAF VDLNIDPLEN ADFTLLELYT 600 KDELSKANVF DLETILREYN SYKSALHHIE TKIATVTPTY IGGIDTFKG 650 LGALGLGLGA VLGVTAGALG DVVNGVFSFL KNPFGGALTI LLTLGVIGLV 700 IFLFLRHHKRL AQTPIDILFP YTSKSTNSVL QATQSVQAQV KEPLDSSPPY 750 LKTNKDTEPQ GDDITHTNEY SQVEALKMLK AIKLLDESYK KAEIAEAKKS 800 QRPSLLERIQ YRGYQKLSTE EL

[0153] Alphaherpesviruses: human herpesvirus type 1 (HHV-1), human herpesvirus type 2 (HHV-2), and varicella zoster virus (VZV, HHV-3) HHV-1, or herpes simplex virus 1 (HSV-1), causes oral herpes, HHV-2, or herpes simplex virus 1 (HSV-2), causes genital herpes, and HHV-3, or VZV, causes chickenpox and shingles. Each of these viruses belongs to the Alphaherpesvirinae subfamily of the Herpesviridae family and is a neurotropic virus. VZV infects nearly all humans, and primary infection causes chickenpox (varicella). Latent VZV is most commonly present along nerve axons in cranial nerve ganglia, dorsal root ganglia, and autonomic ganglia. Viruses of this subfamily can spontaneously reactivate to cause shingles (herpes zoster). Herpes zoster skin lesions usually last for more than a week, but in some individuals, the infection may lead to chronic pain or postherpetic neuralgia (PHN, pain lasting more than 3 months), as well as vasculopathy, which may occur in approximately 40% of patients over the age of 60. Zoster paresis (herpes zoster with lower motor neuron type weakness) may also occur in the arms, legs, diaphragm, and / or abdominal muscles. Pathological features of herpes zoster include inflammation and hemorrhagic necrosis with associated neuritis, focal leptomeningeal inflammation, unilateral segmental acute poliomyelitis, and associated degeneration of motor and sensory roots. Demyelination is seen in areas with mononuclear cell (MNC) infiltration and microglial proliferation. Intranuclear inclusions, viral antigens, and herpes virus particles have been found in acutely infected ganglia. Vascular disease (or stroke) may be caused by proliferative viral infection of cerebral arteries, and may include conditions called granulomatous vasculitis, VZV vasculitis / encephalitis, post-varicella arteriopathy, and herpes zoster ophthalmicus with delayed contralateral hemiparesis. Symptoms include fever, altered mental status, headache, and focal neurological deficits (Gilden et al., Neuropathol. Appl. Neurobiol., 37(5):441-463, 2012). Other serious complications of infection include Mollare meningitis, Zoster multiplex, myelitis, herpes ophthalmicus (herpes serotinus zoster) and Ramsay's disease. -Hunt syndrome. Studies have shown an increased risk of stroke after herpes zoster (Kang et al., Stroke, 40(11):3443-3448, 2009, and Lin et al., Neurology, 74(10):792-797, 2010). Acute infection with VZV can lead to meningitis, meningoencephalitis, meningoradiculitis, and cerebellitis (Habib et al., J. Neurovirol., 15(2):206-208, 2009; Klein et al., Scan. J. Infect. Dis., 42(8):631-633, 2010; Gunson et al., J. Clin. Virol., 50(3):191-193, 2011; and Moses et al., Lancet Neurol., 5(11):984-988, 2006).

[0154] The VZV genome was the first herpesvirus genome to be completely sequenced in 1986. The VZV genome is remarkably stable, having undergone only three point mutations over more than 1200 passages (Liu et al., Arch. Virol., 153(10):1943-7, 2008). Infection proceeds from Langerhans cells to resident T cells in nearby draining lymph nodes. T cells are induced to express skin homing factors that transport virus-loaded T cells to the dermis, where fibroblasts and keratinocytes are exposed to infection and produce proinflammatory cytokines, resulting in chickenpox (Taylor et al., J. Virol., 79(17):11501-6, 2005, and Huch et al., J. Virol., 84(8):4060-72, 2010). VZV infects kidney cells, melanomas, and It induces apoptosis in several cell types, including tumor cells, fibroblasts, etc. (Pugazhenthi et al., J. Virol., 83(18):9273-82, 2009).

[0155] A variety of pharmaceutical treatments are available for VZV infection, including acyclovir for chickenpox, famciclovir, valacyclovir, zoster immune globulin (ZIG), and vidarabine for varicella, and VZV immune globulin is also a treatment (Centers for Disease Control and Prevention (CDC), March 2012, "FDA approval of an extended period for administering VariZIG for postexposure prophylaxis of varicella," Morb. Mortal. Wkly. Rep., 61(12):212, PMID 2245612).

[0156] VZV and HSV-1 / HSV-2 contain known envelope glycoproteins, gB, which correspond in function to the same or similar glycoproteins and related proteins found in other HHV species. It produces gH, gL, gM, and gN. VZV has no equivalent of HHV-1 / HHV-2 glycoprotein gD, but VZV glycoprotein gE performs a similar function (Cohen, JI, Curr. Top. Microbiol. Immunol., 342:1-14, 2010). Expression of gB, gH, and gL is necessary and sufficient to induce membrane fusion prior to entry of the viral particle into the host cell, allowing the nucleocapsid to gain access to the cytoplasm. Other accessory glycoproteins similar to gp42, gD, gO, or UL128-130 are not required for fusion (Vleck et al., Proc. Natl. Acad. Sci. USA, 108:18412-7, 2011, and Oliver et al., Proc. Natl. Acad. Sci. USA, 110:1911-6, 2013).

[0157] At least two cellular proteins, insulin-degrading enzyme (IDE) and myelin-associated glycoprotein (MAG), are thought to function as receptors for VZV entry into host cells, while other studies have shown that the αV subunit of integrin plays a role in membrane fusion for VZV (Yang et al., J. Virol., 90(16):7567-78, 2016).

[0158] The antigenic compositions and methods of the present application typically include two or more HHV proteins involved in mediating HHV binding, fusion and entry into host cells. In certain embodiments, two or more VZV proteins disclosed herein are combined in an antigenic composition. Two or more VZV proteins can be administered simultaneously or separately to induce an immune response or treat or prevent VZV infection in a subject. In certain embodiments, the antigenic composition (or administration method) includes two or more of the following VZV polypeptides (or nucleic acids encoding same): gB, gH and gL. In some embodiments, the gB polypeptide is a monomer, dimer or trimer. In some embodiments, the gH and gL polypeptides are a monomer, dimer, trimer or tetramer. Typically, gH and gL form a gH / gL heterodimer.

[0159] In certain embodiments, the two or more VZV proteins (or nucleic acids encoding same) include a monomeric or multimeric gB and a monomeric or multimeric gH / gL heterodimer. In certain embodiments, gB is a monomer, a dimer, or a trimer, and the gH / gL heterodimer is a monomer or a trimer. In certain embodiments, gB is a monomer and the gH / gL heterodimer is a monomer. In certain embodiments, gB is a trimer and the gH / gL heterodimer is a trimer. In certain embodiments, gB is a trimer and the gH / gL heterodimer is a monomer or a trimer. In certain embodiments, when mixed together, the gB, gH, and gL polypeptides of VZV form a protein complex. In certain embodiments, the gB, gH, and gL polypeptides of VZV are not administered as a protein complex comprising gB, gH, and gL polypeptides. For example, gB can be administered separately from gH and / or gL, or can be administered together with gH and gL but not as a protein complex. In certain embodiments, the two or more VZV proteins further comprise one or more of the following glycoproteins: gI, gC, and / or gE, which may be monomeric or multimeric (e.g., dimers, trimers, or tetramers).

[0160] The amino acid sequence of VZV gH is as follows (SEQ ID NO:35): MFALVLAVVI LPLWTTANKS YVTPTPATRS IGHMSALLRE YSDRNMSLKL 50 EAFYPTGFDE ELIKSLHWGN DRKHVFLVIV KVNPTTHEGD VGLVIFPKYL 100 LSPYHFKAEH RAPFPAGRFG FLSHPVTPDV SFFDSSFAPY LTTQHLVAFT 150 TFPPNPLVWH LERAETAATA ERPFGVSLLP ARPTVPKNTI LEHKAHFATW 200 DALARHTFFS AEAIITNSTL RIHVPLFGSV WPIRYWATGS VLLTSDSGRV 250 EVNIGVGFMS SLISLSSGPP IELIVVPHTV KLNAVTSDTT WFQLNPPGPD 300 PGPSYRVYLL GRGLDMNFSK HATVDICAYP EESLDYRYHL SMAHTEALRM 350 TTKADQHDIN EESYYHIAAR IATSIFALSE MGRTTEYFLL DEIVDVQYQL 400 KFLNYILMRI GAGAHPNTIS GTSDLIFADP SQLHDELSLL FGQVKPANVD 450 YFISYDEARD QLKTAYALSR GQDHVNALSL ARRVIMSIYK GLLVKQNLNA 500 TERQALFFAS MILLNFREGL ENSSRVLDGR TTLLLMTSMC TAAHATQAAL 550 NIQEGLAYLN PSKHMFTIPN VYSPCMGSLR TDLTEEIHVM NLLSAIPTRP 600 GLNEVLHTQL DESEIFDAAF KTMMIFTTWT AKDLHILHTH VPEVFTCQDA 650 AARNGEYVLI LPAVQGHSYV ITRNKPQRGL VYSLADVDVY NPISVVYLSR 700 DTCVSEHGVI ETVALPHPDN LKECLYCGSV FLRYLTTGAI MDIIIIDSKD 750 TERQLAAMGN STIPPFNPDM HGDDSKAVLL FPNGTVVTLL GFERRQAIRM 800 SGQYLGASLG GAFLAVVGFG IIGWMLCGNS RLREYNKIPL T

[0161] The amino acid sequence of VZV gL is as follows (SEQ ID NO:36): MASHKWLLQI VFLKTITIAY CLHLQDDTPL FFGAKPLSDV SLIITEPCVS 50 SVYEAWDYAA PPVSNLSEAL SGIVVKTKCP VPEVILWFKD KQMAYWTNPY 100 VTLKGLAQSV GEEHKSGDIR DALLDALSGV WVDSTPSSTN IPENGCVWGA 150 DRLFQRVCQ

[0162] The amino acid sequence of VZV gB is as follows (SEQ ID NO:37): MSPCGYYSKW RNRDRPEYRR NLRFRRFFSS IHPNAAAGSG FNGPGVFITS 50 VTGVWLCFLC IFSMFVTAVV SVSPSSFYES LQVEPTQSED ITRSAHLGDG 100 DEIREAIHKS QDAETKPTFY VCPPPTGSTI VRLEPTRTCP DYHLGKNFTE 150 GIAVVYKENI AAYKFKATVY YKDVIVSTAW AGSSYTQITN RYADRVPIPV 200 SEITDTIDKF GKCSSKATYV RNNHKVEAFN EDKNPQDMPL IASKYNSVGS 250 <h2 style=";text-align:left;direction:ltr">KAWHTTNDTY MVAGTPGTYR TGTSVNCIIE EVEARSIFPY DSFGLSTGDI 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> IYMSPFFGLR DGAYREHSNY AMDRFHQFEG YRQRDLDTRA LLEPAARNFL 350<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VTPHLTVGWN WKPKRTEVCS LVKWREVEDV VRDEYAHNFR FTMKTLSTTF 400<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ISETNEFNLN QIHLSQCVKE EARAIINRIY TTRYNSSHVR TGDIQTYLAR 450<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> GGFVVVFQPL LSNSLARLYL QELVRENTNH SPQKHPTRNT RSRRSVPVEL 500<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> RANRTITTTS SVEFAMLQFT YDHIQEHVNE MLARISSSWC QLQNRERALW 550<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SGLFPINPSA LASTILDQRV KARILGDVIS VSNCPELGSD TRIILQNSMR 600<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VSGSTTRCYS RPLISIVSLN GSGTVEGQLG TDNELIMSRD LLEPCVANHK 650<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> RYFLFGHHYV YYEDYRYVRE IAVHDVGMIS TYVDLNLTLL KDREFMPLQV 700<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> YTRDELRDTG LLDYSEIQRR NQMHSLRFYD IDKVVQYDSG TAIMQGMAQF 750<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> FQGLGTAGQA VGHVVLGATG ALLSTVHGFT TFLSNPFGAL AVGLLVLAGL 800<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> VAAFFAYRYV LKLKTSPMKA LYPLTTKGLK QLPEGMDPFA EKPNATDTPI 850<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> EEIGDSQNTE PSVNSGFDPD KFREAQEMIK YMTLVSAAER QESKARKKNK 900<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TSALLTSRLT GLALRNRRGY SRVRTENVTG V<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0163] <h2 style=";text-align:left;direction:ltr"> The amino acid sequence of VZV gI is as follows (SEQ ID NO:38): MFLIQCLISA VIFYIQVTNA LIFKGDHVSL QVNSSLTSIL IPMQNDNYTE 50 IKGQLVFIGE QLPTGTNYSG TLELLYADTV AFCFRSVQVI RYDGCPRIRT 100 SAFISCRYKH SWHYGNSTDR ISTEPDAGVM LKITKPGIND AGVYVLLVRL 150 DHSRSTDGFI LGVNVYTAGS HHNIHGVIYT SPSLQNGYST RALFQQARLC 200 DLPATPKGSG TSLFQHMLDL RAGKSLEDNP WLHEDVVTTE TKSVVKEGIE 250 NHVYPTDMST LPEKSLNDPP ENLLIIIPIV ASVMILTAMV IVIVISVKRR 300 RIKKHPIYRP NTKTRRGIQN ATPESDVMLE AAIAQLATIR EESPPHSVVN 350 PFVK

[0164] The amino acid sequence of VZV gC is as follows (SEQ ID NO:39): MKRIQINLIL TIACIQLSTE SQPTPVSITE LYTSAATRKP DPAVAPTSAA 50 SRKPDPAVAP TSAASRKPDP AVAPTSAASR KPDPAVAPTS AATRKPDPAV 100 APTSASRKP DPAVAPTSAA TRKPDPAVAP TSAASRKPDP AANTQHSQPP 150 FLYENIQCVH GGIQSIPYFH TFIMPCYMRL TTGQQAAFKQ QQKTYEQYSL 200 DPEGSNITRW KSLIRPDLHI EVWFTRHLID PHRQLGNALI RMPDLPVMLY 250 SNSADLNLIN NPEIFTHAKE NYVIPDVKTT SDFSVTILSM DATTEGTYIW 300 RVVNTKTKNV ISEHSITVTT YYRPNITVVG DPVLTGQTYA AYCNVSKYYP 350 PHSVRVRWTS RFGNIGKNFI TDAIQEYANG LFSYVSAVRI PQQKQMDYPP 400 PAIQCNVLWI RDGVSNMKYS AVVTPDVYPF PNVSIGIIDG HIVCTAKCVP 450 RGVVHFVWWV NDSPINHENS EITGVCDQNK RFVNMQSSCP TSELDGPITY 500 SCHLDGYPKK FPPFSAVYTY DASTYATTFS VVAVIIGVIS ILGTLGLIAV 550 IATLCIRCCS

[0165] The amino acid sequence of VZV gE is as follows (SEQ ID NO:40): MGTVNKPVVG VLMGFGIITG TLRITNPVRA SVLRYDDFHT DEDKLDTNSV 50 YEPYYHSDHA ESSWVNRGES SRKAYDHNSP YIWPRNDYDG FLENAHEHHG 100 VYNQGRGIDS GERLMQPTQM SAQEDLGDDT GIHVIPTLNG DDRHKIVNVD 150 QRQYGDVFKG DLNPKPQGQR LIEVSVEENH PFTLRAPIQR IYGVRYTETW 200 SFLPSLTCTG DAAPAIQHIC LKHTTCFQDV VVDVDCAENT KEDQLAEISY 250 RFQGKKEADQ PWIVVNTSTL FDELELDPPE IEPGVLKVLR TEKQYLGVYI 300 WNMRGSDGTS TYATFLVTWK GDEKTRNPTP AVTPQPRGAE FHMWNYHSHV 350 FSVGDTFSLA MHLQYKIHEA PFDLLLEWLY VPIDPTCQPM RLYSTCLYHP 400 NAPQCLSHMN SGCTFTSPHL AQRVASTVYQ NCEHADNYTA YCLGISHMEP 450 SFGLILHDGG TTLKFVDTPE SLSGLYVFVV YFNGHVEAVA YTVVSTVDHF 500 VNAIEERGFP PTAGQPPATT KPKEITPVNP GTSPLLRYAA WTGGLAAVVL 550 LCLVIFLICT AKRMRVKAYR VDKSPYNQSM YYAGLPVDDF EDSESTDTEE 600 EFGNAIGGSH GGSSYTVYID KTR

[0166] The antigenic compositions and methods of the present application typically include two or more HHV proteins involved in mediating HHV binding, fusion and entry into host cells. In certain embodiments, two or more HSV-1 or HSV-2 proteins disclosed herein are combined in an antigenic composition. Two or more HSV-1 or HSV-2 proteins may be administered simultaneously or separately to induce an immune response or treat or prevent HSV-1 or HSV-2 infection in a subject. In certain embodiments, the antigenic composition (or method of administration) includes two or more of the following HSV-1 or HSV-2 polypeptides (or nucleic acids encoding same): gB, gH, and gL. In some embodiments, the gB polypeptide is a monomer, dimer, or trimer. In some embodiments, the gH and gL polypeptides are a monomer, dimer, trimer, or tetramer. Typically, gH and gL form a gH / gL heterodimer.

[0167] In certain embodiments, the two or more HSV-1 or HSV-2 proteins (or nucleic acids encoding same) comprise a monomeric or multimeric gB and a monomeric or multimeric gH / gL heterodimer. In certain embodiments, gB is a monomer, a dimer, or a trimer, and the gH / gL heterodimer is a monomer or a trimer. In certain embodiments, gB is a monomer and the gH / gL heterodimer is a monomer or a trimer. In certain embodiments, gB is a trimer and the gH / gL heterodimer is a monomer. In certain embodiments, gB is a trimer and the gH / gL heterodimer is a trimer. In certain embodiments, when mixed together, the HSV-1 or HSV-2 gB, gH, and gL polypeptides form a protein complex. In certain embodiments, the HSV-1 or HSV-2 gB, gH, and gL polypeptides are For example, gB can be administered separately from gH and / or gL, or can be administered together with gH and gL rather than as a protein complex.

[0168] In certain embodiments, the two or more HSV-1 or HSV-2 proteins further comprise a gD polypeptide, which may be monomeric or multimeric (e.g., dimer, trimer, or tetramer).

[0169] The amino acid sequence of HSV-1 gH is as follows (SEQ ID NO:41): MGNGLWFVGV IILGAAWGQV HDWTEQTDPW FLDGLGMDRM YWRDTNTGRL 50 WLPNTPDPQK PPRGFLAPPD ELNLTTASLP LLRWYEERFC FVLVTTAEFP 100 RDPGQLLYIP KTYLLGRPPN ASLPAPTTVE PTAQPPPAVA PLKGLLHNPT 150 ASVLLRSRAW VTFSAVPDPE ALTFPRGDNV ATASHPSGPR DTPPPRPPVG 200 ARRHPTTELD ITHLHNASTT WLATRGLLRS PGRYVYFSPS ASTWPVGIWT 250 TGELVLGCDA ALVRARYGRE FMGLVISMHD SPPAEVMVVP AGQTLDRVGD 300 PADENPPGAL PGPPGGPRYR VFVLGSLTRA DNGSALDALR RVGGYPEEGT 350 NYAQFLSRAY AEFFSGDAGA EQGPRPPLFW RLTGLLATSG FAFVNAAHAN 400 GAVCLSDLLG FLAHSRALAG LAARGAAGCA ADSVFFNVSV LDPTARLQLE 450 ARLQHLVAEI LEREQSLALH ALGYQLAFVL DSPSAYDAVA PSAAHLIDAL 500 YAEFLGGRVV TTPVVHRALF YASAVLRQPF LAGVPSAVQR ERARRSLLIA 550 SALCTSDVAA ATNADLRTAL ARADHQKTLF WLPDHFSPCA ASLRFDLDES 600 VFILDALAQA TRSETPVEVL AQQTHGLAST LTRWAHYNAL IRAFVPEASH 650 RCGGQSANVE PRILVPITHN ASYVVTHSPL PRGIGYKLTG VDVRRPLFLT 700 YLTATCEGST RDIESKRLVR TQNQRDLGLV GAVFMRYTPA GEVMSVLLVD 750 TDNTQQQIAA GPTEGAPSVF SSDVPSTALL LFPNGTVIHL LAFDTQPVAA 800 IAPGFLAASA LGVVMITAAL AGILKVLRTS VPFFWRRE

[0170] The amino acid sequence of HSV-1 gL is as follows (SEQ ID NO:42): MGILGWVGLI AVGVLCVRGG LPSTEYVIRS RVAREVGDIL KVPCVPLPSD 50 DLDWRYETPS AINYALIDGI FLRYHCPGLD TVLWDRHAQK AYWVNPFLFV 100 AGFLEDLSYP AFPANTQETE TRLALYKEIR QALDSRKQAA SHTPVKAGCV 150 NFDYSRTRRC VGRQDLGPTN GTSGRTPVLP PDDEAGLQPK PLTTPPPIIA 200 TSDPTPRRDA ATKSRRRRPH SRRL

[0171] The amino acid sequence of HSV-1 gB is as follows (SEQ ID NO:43): MHQGAPSWGR RWFVVWALLG LTLGVLVASA APTSPGTPGV AAATQAANGG 50 PATPAPPPLG AAPTGDPKPK KNKKPKNPTP PRPAGDNATV AAGHATLREH 100 LRDIKAENTD ANFYVCPPPT GATVVQFEQP RRCPTRPEGQ NYTEGIAVVF 150 KENIAPYKFK ATMYYKDVTV SQVWFGHRYS QFMGIFEDRA PVPFEEVIDK 200 INAKGVCRST AKYVRNNLET TAFHRDDHET DMELKPANAA TRTSRGWHTT 250 DLKYNPSRVE AFHRYGTTVN CIVEEVDARS VYPYDEFVLA TGDFVYMSPF 300 YGYREGSHTE HTTYAADRFK QVDGFYARDL TTKARATAPT TRNLLTTPKF 350 TVAWDWVPKR PSVCTMTKWQ EVDEMLRSEY GGSFRFSSDA ISTTFTTNLT 400 EYPLSRVDLG DCIGKDARDA MDRIFARRYN ATHIKVGQPQ YYQANGGFLI 450 AYQPLLSNTL AELYVREHLR EQSRKPPNPT PPPPGASANA SVERIKTTSS 500 IEFARLQFTY NHIQRHVNDM LGRVAIAWCE LQNHELTLWN EARKLNPNAI 550 ASVTVGRRVS ARMLGDVMAV STCVPVAADN VIVQNSMRIS SRPGACYSRP 600 LVSFRYEDQG PLVEGQLGEN NELRLTRDAI EPCTVGHRRY FTFGGGYVYF 650 EEYAYSHQLS RADITTVSTF IDLNITMLED HEFVPLEVYT RHEIKDSGLL 700 DYTEVQRRNQ LHDLRFADID TVIHADANAA MFAGLGAFFE GMGDLGRAVG 750 KVVMGIVGGV VSAVSGVSSF MSNPFGALAV GLLVLAGLAA AFFAFRYVMR 800 LQSNPMKALY PLTTKELKNP TNPDASGEGE EGGDFDEAKL AEAREMIRYM 850 ALVSAMERTE HKAKKKGTSA LLSAKVTDMV MRKRRNTNYT QVPNKDGDAD 900 EDDL

[0172] The amino acid sequence of HSV-1 gD is as follows (SEQ ID NO:44): MGGAAARLGA VILFVVIVGL HGVRGKYALA DASLKMADPN RFRGKDLPVP 50 DRLTDPPGVR RVYHIQAGLP DPFQPPSLPI TVYYAVLERA CRSVLLNAPS 100 EAPQIVRGGS EDVRKQPYNL TIAWFRMGGN CAIPITVMEY TECSYNKSLG 150 ACPIRTQPRW NYYDSFSAVS EDNLGFLMHA PAFETAGTYL RLVKINDWTE 200 ITQFILEHRA KGSCKYALPL RIPPSACLSP QAYQQGVTVD SIGMLPRFIP 250 ENQRIVAVYS LKIAGWHGPK APYTSTLLP ELSETPNATQ PELAPEDPED 300 SALLEDPVGT VAPQIPPNWH IPSIQDAATP YHPPATPNNM GLIAGAVGGS 350 LLAALVICGI VYWMRRRTQK GPKRIRLPHI REDDQPSSHQ PLFY

[0173] The amino acid sequence of HSV-2 gH is as follows (SEQ ID NO:45): MGPGLWVVMG VLVGVAGGHD TYWTEQIDPW FLHGGLGLART YWRDTNTGRL 50 WLPNTPDASD PQRGRLAPPG ELNLTTASVP MLRWYAERFC FVLVTTAEFP 100 RDPGQLLYIP KTYLLGRPRN ASLPELPEAG PTSRPPAEVT QLKGLSHNPG 150 ASALLRSRAW VTFAAAPDRE GLTFPRGDDG ATERHPDGRR NAPPPGPPAG 200 APRHPTTNLS IAHLHNASVT WLAARGLLRT PGRYVYLSPS ASTWPVGVWT 250 TGGLAFGCDA ALVRARYGKG FMGLVISMRD SPPAEIIVVP ADKTLARVGN 300 PTDENAPAVL PGPPAGPRYR VFVLGAPTPA DNGSALDALR RVAGYPEEST 350 NYAQYMSRAY AEFLGEDPGS GTDARPSLFW RLAGLLASSG FAFINAAHAH 400 DAIRLSDLLG FLAHSRVLAG LAARGAAGCA ADSVFLNVSV LDPAARLRLE 450 ARLGHLVAAI LEREQSLAAH ALGYQLAFVL DSPAAYGAVA PSAARLIDAL 500 YAEFLGGRAL TAPMVRRALF YATAVLRAPF LAGAPSAEQR ERARRGLLIT 550 TALCTSDVAA ATHADLRAAL ARTDHQKNLF WLPDHFSPCA ASLRFDLAEG 600 GFILDALAMA TRSDIPADVM AQQTRGVASA LTRWAHYNAL IRAFVPEATH 650 QCSGPSHNAE PRILVPITHN ASYVVTHTPL PRGIGYKLTG VDVRRPLFIT 700 YLTATCEGHA REIEPKRLVR TENRRDLGLV GAVFLRYTPA GEVMSVLLVD 750 TDATQQQLAQ GPVAGTPNVF SSDVPSVALL LFPNGTVIHL LAFDTLPIAT 800 IAPGFLAASA LGVVMITAAL AGILRVVRTC VPFLWRRE

[0174] The amino acid sequence of HSV-2 gL is as follows (SEQ ID NO:46): MGFVCLFGLV VMGAWGAWGG SQATEYVLRS VIAKEVGDIL RVPCMRTPAD 50 DVSWRYEAPS VIDYARIDGI FLRYHCPGLD TFLWDRHAQR AYLVNPFLFA 100 AGFLEDLSHS VFPADTQETT TRRALYKEIR DALGSRKQAV SHAPVRAGCV 150 NFDYSRTRRC VGRRDLRPAN TTSTWEPPVS SDDEASSQSK PLATQPPVLA 200 LSNAPHGGSP RREVGAGILA SDATSHVCIA SHPGSGAGQP TRLAAGSAVQ 250 RRRPRGCPPG VMFSASTTPE QPLGLSGDAT PPLPTSVPLD WAAFRRAFLI 300 DDAWRPLLEP ELANPLTARL LAEYDRRCQT EEVLPPREDV FSWTRYCTPD 350 DVRVVIIGQD PYHHPGQAHG LAFSVRADVP VPPSLRNVLA AVKNCYPDAR 400 MSGRGCLEKW ARDGVLLLNT TLTVKRGAAA SHSKLGWDRF VGGVVRRLAA 450 RRPGLVFMLW GAHAQNAIRP DPRQHYVLKF SHPSPLSKVP FGTCQHFLAA 500 NRYLETRDIM PIDWSV

[0175] The amino acid sequence of HSV-2 gB is as follows (SEQ ID NO:47): MRGGGLICAL VVGALVAAVA SAAPAAPAAP RASGGVAATV AANGGPASRP 50 PPVPSPATTK ARKRKTKKPP KRPEATPPPD ANATVAAGHA TLRAHLREIK 100 VENADAQFYV CPPPTGATVV QFEQPRRCPT RPEGQNYTEG IAVVFKENIA 150 PYKFKATMYY KDVTVSQVWF GHRYSQFMGI FEDRAPVPFE EVIDKINTKG 200 VCRSTAKYVR NNMETTAFHR DDHETDMELK PAKVATRTSR GWHTTDLKYN 250 PSRVEAFHRY GTTVNCIVEE VDARSVYPYD EFVLATGDFV YMSPFYGYRE 300 GSHTEHTSYA ADRFKQVDGF YARDLTTKAR ATSPTTRNLL TTPKFTVAWD 350 WVPKRPAVCT MTKWQEVDEM LRAEYGGSFR FSSDAISTTF TTNLTEYSLS 400 RVDLGDCIGR DAREAIDRMF ARKYNATHIK VGQPQYYLAT GGFLIAYQPL 450 LSNTLAELYV REYMREQDRK PRNATPAPLR EAPSANASVE RIKTTSSIEF 500 ARLQFTYNHI QRHVNDMLGR IAVAWCELQN HELTLWNEAR KLNPNAIASA 550 TVGRRVSARM LGDVMAVSTC VPVAPDNVIV QNSMRVSSRP GTCYSRPLVS 600 FRYEDQGPLI EGQLGENNEL RLTRDALEPC TVGHRRYFIF GGGYVYFEEY 650 AYSHQLSRAD VTTVSTFIDL NITMLEDHEF VPLEVYTRHE IKDSGLLDYT 700 EVQRRNQLHD LRFADIDTVI RADANAAMFA GLCAFFEGMG DLGRAVGKVV 750 MGVVGGVVSA VSGVSFMSN PFGALAVGLL VLAGLVAAFF AFRYVLQLQR 800 NPMKALYPLT TKELKTSDPG GVGGEGEEGA EGGGFDEAKL AEAREMIRYM 850 ALVSAMERTE HKARKKGTSA LLSSKVTNMV LRKRNKARYS PLHNEDEAGD 900 EDEL

[0176] The amino acid sequence of HSV-2 gD is as follows (SEQ ID NO:48): MGRLTSGVGT AALLVVAVGL RVVCAKYALA DPSLKMADPN RFRGKNLPVL 50 DRLTDPPGVK RVYHIQPSLE DPFQPPSIPI TVYYAVLERA CRSVLLHAPS 100 EAPQIVRGAS DEARKHTYNL TIAWYRMGDN CAIPITVMEY TECPYNKSLG 150 VCPIRTQPRW SYYDSFSAVS EDNLGFLMHA PAFETAGTYL RLVKINDWTE 200 ITQFILEHRA RASCKYALPL RIPPAACLTS KAYQQGVTVD SIGMLPRFIP 250 ENQRTVALYS LKIAGWHGPK PPYTSTLLPP ELSDTTNATQ PELVPEDPED 300 SALLEDPAGT VSSQIPPNWH IPSIQDVAPH HAPAAPSNPG LIIGALAGST 350 LAVLVIGGIA FWVRRRAQMA PKRLRLPHIR DDDAPPSHQP LFY

[0177] HHV Proteins. The present application demonstrates that various combinations of HHV proteins involved in mediating viral binding, fusion and host cell entry unexpectedly induce synergistic or additive neutralizing antibody responses, despite concerns in the art about vaccines or immune interference. The HHV proteins that are combined in the antigenic compositions disclosed herein (e.g., gB, gH, gL, gp350) or administered (simultaneously or separately) to prevent or treat HHV infection or induce immunity in a subject can be made using any conventional technique.

[0178] For example, in certain embodiments, one or more of the HHV proteins are naturally occurring. In other embodiments, one or more of the HHV proteins are recombinant (i.e., prepared using recombinant DNA technology). In certain embodiments, the recombinant HHV proteins have one or more differences in the glycosylation pattern of naturally occurring HHV proteins. In certain embodiments, one or more of the HHV proteins are modified and are not naturally occurring proteins. In certain embodiments, all of the HHV proteins are modified and are not naturally occurring proteins. For example, the HHV proteins may be mutant forms of the wild-type proteins, truncated forms of the wild-type proteins, multimerized proteins, or fusion proteins.

[0179] In certain embodiments, the modified HHV protein is a protein that binds to a specific target molecule, and the modified HHV protein retains its ability to bind to the target molecule. In certain embodiments, the truncated HHV protein is an extracellular domain of an HHV protein, for example, including one or more extracellular domains of gB, gp350, gL, or gH; or portions thereof that retain the ability to bind to their target molecules. For example, gp350 binds to CD21 (also known as CR2) on the surface of B cells, gp42 binds to HLA class II molecules, gD binds to nectin-1 (HveC, CD111) and herpes virus entry mediator (HVEM), and gpK8.1A and gpK8.1B bind to cell surface heparin sulfate molecules.

[0180] In certain embodiments, the HHV polypeptide is a variant HHV polypeptide that contains one or more deletions, insertions, or substitutions. For example, gp350 and gp220 polypeptides that bind to CR2 include naturally occurring or synthetically programmed variant polypeptides that are substantially identical to either the gp350 or gp220 polypeptides, but have amino acids that differ from gp350 or gp220 due to one or more deletions, insertions, or substitutions. Several gp350 / 220 variant sequences have already been identified by sequencing DNA from different strains of EBV and are readily available to those skilled in the art (Beisel et al., J. Viriol., 1985, 54(3):665-74). .

[0181] Similarly, variant gH, gL, gB, gp42, gM, gN, gI, gC, gE, gD, ORF68, BMRF-2, UL128, UL130, UL131A, and gpK8.1 polypeptides can include naturally occurring or synthetically programmed variant polypeptides that are substantially identical to any of the gH, gL, gB, gp42, gM, gN, gI, gC, gE, gD, ORF68, BMRF-2, UL128, UL130, UL131A, and gpK8.1 polypeptides, but have an amino acid sequence that differs from gH, gL, gB, gp42, gM, gN, gI, gC, gE, gD, ORF68, BMRF-2, UL128, UL130, UL131A, and gpK8.1 due to one or more deletions, insertions, or substitutions.

[0182] The variant amino acid sequence is preferably at least 60%, 65%, 70%, or 80% identical to gp350, gp220 polypeptide or gH, gL, gB, gp42, gM, gN, gI, gC, gE, gD, ORF68, BMRF-2, UL128, UL130, UL131A, and gpK8.1, more preferably at least 85% identical, even more preferably at least 90% identical, and most preferably at least 95% identical. Percent identity can be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, described by Devereux et al. (Nucl. Acids Res., 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program is an adaptation of Needleman and Wunsch's algorithm as revised by Smith and Waterman (Adv. Appl. Math, 2:482, 1981). The method of quantification (J. Mol. Biol., 48:443, 1970) is used. Preferred default parameters include: (1) a single element comparison matrix for nucleotides (with the value 1 representing a match and the value 0 representing a mismatch) and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res., 14:6745, 1986, as described in Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for terminal gaps.

[0183] Variant polypeptides can be obtained by mutation of the nucleotide sequence encoding the gp350, gp220, gH, gL, gB, gp42, gM, gN, gI, gC, gE, gD, ORF68, BMRF-2, UL128, UL130, UL131A, and / or gpK8.1 polypeptides. The amino acid sequence alterations can occur naturally or can be accomplished by any of a number of conventional methods. Mutations can include modifications to fragments of the native sequence. Mutations can be introduced into a particular locus by synthesizing oligonucleotides containing the mutated sequence flanked by restriction enzyme sites that allow for gating. Following ligation, the resulting reconstituted sequence encodes an analog with the desired amino acid insertion, substitution, or deletion.

[0184] Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be utilized to provide altered genes in which prespecified codons can be altered by substitution, deletion, or insertion. Examples of the alternative methods described above are those described by Walder et al. (Gene, 42:133, 1986), Bauer et al. (Gene 37:73, 1985), Craik, (BioTechniques, Jan. 12-19, 1985). , Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981 ), Kunkel (Proc. Natl. Acad. Sci. USA, 82:488, 1985), Kunkel et al. (Methods in Enzymol., 154:367, 1987), and U.S. Pat. Nos. 4,518,584 and 4,712,585. 37,462, the entireties of which are incorporated herein by reference.

[0185] Although it has been shown that multimerizing HHV proteins enhances their immunogenicity (see US Patent Publication Nos. 2015-0174237 and 2016-0303225, which are incorporated herein by reference in their entirety), unexpected additive and synergistic antibody responses were observed when combining both multimeric and / or monomeric HHV proteins. Thus, in certain embodiments, one or more HHV proteins are in monomeric form of the protein. In certain embodiments, one or more HHV proteins are in multimeric form of the protein. In certain embodiments, one or more HHV proteins are monomeric and one or more HHV proteins are multimeric. In certain embodiments, the antigenic composition comprises an HHV gB polypeptide that is monomeric or multimeric. In certain embodiments, the multimeric gB polypeptide is a dimer or trimer, preferably a trimer. In certain embodiments, the gp350 polypeptide is a monomer or a multimer. In certain embodiments, the multimeric gp350 is a dimer, trimer, or tetramer, preferably a tetramer. Methods for multimerizing HHV proteins are known in the art and are discussed elsewhere in this application.

[0186] The gH and gL polypeptides of HHV may be combined as individual polypeptides in the antigenic compositions and methods described herein. In other embodiments, gH and gL form a gH / gL heterodimer. In certain embodiments, the gH / gL heterodimer is a non-covalently associated protein complex, such as a gH / gL protein complex that occurs naturally and may form spontaneously under certain in vitro conditions. In other embodiments, the gH / gL heterodimer is a fusion protein. When the HHV antigenic composition comprises a gH and a gL polypeptide in the form of a gH / gL heterodimer, the antigenic composition further comprises a gB polypeptide, or for EBV, the antigenic composition further comprises a gB and / or a gp350 polypeptide. In certain embodiments, the gH or gL polypeptide is a monomer or multimer. In certain embodiments, the gH or gL polypeptide is a dimer, trimer, or tetramer, preferably a trimer. In certain embodiments, the gH / gL heterodimer is a monomer or a multimer. In certain embodiments, the multimeric gH / gL heterodimer is a dimer, trimer or tetramer, preferably a trimer.

[0187] Multimerization of HHV proteins. As discussed above, in the disclosed antigenic compositions, two or more HHV polypeptides may be multimerized, or they may be monomeric. For example, it is known that under certain conditions, at least the gH and gL polypeptides form heterodimers. Furthermore, it is known that under certain conditions, the gB polypeptide exists as a multimer, e.g., at least a homotrimer (Ma, A., Virology, 178(2):588-592, 1990). Furthermore, the polypeptide gB forms a heterodimer under certain conditions. It is known that HHV associates with gH / gL to form a heterotrimer of gB / gH / gL. Thus, by introducing such an HHV polypeptide into a composition, spontaneous multimerization can occur under certain conditions.

[0188] Multimerization of HHV polypeptides can occur spontaneously for some polypeptides under appropriate conditions, whereas others do not form multimers under natural conditions. In certain embodiments, it is advantageous to modify two or more HHV polypeptides to form multimers to enhance their immunogenicity. In certain embodiments, a trimeric HHV gB polypeptide is formed by expressing a modified HHV gB polypeptide in a host cell. In the modified gB polypeptide, the furin cleavage site in the extracellular domain of the gB polypeptide is replaced by a linker sequence, as described in US Patent Publication No. 2016-0303225 (also published in US Patent Application Publication No. 2016-0303225, which is incorporated herein by reference in its entirety). Figure 1, right panel, and Figure 7 show exemplary modified EBV and HCMV gB constructs that form homotrimeric gB complexes when expressed in a host cell. In these embodiments, a linker sequence (e.g., (Gly4Ser)3 (SEQ ID NO:3)) replaces the furin cleavage site in the extracellular domain of the EBV or HCMV gB polypeptide. An optional leader sequence may be added to the construct to direct secretion of the recombinant polypeptide. Although these embodiments are illustrated using EBV and HCMV gB polypeptides, any HHV gB sequence may be substituted in the construct to produce a desired modified gB polypeptide.

[0189] In certain embodiments, multimeric HHV proteins can be synthesized using recombinant cloning techniques to combine oligomerization domains and HHV polypeptides, and optionally expressed as fusion proteins, for example, as described in Patent Document 1 (also published as U.S. Patent Application Publication No. 2015-0174237, which is incorporated herein by reference in its entirety).

[0190] Fusion proteins. Fusion proteins used to generate multimeric HHV proteins can be synthesized using standard recombinant cloning techniques. For example, one strategy for generating fusion proteins involves creating a nucleic acid construct that includes an oligomerization motif sequence and a linker sequence that separates two or more antigens, so that the encoded fusion protein can form a dimer, trimer, tetramer, hexamer, heptamer, or octamer complex from a single nucleic acid construct (see U.S. Patent No. 5,333,399, which is incorporated by reference for all purposes). This platform can be used to generate multimeric fusion proteins that include multiple copies of a single antigen of interest, including, for example, gp350, gp220 polypeptide, or gB. For example, homodimers, homotrimers, or homotetramers can be generated using two, three, or four copies of the same polypeptide that has a dimerization, trimerization, or tetramerization domain, respectively. When the oligomerization domains associate with each other, the constructs form tetramers containing four copies of the same polypeptide (if a dimerization domain is used), hexamers containing six copies of the same polypeptide (if a trimerization domain is used), or octamers containing eight copies of the same polypeptide (if a tetramerization domain is used).

[0191] Alternatively, this platform can be used to create multimeric fusion proteins containing two or more different antigens of interest. For example, heterodimers can be created using a first HHV polypeptide linked to a second different HHV polypeptide (or a heterotrimer containing two or three different antigens), such as a heterodimer formed between gH and gL of HHV. When the oligomerization domains are associated together, the construct forms a tetramer that is a dimer with both the first and second HHV polypeptides (if a dimerization domain is used) and a trimerization domain with at least the first and second HHV polypeptides (if a trimerization domain is used in the construct). It forms a hexamer that is a dimer with a second HHV polypeptide, or a trimer with a first, second and third HHV polypeptide, or (if a tetramerization domain is used) forms an octamer.

[0192] In one embodiment, a trimeric protein may be formed when the native polypeptide is presented in a monomeric form in association with the trimerization domain. The fusion protein may optionally further comprise a third polypeptide and a second linker sequence, where the second linker sequence connects the second polypeptide to the third polypeptide, the first polypeptide, or the oligomerization domain. In another embodiment, the fusion protein comprises four or more polypeptides and additional linkers. In one embodiment, the fusion protein forms a multimeric polypeptide when expressed in a host cell. In another embodiment, the first and second polypeptides do not naturally occur as multimeric proteins.

[0193] In some embodiments, only a portion of the extracellular domain of each HHV polypeptide is engineered into a nucleic acid construct encoding a fusion protein. Shorter polypeptides are easier to express in larger amounts, and in some embodiments, only a portion of the HHV polypeptide is necessary or desirable to achieve the desired immunological effect, i.e., those portions of the HHV polypeptide that elicit an immune response.

[0194] Optionally, the nucleic acid construct is capable of infecting mammalian host cells, such as HeLa cells, yeast cells, insect cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, COS cells, Vero cells, NS0 mouse myeloma cells, and other cells disclosed in the art, such as Khan, K., Adv. Pharm. Bull., 3(2):257-263, 2013. The nucleic acid encoding a signal peptide is included such that it is secreted from tissue culture containing one or more host cells such as, for example, a fusion protein. Secretion of the fusion protein provides an easy means for harvesting and purifying the protein by known methodologies.

[0195] In one embodiment, the fusion protein is formed from the expression of a nucleic acid construct that includes a nucleic acid sequence that encodes one or more gp350 polypeptides, such as two such sequences, so that when expressed together with a dimerization domain, such as a leucine zipper oligomerization domain, a gp350 tetramer is formed (see FIG. 1, left panel). The gp350 nucleic acid sequence can be derived from any HHV genome that includes such a sequence. Alternatively, the gp350 sequence can be replaced by one or more other HHVs disclosed herein.

[0196] As shown in the center panel of FIG. 1, in another embodiment, the fusion protein can be encoded by a first nucleic acid construct encoding gH and a second nucleic acid encoding gL, and a trimerization domain, such as a T4 Foldon oligomerization domain, to result in, for example, a trimeric gH / gL heterodimer upon expression. The gH and gL polypeptides can be any gH / gL polypeptide sequence found in any of the known HHV genomes. Alternatively, in another embodiment, the gH and / or gL polypeptides can be replaced by one or more other HHV polypeptides to form the desired HHV proteins described herein.

[0197] In such an embodiment, the nucleic acid construct need not contain the full length HHV polypeptide sequence. The sequence may be modified. For example, the modified sequence may be a portion, truncated, or altered or mutated sequence. Such modified sequences may improve protein expression, for example, by removing sequences of the transmembrane and intracellular domains, or may elicit a more robust immune response, for example, by strategic placement of highly immunogenic epitopes of the HHV polypeptides discussed herein.

[0198] Linker sequence. A linker sequence is used in the modified gB polypeptide to replace the furin cleavage site in the extracellular domain of the gB polypeptide. A linker sequence is also used in the fusion protein to separate the various components of the fusion protein. Thus, in certain embodiments, the amino terminus of the linker sequence is linked to the first polypeptide by a peptide bond, and the carboxy terminus of the linker sequence is linked to the second polypeptide by a peptide bond. The first and second polypeptides are one of the fusion and host cell entry proteins of HHV, respectively, or one of the HHV accessory proteins. In certain embodiments, the first and second polypeptides are the same (e.g., gp350). In other embodiments, the first and second polypeptides are different (e.g., gH and gL). In contrast to the first and second polypeptides being linked together without an intervening polypeptide sequence, such a linker sequence links the first and second polypeptides. It is understood that the linker sequence is not a sequence that naturally separates the first and second polypeptides when the first and second polypeptides fortuitously occur together in nature.

[0199] In one embodiment, the linker sequence is a polypeptide having a length of 5 to 25 amino acids, in particular 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In another embodiment, the linker sequence is a polypeptide having a length of 10 to 25 amino acids. The linker sequence preferably comprises the amino acids glycine and serine. In one embodiment, the linker sequence is 15 amino acids long and has the amino acid sequence (Gly4Ser)3 (SEQ ID NO: 3).

[0200] Other suitable peptide linkers are those described in U.S. Patent Nos. 4,751,180, 4,935,233 and 5,073,627, each of which is incorporated herein by reference in its entirety. A DNA sequence encoding a desired linker sequence is inserted between and in the same reading frame as the DNA sequences encoding the first and second polypeptides, for example, using conventional techniques known in the art. For example, a chemically synthesized oligonucleotide encoding the linker can be ligated between the sequences encoding the first and second polypeptides.

[0201] Oligomerization domain. In certain embodiments, an oligomerization domain is used to generate multimeric HHV polypeptides. An oligomerization domain is a stretch of amino acid residues that causes a polypeptide that contains the oligomerization domain to oligomerize, i.e., form a covalent and / or non-covalent association with another polypeptide that contains a corresponding or homologous oligomerization domain. Thus, two or more polypeptides are "oligomerized" when they are linked to each other by an oligomerization domain. Any oligomerization domain known in the art can be used. Examples include leucine zipper domains, complement C1q domains, α-helical coiled-coil domains, thrombospondin domains, and fibritin domains (see Engel et al., Matrix Biol., 19(4):283-288, 2000). Polypeptides in an oligomer may be bound to each other by a covalent or non-covalent association with another polypeptide that contains a corresponding or homologous oligomerization domain. The peptides may have identical, similar, or different polypeptide sequences.

[0202] Homodimerization and homooligomerization refer to the association of the same polypeptide components to form a dimer or oligomer. Heterodimerization and heterooligomerization refer to the association of different polypeptides to form a dimer or oligomer. Thus, a homooligomer comprises the association of multiple copies of a particular polypeptide, whereas a heterooligomer comprises the association of copies of different polypeptides. "Oligomerization," "oligomerize," and "oligomer," with or without a prefix, are intended to encompass "dimerization," "dimerize," and "dimer." Thus, in one embodiment, the oligomerization domain is intended to facilitate the self-assembly of two HHV polypeptides and / or two HHV fusion proteins. In another embodiment, the oligomerization domain is a dimerization domain that mediates the self-association of three HHV polypeptides and / or three HHV fusion proteins. In another embodiment, the oligomerization domain is a tetramerization domain that mediates the self-association of four HHV polypeptides and / or four HHV fusion proteins. In one embodiment, the trimerization domain is a fibritin motif or a eukaryotic GCN4 transcription factor motif, or a derivative thereof.

[0203] In one embodiment, the oligomerization domain comprises a leucine zipper domain. Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found. Leucine zippers were first identified in several DNA-binding proteins (Landschulz et al., Science, 240:1759, 1988) and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives that dimerize or trimerize. For example, the yeast GCN4 leucine zipper can be used to dimerize a polypeptide of interest (Czerwinski et al., Transfusion, 35(2):137-44, 1995, and O'Shea et al., Science, 243(4890):538-42, 1989). Other examples of leucine zipper domains suitable for the production of soluble multimeric proteins are described in International Patent Application WO 94 / 10308, and the leucine zipper derived from pulmonary surfactant protein D (SPD) is described in Hoppe et al. FEBS Lett. 344:191, 1994. The use of a modified leucine zipper that allows for stable trimerization of modified heterologous proteins is described in Fanslow et al., Semin. Immunol., 6:267, 1994.

[0204] In yet another embodiment, the oligomerization domain is a fibritin trimerization motif, in particular a bacteriophage fibritin trimerization motif, more particularly the fibritin trimerization domain from bacteriophage T4 (also referred to as T4 Foldon or Foldon domain) or the fibritin trimerization domain from phage RB69 or the fibritin trimerization domain from phage AR1, or derivatives thereof. The T4 fibritin trimerization domain and variants thereof are described in U.S. Patent No. 6,911,205, U.S. Patent No. 8,147,843, and WO 01 / 19958, which are hereby incorporated by reference in their entirety.

[0205] Protein complex. In certain embodiments, the HHV polypeptides disclosed herein are present in the antigenic composition as a protein complex. For example, in certain embodiments, HHV gB, gL and gH are present in the antigenic composition as a protein complex. In other embodiments, HHV gH, gL, UL128, UL130 and UL131A polypeptides are present in the antigenic composition as a protein complex. In yet another embodiment, HHV gH, gL and gO polypeptides are present in the antigenic composition as a protein complex.

[0206] Proteins in a protein complex are typically linked by non-covalent protein-protein interactions, including but not limited to hydrogen bonds and salt bridges. Corresponding to the arrangement or shape resulting from the assembly and interaction of individual proteins, the protein complex has a quaternary structure, which is useful for inducing neutralizing antibodies against structural epitopes on the HHV protein complex. In some embodiments, the protein complex as used herein is present on the surface of a herpesvirus, and therefore does not refer to a native protein complex. Rather, the protein complex is formed by incubating individual proteins in vitro to produce a reconstituted protein complex.

[0207] Nucleic Acids, Cloning and Expression Systems. The present disclosure further provides isolated nucleic acids encoding the disclosed monomeric or multimeric HHV polypeptides. The nucleic acids may comprise DNA or RNA and may be wholly or partially synthetic or recombinant. Nucleic acids as defined herein may be used in the context of Reference to a nucleic acid sequence includes DNA molecules having the specified sequence, unless the context requires otherwise, and includes RNA molecules having the specified sequence in which T is replaced by U.

[0208] The disclosure also provides constructs in the form of plasmids, vectors, phagemids, transcription or expression cassettes that contain at least one nucleic acid encoding a monomeric or multimeric HHV fusion or host entry protein, or a portion thereof. The disclosure further provides host cells that contain one or more of the above constructs.

[0209] Also provided are methods for producing monomeric or multimeric HHV polypeptides encoded by these nucleic acids. Monomeric or multimeric HHV polypeptides can be produced using recombinant techniques. Recombinant protein production and expression are well known in the art and can be performed using conventional procedures, such as those disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual (4th Ed. 2012), Cold Spring Harbor Press. For example, expression of fusion proteins can be achieved by culturing recombinant host cells containing nucleic acids encoding monomeric or multimeric HHV polypeptides under appropriate conditions. Following production by expression, monomeric or multimeric HHV polypeptides can be isolated and / or purified using any suitable technique and then used as needed. As discussed herein, under certain conditions, two or more HHV fusion and host cell entry proteins, and optionally one or more HHV accessory proteins, form a protein complex when incubated in vitro.

[0210] Systems for cloning and expressing polypeptides in a variety of different host cells are known in the art. Any protein expression system compatible with the constructs disclosed herein can be used to produce the disclosed monomeric or multimeric HHV polypeptides.

[0211] Suitable vectors can be chosen or constructed to contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.

[0212] A further aspect of the disclosure provides a host cell comprising a nucleic acid as disclosed herein. Yet a further aspect provides a method comprising introducing such a nucleic acid into a host cell. The introduction can use any available technique. For eukaryotic cells, suitable techniques can include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction with retroviruses or other viruses, such as vaccinia virus, or, for insect cells, baculovirus. For bacterial cells, suitable techniques can include calcium chloride transformation, electroporation, and transfection with bacteriophage. These techniques are known in the art (see, e.g., "Current Protocols in Molecular Biology," Ausubel et al. eds., John Wiley & Sons (2010)). Following DNA introduction, a selection method (e.g., antibiotic resistance) can be used to select for cells that contain the vector.

[0213] gH / gL / UL128 / UL130 / UL131A. A recombinant nucleic acid construct was designed to produce an HHV protein complex including gH, gL, UL128, UL130, and UL131A. In one embodiment, the recombinant nucleic acid construct comprises a first nucleic acid encoding an HHV gH polypeptide, a second nucleic acid encoding an HHV gL polypeptide, a third nucleic acid encoding an HHV UL128 polypeptide, a fourth nucleic acid encoding an HHV UL130 polypeptide, and a fifth nucleic acid encoding an HHV UL131A polypeptide. In certain embodiments, when the recombinant nucleic acid is expressed in a host cell, a pentameric complex is formed. In certain embodiments, any of the encoded polypeptides is membrane-associated. It does not include a transmembrane domain or an intracellular domain. In certain embodiments, the recombinant nucleic acid includes one or more internal ribosome entry sites (IRES) to facilitate expression of multiple proteins from a single transcript. In certain embodiments, the recombinant nucleic acid includes a first IRES between the first and second nucleic acids, a second IRES between the second and third nucleic acids, and / or a third IRES between the fourth and fifth nucleic acids. In certain embodiments, the recombinant nucleic acid includes one or more promoter sequences to facilitate expression of an HHV polypeptide. In certain embodiments, the recombinant nucleic acid includes a first promoter operably linked to the first nucleic acid and a second promoter operably linked to the third nucleic acid. In one embodiment, the promoter is a CMV promoter. In certain embodiments, the HHV is a member of the Betaherpesvirinae subfamily, including, for example, HCMV. Non-limiting exemplary embodiments of such recombinant nucleic acids are shown in FIG. 13. Such nucleic acid sequences may include additional nucleic acid sequences, such as a protein purification tag to aid in purification (e.g., a his-tag sequence) or a leader sequence to facilitate secretion from the host cell (e.g., an immunoglobulin kappa light chain leader sequence). In certain embodiments, a leader sequence is inserted in frame with each of the first, second, third, fourth and fifth nucleic acids.

[0214] gH / gL / gO. The recombinant nucleic acid construct is designed to produce an HHV complex comprising gH, gL and gO. In one embodiment, the recombinant nucleic acid construct comprises a first nucleic acid encoding an HHV gH polypeptide, a second nucleic acid encoding an HHV gL polypeptide, and a third nucleic acid encoding an HHV gO polypeptide. In certain embodiments, a trimeric complex is formed when the recombinant nucleic acid is expressed in a host cell. In certain embodiments, none of the encoded polypeptides comprises a transmembrane domain or an intracellular domain. In certain embodiments, the recombinant nucleic acid comprises one or more internal ribosome entry sites (IRES) to facilitate expression of multiple proteins from a single transcript. In certain embodiments, the recombinant nucleic acid comprises an IRES between the first and second nucleic acids. In certain embodiments, the recombinant nucleic acid comprises one or more promoter sequences to facilitate expression of the HHV polypeptides. In certain embodiments, the recombinant nucleic acid comprises a first promoter operably linked to the first nucleic acid and a second promoter operably linked to the third nucleic acid. In one embodiment, the promoter is a CMV promoter. In certain embodiments, the HHV is a member of the Beta-Herpesvirinae subfamily, including, for example, HCMV. An exemplary embodiment of such a recombinant nucleic acid is shown in FIG. 14. To aid in purification, additional nucleic acid sequences, such as a protein purification tag (e.g., a his-tag sequence) or a leader sequence (e.g., an immunoglobulin kappa light chain leader sequence), may be included in such a nucleic acid sequence. In certain embodiments, the leader sequence is inserted in frame with each of the first, second, and third nucleic acids.

[0215] Vaccine Compositions The combinations of monomeric and / or multimeric HHV polypeptides, and the nucleic acids encoding them, described herein provide an improved platform for developing HHV vaccines.

[0216] Thus, one aspect relates to an antigenic composition as described herein, comprising two or more HHV fusion and host cell entry proteins (or nucleic acids encoding them). In certain embodiments, the vaccine comprises a virus-like particle. In certain embodiments, the antigenic composition further comprises at least one pharma- ceutically acceptable excipient, and optionally an adjuvant (hereinafter referred to as a "vaccine composition"). In certain embodiments, the vaccine composition does not comprise an adjuvant.

[0217] In certain embodiments, the vaccine is a nucleic acid vaccine comprising a nucleic acid encoding two or more HHV fusion and host cell entry proteins. In certain embodiments, the nucleic acid vaccine is a DNA vaccine. In other embodiments, the nucleic acid vaccine is an RNA vaccine. In certain embodiments, the nucleic acid vaccine is a viral vector vaccine.

[0218] Pharmaceutically acceptable excipients include, for example, diluents such as starch, microcrystalline cellulose, dicalcium phosphate, lactose, sorbitol, mannitol, sucrose, methyldextrin, binders such as povidone, hydroxypropylmethylcellulose, dihydroxypropylcellulose, and sodium carboxylmethylcellulose. The pharmacopovidone may be selected from disintegrants, such as crospovidone, sodium starch glycolate, croscarmellose sodium, and mixtures of any of the above. The pharmacopovidone may be further selected from lubricants, such as magnesium stearate, calcium stearate, stearic acid, glyceryl behenate, hydrogenated vegetable oils, glyceryl fumarate, and glidants, such as colloidal silicon dioxide, and mixtures thereof. In some embodiments, the pharmacopovidone may be selected from microcrystalline cellulose, starch, talc, povidone, crospovidone, magnesium stearate, colloidal silicon dioxide, sodium dodecyl sulfate, and mixtures of any of the above. The excipient may be intragranular, intergranular, or mixtures thereof.

[0219] The vaccine composition can be formulated as a lyophilized or liquid preparation according to any means suitable in the art. Non-limiting examples of liquid form preparations include solutions, suspensions, syrups, slurries and emulsions. Suitable liquid carriers include any suitable organic or inorganic solvent, preferably in sterile form, such as water, alcohol, saline solution, buffered saline solution, physiological saline solution, dextrose solution, aqueous propylene glycol, etc. After formulation, the vaccine composition can be placed in a sterile container, which can then be sealed and stored at low temperature (e.g., 4°C), or can be lyophilized.

[0220] Vaccine compositions can be formulated in neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the active polypeptide) formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0221] The vaccine composition may optionally include an agent that enhances the prophylactic effect of the vaccine, such as an adjuvant. Adjuvants include any compound(s) that act to increase the immune response to two or more HHV fusion and host cell entry proteins, thereby reducing the amount of protein (or nucleic acid encoding same) required for the vaccine and / or the frequency of administration required to generate a protective immune response. Adjuvants may include, for example, emulsifiers, aqueous adjuvants such as muramyl dipeptide, avridine, aluminum hydroxide, chitosan-based adjuvants and any of a variety of saponins, oils, and other substances known in the art, such as Amphigen, LPS, bacterial cell wall extracts, bacterial DNA, CpG sequences, synthetic oligonucleotides, and combinations thereof (Schijns et al. (2000) Curr. Opin. Immunol. 12:456), Mycobacterial phlei (M. phlei) cell wall extract (MCWE) (U.S. Pat. No. 4,744,984), Mycobacterium phlei DNA (M-DNA), and Mycobacterium phlei cell wall complex (MCC). Compounds that may function as emulsifiers include natural and synthetic emulsifiers, as well as anionic, cationic, and nonionic compounds. Among the synthetic compounds, anionic emulsifiers include, for example, potassium, sodium and ammonium salts of lauric acid and oleic acid, calcium, magnesium and aluminum salts of fatty acids, and organic sulfonates such as sodium lauryl sulfate. Synthetic cationic agents include, for example, cetyltrimethylammonium bromide, and synthetic nonionic agents include, for example, ethylhexyl ammonium bromide. are exemplified by glyceryl esters (e.g., glyceryl monostearate), polyoxyethylene glycol esters and ethers, and sorbitan fatty acid esters (e.g., sorbitan monopalmitate) and their polyoxyethylene derivatives (e.g., polyoxyethylene sorbitan monopalmitate). Natural emulsifiers include gum arabic, gelatin, lecithin, and cholesterol.

[0222] Other suitable adjuvants can be formed by an oil component, such as a single oil, a mixture of oils, a water-in-oil emulsion, or an oil-in-water emulsion. The oil can be a mineral oil, a vegetable oil, or an animal oil. Mineral oil is a liquid hydrocarbon obtained by distillation from petroleum, and is also referred to in the art as liquid paraffin, liquid petrolatum, or white mineral oil. Suitable animal oils include, for example, cod liver oil, halibut oil, menhaden oil, orange roughy oil, and shark liver oil, all of which are commercially available. Suitable vegetable oils include, for example, rapeseed oil, almond oil, cottonseed oil, corn oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil, and the like. Freund's complete adjuvant (FCA) and Freund's incomplete adjuvant (FIA) are two common adjuvants commonly used in vaccine preparations and suitable for use in the present invention. Both FCA and FIA are water-in-mineral oil emulsions, but FCA also contains killed Mycobacterium sp.

[0223] Immunomodulatory cytokines can also be used in the vaccine composition, for example as adjuvants, to enhance the effectiveness of the vaccine, non-limiting examples of such cytokines include interferon alpha (IFN-α), interleukin-2 (IL-2), and granulocyte-macrophage colony-stimulating factor (GM-CSF), or combinations thereof.

[0224] The vaccine composition can be prepared using techniques known to those of skill in the art, including, but not limited to, mixing, sonication, and microfluidization. The adjuvant may comprise from about 10% to about 80% (v / v), more preferably from about 20% to about 50% (v / v), more preferably from about 20% to about 30% (v / v), or any integer value within these ranges, of the vaccine composition.

[0225] The vaccine composition can be administered to any animal, preferably a mammal such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig, etc. Humans are most preferred.

[0226] Administration of the vaccine composition may be by infusion or injection (e.g., intravenous, intramuscular, intradermal, subcutaneous, intrathecal, intraduodenal, intraperitoneal, etc.). The vaccine composition may also be administered intranasally, vaginally, rectally, orally, intratonsillarly, or transdermally. Additionally, the vaccine composition may be administered by a "needle-free" delivery system.

[0227] An effective amount of a vaccine composition may depend on a variety of variables, including, but not limited to, the patient's species, breed, size, height, weight, age, general health, type of formulation, or mode or method of administration. An appropriate effective amount can be routinely determined by one of skill in the art using routine optimization procedures and informed judgment of the expert and other factors apparent to one of skill in the art. A therapeutically effective dose of the vaccine composition described herein is preferably one that provides a therapeutic prophylactic effect without causing substantial toxicity to the subject.

[0228] The vaccine composition can be administered to a patient in any suitable regimen that induces and / or sustains an immune response against two or more HHV fusion and host cell entry proteins. For example, a patient can be administered a vaccine composition as described and exemplified herein as a primary immunization, followed by a secondary or booster immunization to strengthen and / or maintain protective immunity.

[0229] The vaccination regimen, including administration of primary and booster immunizations, can be continued for as long as required by the patient, for example, over several years, or even throughout the patient's lifetime. The frequency of administration of primary vaccines and booster immunizations and the dosage administered can be modified and / or tailored to meet the particular needs of an individual patient, according to any means suitable in the art, as determined by the managing physician.

[0230] Vaccine compositions can be administered prophylactically (prior to exposure to an antigen or pathogen of interest) or therapeutically (post-exposure to an antigen or pathogen of interest).

[0231] Methods of inducing an immune response. In another embodiment, two or more HHV fusion and host cell entry proteins (or nucleic acids encoding them) can be used in a method of inducing an immune response or treating or preventing HHV infection in a subject. The immune response can be induced in a naive subject that has not been previously exposed to HHV. Alternatively, the immune response can be induced in a subject that has been previously exposed to HHV and used to boost an existing immune response.

[0232] In one embodiment, the method of inducing an immune response comprises administering to a subject two or more HHV fusion or host cell entry proteins described herein in an amount sufficient to induce an immune response in the subject against two or more HHV fusion and host cell entry proteins. In another embodiment, the method of inducing an immune response comprises administering to a subject one or more nucleic acid constructs encoding two or more HHV fusion and host cell entry proteins described herein in an amount sufficient to induce an immune response in the subject against two or more HHV polypeptides. In certain embodiments, the method induces an additive antibody response against two or more HHV fusion or host cell entry proteins. In certain embodiments, the method induces a synergistic antibody response against two or more HHV fusion and host cell entry proteins.

[0233] In these methods of inducing an immune response, the immune response can be measured using methods routine in the art, such as those disclosed herein, including, but not limited to, measuring antibody responses, such as antibody responses to HHV proteins, and measuring cell proliferation, including, for example, measuring tritiated thymidine incorporation or cytokine (e.g., IFN-γ) production.

[0234] In certain embodiments, a method for treating or preventing an HHV infection comprises administering to a subject a therapeutically effective amount of two or more HHV polypeptides, or one or more nucleic acid constructs encoding them, as described herein.

[0235] In those methods that include administering two or more HHV fusion and host cell entry proteins, the proteins can be administered simultaneously or sequentially. In certain embodiments, the HHV proteins that make up the antigenic compositions disclosed herein are administered simultaneously (concomitantly), e.g., as part of the same composition or as part of different compositions that are administered simultaneously. In other embodiments, the HHV proteins that make up the antigenic compositions disclosed herein are administered separately (sequentially), e.g., as separate compositions at different times. That is, at least two HHV polypeptides in the composition can be administered simultaneously or separately to achieve the effects disclosed herein. Furthermore, the compositions can be administered in one or more doses to achieve the desired results.

[0236] Typically, the subject is a human. In certain embodiments, the subject is at risk of developing PTLD after transplantation, such as hematopoietic stem cell or solid organ transplantation. In certain embodiments, the subject suffers from a primary immune deficiency syndrome, including, for example, AIDS. In certain embodiments, the subject is at risk of developing nasopharyngeal carcinoma.

[0237] In some embodiments, the subject is administered one or more of anti-CD20 antibody, antiviral therapy, interferon alpha, radiation therapy, and / or chemotherapy simultaneously. CD20 antibody therapy and related biologics are known in the art, as are radiation therapy and chemotherapy. Any of the known therapeutic regimens in these categories can be administered simultaneously to a subject in need thereof.

[0238] Passive immunotherapy and adoptive transfer of cell-mediated immunity. Passive immunotherapy methods for a variety of indications are known in the art and have been utilized in various forms for over 120 years (see Waldman, TA, Nature Medicine, 9(3):269-277, 2003, and Chippeaux et al., J. Venom. Anim. Toxins Incl. Trop. Dis., 21:3, 2013; see also Casadevall et al., Clin. Infect. Dis., 21(1):150-61, 1995). The benefits of passive transfer of antibodies against inflammation, immune deficiencies, acute and chronic autoimmune diseases, and cancer are well established (Kivity et al., Clin. Rev. Allergy Immunol., 38:201-69, 2010, and Toubi et al., Clin. Rev. Allergy Immunol., 29:167-72, 2005). We have demonstrated the multifunctional mechanisms of passively transferred antibodies, including mediation of humoral and cellular immune responses by both the Fab and Fc portions of the antibody, as well as enhanced clearance of pathogens. Passive immunotherapy is also sometimes arbitrarily referred to as cell transfer therapy, immunoglobulin therapy, antiserum therapy, passive transfer, or passive immunity. When immune cells are the immune components or neutralizing agents administered to a subject in need thereof, the method is often referred to as adoptive transfer, adoptive cell therapy (ACT), or adoptive immunotherapy.

[0239] In passive immunotherapy, antibodies (or immunoglobulins) or other immune system components, i.e., substances with antigen-neutralizing activity, such as immune cells, are generated outside the subject to which they are administered, typically generated in a laboratory and / or produced ex vivo by a second subject (or several other subjects). In some embodiments, the immune system components administered to the subject are monoclonal antibodies. In other embodiments, the immune components are polyclonal antibodies. In yet other embodiments, the immune components are one or more immune cells. In all instances, the immune components include antibodies or cells that specifically recognize target antigens, such as those present on HHV fusion and host cell entry proteins.

[0240] Since various combinations of HHV fusion and host cell entry proteins have been shown to induce high titer neutralizing antibodies, it is contemplated that such high titer neutralizing antibodies can be used to passively transfer immunity to HHV. Thus, antibodies generated by a subject immunized with two or more HHV fusion and host cell entry proteins described herein can be harvested and isolated from the subject. A donor subject can be immunized with any combination of fusion or host cell entry proteins of HHV (e.g., EBV, HCMV, HSV-1 or HSV-2, VZV, HHV-6, HHV-7, or KSVH) described herein to induce high titer anti-HHV antibodies.

[0241] In an embodiment of EBV passive immunization or adoptive transfer, a donor subject is immunized, for example, with a tetrameric EBV gp350 protein, and the high titer neutralizing antibodies induced therefrom are utilized for passive transfer of immunity to an acceptor subject that will benefit therefrom. In a further EBV embodiment, a donor subject is immunized, for example, with a trimeric EBV gH / gL protein, and the high titer neutralizing antibodies induced therefrom are utilized for passive transfer of immunity to an acceptor subject that will benefit therefrom. In another exemplary EBV embodiment, a donor subject is immunized, for example, with a trimeric gB protein, and the high titer neutralizing antibodies induced therefrom are utilized for passive transfer of immunity to an acceptor subject that will benefit therefrom. It is used for.

[0242] In an HCMV passive immunization or adoptive transfer embodiment, a donor subject is immunized with, for example, a trimeric HCMV gB protein, and the high titer neutralizing antibodies induced therefrom are used for passive transfer of immunity to an acceptor subject that will benefit therefrom. In a further HCMV embodiment, a donor subject is immunized with, for example, a trimeric HCMV gH / gL protein, and the high titer neutralizing antibodies induced therefrom are used for passive transfer of immunity to an acceptor subject that will benefit therefrom.

[0243] In an embodiment of passive immunization or adoptive transfer of HSV, a donor subject is immunized, for example, with trimeric HSV gB protein, and the high titer neutralizing antibodies induced therefrom are used for passive transfer of immunity to an acceptor subject that will benefit therefrom. In a further HSV embodiment, a donor subject is immunized, for example, with trimeric HSV gH / gL protein, and the high titer neutralizing antibodies induced therefrom are used for passive transfer of immunity to an acceptor subject that will benefit therefrom.

[0244] In a VZV passive immunization or adoptive transfer embodiment, a donor subject is immunized, for example, with a trimeric HSV gB protein, and the high titer neutralizing antibodies induced therefrom are used for passive transfer of immunity to an acceptor subject that will benefit therefrom. In a further VZV embodiment, a donor subject is immunized, for example, with a trimeric VZV gH / gL protein, and the high titer neutralizing antibodies induced therefrom are used for passive transfer of immunity to an acceptor subject that will benefit therefrom.

[0245] In KSHV passive immunization or adoptive transfer embodiments, a donor subject is immunized, for example, with a trimeric KSHV gB protein, and the resulting induced high titer neutralizing antibodies are used to passively transfer immunity to an acceptor subject that will benefit therefrom. In further KSHV embodiments, a donor subject is immunized, for example, with a trimeric KSHV gH / gL protein, and the resulting induced high titer neutralizing antibodies are used to passively transfer immunity to an acceptor subject that will benefit therefrom.

[0246] Immunization with two or more HHV fusion and host cell entry proteins may be simultaneous, multiple, or staggered, so long as the desired neutralizing activity is obtained in the donor subject. The antibodies can then be administered to another subject in need thereof. Alternatively, high titer neutralizing antibodies against HHV fusion or host cell entry proteins can be obtained from one or more blood, serum or plasma samples selected for high titer antibodies. In certain embodiments, one or more blood, serum or plasma samples are obtained from a human donor.

[0247] The immune components may also be synthetically produced in tissue culture or obtained by animals as monoclonal antibodies, or may be obtained from another donor subject that is either seropositive for immune components that specifically recognize the desired antigen or has become seropositive due to exposure to the antigen. In certain embodiments, the donor subject has a high degree of reactivity to the antigen, i.e., high concentrations or titers of antigen-neutralizing immune components (e.g., antibodies or immune cells). These immune components are then removed from the donor subject or obtained from tissue culture or animals, purified or otherwise manipulated in the laboratory as necessary to avoid potential graft-versus-host reactions or other adverse reactions, and then administered to a subject in need thereof.

[0248] Steps for carrying out a passive immunotherapy or adoptive transfer protocol or methodology, in one embodiment, include first identifying a donor subject with high neutralizing activity against HHV. In certain embodiments, high titer anti-HHV antibodies or immune cells are obtained from blood, serum and / or plasma samples collected from a donor subject. These immune components are then transferred to a second subject in need thereof to induce an immune protective effect in the second subject, thereby preventing or treating HHV infection. The second subject may be infected with HHV or susceptible to HHV infection. The antibodies may optionally be extracted and / or purified prior to administration to the subject in need thereof. Additionally, in other embodiments, optionally the donor subject is histocompatible with the subject in need thereof such that the blood, serum and / or plasma may be administered to the subject in need thereof. In some embodiments, the blood, serum and / or plasma is obtained from a human donor.

[0249] The term "high titer" as used herein refers to an antibody having a titer specific for a desired HHV polypeptide that is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 25-fold, or in some embodiments, 30-fold greater than the average titer from unselected plasma, serum, or blood samples from the general population of donor subjects containing antibodies of the same specificity. In certain embodiments, the donor subject has been exposed to an HHV polypeptide antigen and is not seronegative or naive. In certain embodiments, the donor subject has been administered two or more HHV fusion and host cell entry proteins to generate a high titer antibody response in the donor subject(s). High titer antibodies can be identified or selected using methods described herein (e.g., Raji B cell neutralization assay or HeLa cell neutralization assay) or any method known in the art. Antibody titers can be determined by various art-recognized screening methods or by the methods disclosed herein. In one embodiment, high titer antibodies are identified or selected using, for example, the Raji B cell neutralization assay or the HeLa cell neutralization assay described in the Examples of the present application. In certain embodiments, the HeLa cell neutralization assay comprises infecting HeLa cells with a labeled EBV (e.g., an EBV having a fluorescent label such as green fluorescent protein) to generate EBV-infected HeLa cells, incubating a blood, plasma or serum sample with the EBV-infected HeLa cells, analyzing the neutralizing activity of the blood, plasma or serum sample (e.g., using flow cytometry or an ELISpot assay), and, optionally, measuring the IC of the blood, plasma or serum sample. 50 and calculating

[0250] The subject in need of treatment is a naive (seronegative to HHV), immunocompromised, or susceptible to infection, or a subject already infected with one or more HHV. In certain embodiments, the subject is administered high titer anti-EBV antibodies, and the subject is at risk of developing post-transplant lymphoproliferative disorder (PTLD) after hematopoietic stem cell or solid organ transplantation, or has or is at risk of developing nasopharyngeal carcinoma (NPC), Burkitt's lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastric cancer, severe infectious mononucleosis, chronic active EBV infection, multiple sclerosis, systemic lupus erythematosus, or rheumatoid arthritis. In certain embodiments, the subject is at risk of developing PTLD, for example, after hematopoietic stem cell or solid organ transplantation. In certain embodiments, the subject is at risk of developing nasopharyngeal carcinoma.

[0251] In another embodiment, the subject is administered high titer anti-HCMV antibodies and the subject is a pregnant woman, a transplant patient, a patient immunosuppressed during chemotherapy or radiation therapy, or a patient infected with human immunodeficiency virus (HIV). In another embodiment, the subject is administered high titer anti-HSV-1 or HSV-2 antibodies and the subject is at risk of developing encephalitis caused by HSV-1 or HSV-2 infection, or is a pregnant woman with active HSV-2 or HSV-1 infection and / or HSV encephalitis. In another embodiment, the subject is administered high titer anti-ZVZ antibodies and the subject is at risk of developing herpes zoster (shingles) or chickenpox (varicella). In a further embodiment, the subject is administered high titer anti-HSV-1 or HSV-2 antibodies and the subject is at risk of developing encephalitis caused by HSV-1 or HSV-2 infection, or is a pregnant woman with active HSV-2 or HSV-1 infection and / or HSV encephalitis. A monovalent anti-KSHV antibody is administered to the subject, the subject being at risk of developing KSHV-associated Kaposi's sarcoma, primary effusion lymphoma, multicentric Castleman's disease, KSHV-associated inflammatory cytokine syndrome, or KSHV immune reconstitution inflammatory syndrome.

[0252] In another embodiment, the subject in need of treatment is concurrently receiving anti-viral therapy, an anti-CD20 antibody composition, interferon-alpha, radiation therapy, and / or chemotherapy.

[0253] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, shall control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. [Section 1] The following human herpesvirus polypeptides: a glycoprotein B (gB) polypeptide comprising the extracellular domain of human herpesvirus gB; glycoprotein 350 (gp350) polypeptide comprising the extracellular domain of human herpesvirus gp350; glycoprotein L (gL) polypeptide, and a glycoprotein H (gH) polypeptide comprising the extracellular domain of human herpesvirus gH; or one or more nucleic acids encoding said at least two human herpesvirus polypeptides, When the antigenic composition comprises a gH polypeptide and a gL polypeptide in the form of a gH / gL heterodimer, the antigenic composition further comprises the gp350 polypeptide and / or the gB polypeptide. [Section 2] Item 2. The composition according to Item 1, wherein the human herpes virus is human cytomegalovirus (HCMV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human herpes virus 6 (HHV-6), human herpes virus 7 (HHV-7), or Kaposi's sarcoma-associated herpes virus (KSHV). [Section 3] 3. The composition of claim 1 or 2, wherein the gB polypeptide, the gp350 polypeptide, and / or the gH polypeptide, when present, each further comprises the corresponding gB, gp350, and / or gH intracellular domain. [Section 4] The composition of claim 3, wherein the extracellular domain is fused to the intracellular domain via a polypeptide linker sequence. [Section 5] Item 5. The composition according to item 4, wherein the polypeptide linker sequence is about 6 to about 70 amino acids in length, or the peptide linker is about 15 amino acids in length. [Section 6] The composition of any one of the preceding claims, wherein at least two of the at least two human herpesvirus polypeptides form a fusion protein, the fusion protein optionally comprising a polypeptide linker sequence linking the at least two human herpesvirus polypeptides. [Section 7] The composition of any one of the preceding claims, wherein at least three of the at least two human herpesvirus polypeptides form a fusion protein, the fusion protein optionally comprising one or more polypeptide linker sequences linking the at least three human herpesvirus polypeptides. [Section 8] The composition of any one of the preceding claims, wherein the at least two human herpesvirus polypeptides comprise the gB polypeptide and one or more of the gp350 polypeptide, the gL polypeptide, and the gH polypeptide, and wherein the gB polypeptide is monomeric or multimeric. [Section 9] The composition of any one of the preceding claims, wherein the at least two Human Herpesvirus polypeptides comprise the gB polypeptide, the gL polypeptide, and the gH polypeptide. [Section 10] The composition of claim 9, wherein the gB polypeptide is a monomer, dimer, or trimer, and the gL polypeptide and the gH polypeptide form a gH / gL heterodimer, and optionally, the gB polypeptide is a monomer, and the gH polypeptide and the gL polypeptide form a monomeric gH / gL heterodimer. [Section 11] The composition of any one of the preceding clauses, wherein the at least two Human Herpesvirus polypeptides are HCMV polypeptides. [Section 12] The composition of claim 11, wherein the composition further comprises an HCMV glycoprotein O (gO) polypeptide. [Section 13] 13. The composition of claim 11 or 12, wherein the composition further comprises an HCMV unique long 128 (UL128) polypeptide, an HCMV unique long 130 (UL130) polypeptide, an HCMV unique long 131A (UL131A) polypeptide, and optionally an HCMV glycoprotein M (gM) polypeptide and / or an HCMV glycoprotein N (gN) polypeptide. [Section 14] Item 11. The composition according to any one of Items 1 to 10, wherein the at least two human herpesvirus polypeptides are human EBV polypeptides. [Section 15] 15. The composition of claim 14, wherein the at least two human herpesvirus polypeptides comprise the gp350 polypeptide and the gB polypeptide. [Section 16] 16. The composition of claim 15, wherein the gp350 polypeptide is a monomeric, dimeric, trimeric or tetrameric gp350 and the gB polypeptide is a monomeric, dimeric or trimeric gB, optionally, the gp350 polypeptide is a monomeric or tetrameric and the gB polypeptide is a trimer. [Section 17] 15. The composition of claim 14, wherein the at least two human herpesvirus polypeptides comprise the gp350 polypeptide, the gH polypeptide, and the gL polypeptide. [Section 18] 18. The composition of claim 17, wherein the gp350 polypeptide is monomeric and the gH polypeptide and the gL polypeptide form a monomeric gH / gL heterodimer, or wherein the gp350 polypeptide is tetrameric and the gH polypeptide and the gL polypeptide form a trimeric gH / gL heterodimer. [Section 19] 15. The composition of claim 14, wherein the at least two human herpesvirus polypeptides comprise the gB polypeptide, the gH polypeptide, and the gL polypeptide. [Section 20] 20. The composition of claim 19, wherein the gB polypeptide is a trimeric gB, and the gH polypeptide and the gL polypeptide form a monomeric or trimeric gH / gL heterodimer. [Section 21] 15. The composition of claim 14, wherein the at least two human herpesvirus polypeptides comprise a monomeric gp350 polypeptide, a trimeric gB polypeptide, and a monomeric gH / gL heterodimer formed by the gH polypeptide and the gL polypeptide. [Section 22] 15. The composition of claim 14, wherein the at least two human herpesvirus polypeptides comprise a tetrameric gp350 polypeptide, a trimeric gB polypeptide, and a trimeric gH / gL heterodimer formed by the gH polypeptide and the gL polypeptide. [Section 23] 23. The composition according to any one of items 14 to 22, further comprising a human EBV glycoprotein 42 (gp42) polypeptide, a BDFL2 polypeptide, and / or a human EBV BMRF-2 polypeptide. [Section 24] Item 11. The composition according to any one of Items 1 to 10, wherein the at least two human herpesvirus polypeptides are human HSV-1 polypeptides or human HSV-2 polypeptides. [Section 25] 25. The composition of claim 24, wherein the at least two human herpesvirus polypeptides comprise the gH polypeptide, the gL polypeptide, and the gB polypeptide, wherein the gH polypeptide and the gL polypeptide are monomeric, dimeric, trimer, or tetrameric, and the gB polypeptide is monomeric, dimeric, or trimer, and optionally, the at least two human herpesvirus polypeptides comprise a monomeric gH / gL heterodimer formed by the gH polypeptide and the gL polypeptide, and a monomeric gB polypeptide. [Section 26] 26. The composition of claim 25, further comprising an HSV-1 or HSV-2 glycoprotein D (gD) polypeptide, wherein the gD polypeptide is a monomer, dimer, trimer, or tetramer. [Section 27] Item 11. The composition according to any one of Items 1 to 10, wherein the at least two human herpesvirus polypeptides are human VZV polypeptides. [Section 28] 28. The composition of claim 27, wherein the at least two herpesvirus polypeptides comprise the gH polypeptide, the gL polypeptide, and the gB polypeptide, wherein the gH polypeptide and the gL polypeptide are monomeric, dimeric, trimer, or tetrameric, and the gB polypeptide is monomeric, dimeric, or trimer, and optionally, the at least two human herpesvirus polypeptides comprise a monomeric gH / gL heterodimer formed by the gH polypeptide and the gL polypeptide, and a monomeric gB polypeptide. [Section 29] 30. The composition of claim 28, further comprising one or more of a human VZV glycoprotein C (gC) polypeptide, a human glycoprotein E (gE) polypeptide, and a human VZV glycoprotein I (gI) polypeptide. [Section 30] Item 11. The composition according to any one of Items 1 to 10, wherein the at least two human herpesvirus polypeptides are human HHV-6 polypeptide or human HHV-7 polypeptide. [Section 31] 31. The composition of claim 30, wherein the at least two herpesvirus polypeptides comprise the gH polypeptide, the gL polypeptide, and the gB polypeptide, wherein the gH polypeptide and the gL polypeptide are monomeric, dimeric, trimer, or tetrameric, and the gB polypeptide is monomeric, dimeric, or trimer, and optionally, the at least two human herpesvirus polypeptides comprise a monomeric gH / gL heterodimer formed by the gH polypeptide and the gL polypeptide, and a monomeric gB polypeptide. [Section 32] Item 11. The composition of any one of items 1 to 10, wherein the at least two human herpesvirus polypeptides are human KSHV polypeptides. [Section 33] 33. The composition of claim 32, wherein the at least two human herpesvirus polypeptides comprise the gH polypeptide, the gL polypeptide, and the gB polypeptide, wherein the gH polypeptide and the gL polypeptide are monomeric, dimeric, trimer, or tetrameric, and the gB polypeptide is monomeric, dimeric, or trimer, and optionally, the at least two human herpesvirus polypeptides comprise a monomeric gH / gL heterodimer formed by the gH polypeptide and the gL polypeptide, and a monomeric gB polypeptide. [Section 34] 34. The composition of claim 33, further comprising one or more of a human KSHV glycoprotein M (gM) polypeptide, a human KSHV glycoprotein N (gN) polypeptide, a human KSHV open reading frame 68 (ORF68) polypeptide, and a human KSHV glycoprotein K8.1 polypeptide. [Section 35] The composition of any one of the preceding claims, wherein the one or more nucleic acids are in a viral vector capable of expressing the at least two human herpesvirus polypeptides. [Section 36] The composition of any one of the preceding claims, further comprising a pharma- ceutically acceptable excipient and / or adjuvant. [Section 37] A method of preventing or treating a human herpes virus infection in a subject, comprising administering to the subject a therapeutically effective amount of a composition of any one of the preceding clauses. [Section 38] A method for inducing immunity against human herpesvirus in a subject, comprising administering a therapeutically effective amount of the composition according to any one of items 1 to 36 to the subject. [Section 39] 39. The method of claim 37 or 38, wherein the subject is at risk of developing post-transplant lymphoproliferative disorder (PTLD) after hematopoietic stem cell or solid organ transplantation and suffers from a primary immunodeficiency syndrome. [Section 40] 40. The method of any one of paragraphs 37 to 39, wherein the at least two Human Herpesvirus polypeptides in the composition are administered sequentially or simultaneously. [Section 41] 1. A recombinant nucleic acid construct encoding a human herpesvirus polypeptide, the nucleic acid construct comprising: a first nucleic acid molecule encoding a human herpesvirus gL polypeptide; a second nucleic acid molecule encoding a human herpesvirus gH polypeptide; a third nucleic acid molecule encoding a human herpesvirus UL128 polypeptide; a fourth nucleic acid molecule encoding a human herpesvirus UL130 polypeptide; and a fifth nucleic acid molecule encoding a human herpesvirus UL131A polypeptide, wherein said polypeptides form a pentameric complex when expressed from said nucleic acid construct in a host cell, and wherein none of said encoded polypeptides comprises a transmembrane domain sequence or an intracellular domain sequence. [Section 42] 42. The recombinant nucleic acid construct of claim 41, further comprising a first promoter operably linked to the first nucleic acid and a second promoter operably linked to the third nucleic acid molecule. [Section 43] 43. The recombinant nucleic acid construct of claim 41 or 42, further comprising a first internal ribosome entry site (IRES) located between the first nucleic acid molecule and the second nucleic acid molecule, a second IRES located between the third nucleic acid molecule and the fourth nucleic acid molecule, and a third IRES located between the fourth nucleic acid molecule and the fifth nucleic acid molecule. [Section 44] Item 44. The recombinant nucleic acid construct according to any one of Items 41 to 43, further comprising a first, second, third, fourth and fifth nucleotide sequence encoding an IgG kappa light chain leader peptide, wherein the first, second, third, fourth and fifth nucleotide sequences encoding the IgG kappa light chain leader peptide are in frame with the first nucleic acid molecule, the second nucleic acid molecule, the third nucleic acid molecule, the fourth nucleic acid molecule and the fifth nucleic acid molecule, respectively. [Section 45] A recombinant nucleic acid construct encoding a human herpesvirus polypeptide, the nucleic acid construct comprising a first nucleic acid molecule encoding a human herpesvirus gL polypeptide, a second nucleic acid molecule encoding a human herpesvirus gH polypeptide, and a third nucleic acid molecule encoding a human herpesvirus gO polypeptide. [Section 46] 46. ​​The recombinant nucleic acid construct of Paragraph 45, further comprising a first promoter operably linked to the first nucleic acid and a second promoter operably linked to the third nucleic acid molecule. [Section 47] 47. The recombinant nucleic acid construct of paragraph 45 or 46, further comprising an internal ribosome entry site (IRES) located between the first nucleic acid molecule and the second nucleic acid molecule. [Section 48] Item 48. The recombinant nucleic acid construct according to any one of Items 45 to 47, further comprising a first, second and third nucleotide sequence encoding an IgG kappa light chain leader peptide, wherein the first, second and third nucleotide sequences encoding the IgG kappa light chain leader peptide are in frame with the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule, respectively. [Section 49] Item 49. The recombinant nucleic acid construct according to any one of Items 41 to 48, wherein the human herpes virus is human cytomegalovirus (HCMV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human herpes virus 6 (HHV-6), human herpes virus 7 (HHV-7), or Kaposi's sarcoma-associated herpes virus (KSHV). [Section 50] A protein complex formed by expression of the recombinant nucleic acid construct according to any one of items 41 to 49 in a host cell. [Section 51] 1. A method for passively transferring immunity to Epstein-Barr Virus (EBV), comprising administering to a subject in need thereof immune cells or high titer anti-EBV immunoglobulin, the immune cells or high titer anti-EBV immunoglobulin being obtained from one or more blood, plasma or serum samples selected for the high titer anti-EBV immunoglobulin. [Section 52] 52. The method of claim 51, wherein the one or more blood, plasma or serum samples are human blood, plasma or serum samples. [Section 53] 53. The method of claim 51 or 52, wherein the titer of the high titer anti-EBV immunoglobulin is up to 25 times higher than the average titer of anti-EBV immunoglobulin obtained from unselected blood, plasma or serum samples. [Section 54] 54. The method according to any one of items 51 and 53, wherein the titer of the high titer anti-EBV immunoglobulin is 4-fold to 25-fold or 10-fold to 20-fold higher than the average titer of anti-EBV immunoglobulin obtained from unselected blood, plasma or serum samples. [Section 55] The method according to any one of claims 51 to 54, wherein the blood, plasma or serum sample is obtained from a donor immunized with the composition according to any one of claims 1 to 10 and 14 to 23, or with a multimeric EBV protein involved in mediating EBV binding, fusion and entry into host cells, such as tetrameric gp350, trimeric gH / gL or trimeric gB. [Section 56] 56. The method according to any one of items 51 to 55, wherein the subject is at risk of developing post-transplant lymphoproliferative disorder (PTLD) after hematopoietic stem cell or solid organ transplantation, or has or is at risk of developing nasopharyngeal carcinoma (NPC), Burkitt's lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastric cancer, severe infectious mononucleosis, chronic active EBV infection, multiple sclerosis, systemic lupus erythematosus, or rheumatoid arthritis. [Section 57] 57. The method of claim 56, wherein the subject is at risk of developing post-transplant lymphoproliferative disorder (PTLD) following hematopoietic stem cell or solid organ transplantation and suffers from a primary immunodeficiency syndrome. [Section 58] 58. The method of any one of clauses 51 to 57, wherein the subject is seronegative for EBV. [Section 59] Item 59. The method according to any one of items 51 to 58, wherein the subject is simultaneously receiving one or more of the following: administration of an anti-CD20 antibody, antiviral therapy, administration of interferon alpha, radiation therapy, and chemotherapy. [Section 60] Prior to administering the high titer anti-EBV immunoglobulin to the subject, identifying a blood, plasma or serum sample obtained from one or more human subjects that has high EBV neutralizing activity; collecting high titer anti-EBV immunoglobulins from said blood, plasma or serum sample having high EBV neutralizing activity; 60. The method according to any one of items 51 to 59, further comprising: [Section 61] The identifying step comprises subjecting the blood, plasma or serum sample to a Raji B cell neutralization assay and / or a HeLa cell neutralization assay, and optionally, the blood, plasma or serum sample is compared to a mean IC50 of unselected blood, plasma or serum samples. 50 IC 4-25 or 10-20 times higher than 50 61. The method of claim 60, wherein the blood, plasma or serum sample is identified as having high EBV neutralizing activity if it has the following: [Section 62] Prior to administering the high titer anti-EBV immunoglobulin to the subject, administering to one or more human donor subjects an immunogenic composition comprising at least two of the following EBV polypeptides: an EBV gp350 polypeptide, an EBV gH / gL heterodimer comprising an EBV gH polypeptide and an EBV gL polypeptide, and an EBV gB polypeptide, in an amount sufficient to produce high titer anti-EBV immunoglobulin; collecting said high titer anti-EBV immunoglobulin from said one or more human donor subjects; Item 62. The method according to any one of items 51 to 61, further comprising: [Section 63] 63. The method of claim 62, wherein the EBV gp350 polypeptide is a monomer, dimer, trimer or tetramer, the EBV gB polypeptide is a monomer, dimer or trimer, and the gH / gL heterodimer is a monomer, dimer, trimer or tetramer. [Section 64] The HeLa cell neutralization assay comprises infecting HeLa cells with GFP-labeled EBV to generate EBV-infected HeLa cells; incubating the blood, plasma or serum sample with the EBV-infected HeLa cells; analyzing the neutralizing activity of the blood, plasma or serum sample by flow cytometry or ELISpot assay; and optionally, measuring the IC 50 62. The method of claim 61, comprising: [Section 65] A method for passively transferring immunity to human cytomegalovirus (HCMV), comprising administering to a subject in need thereof immune cells or high titer anti-HCMV immunoglobulin, the immune cells or high titer anti-HCMV immunoglobulin being obtained from one or more blood, plasma or serum samples selected against the high titer anti-HCMV immunoglobulin. [Section 66] 66. The method of claim 65, wherein the one or more blood, plasma or serum samples are human blood, plasma or serum samples. [Section 67] 67. The method of claim 65 or 66, wherein the blood, plasma or serum sample is obtained from a donor immunized with the composition of any one of claims 1 to 13, or with a multimeric HCMV protein involved in mediating HCMV binding, fusion and entry into host cells, such as trimeric gH / gL or trimeric gB. [Section 68] Item 68. The method according to any one of items 65 to 67, wherein the subject at risk of HCMV infection is a pregnant woman, a transplant patient, a patient immunosuppressed by chemotherapy or radiotherapy, or a patient infected with human immunodeficiency virus (HIV). [Section 69] Prior to administering the high titer anti-HCMV immunoglobulin to the subject, administering to one or more human donor subjects an immunogenic composition comprising at least two of an HCMV gB polypeptide, an HCMV gH / gL heterodimer comprising an HCMV gH polypeptide and an HCMV gL polypeptide, an HCMV glycoprotein O (gO) polypeptide, an HCMV UL128 polypeptide, an HCMV UL130 polypeptide, and an HCMV unique UL131A polypeptide in an amount sufficient to generate a high titer anti-HCMV immunoglobulin response in said subjects; collecting said high titer anti-HCMV immunoglobulin from said one or more human donor subjects; Item 69. The method according to any one of items 65 to 68, further comprising: [Section 70] 70. The method of claim 69, wherein the HCMV gB polypeptide is a monomer, dimer, or trimer and the gH / gL heterodimer is a monomer, dimer, trimer, or tetramer. [Section 71] A method for passively transferring immunity to herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2), comprising administering immune cells or anti-HSV-1 and / or anti-HSV-2 hyperimmune globulin to a subject in need thereof, wherein the immune cells or anti-HSV-1 or anti-HSV-2 hyperimmune globulin are obtained from one or more blood, plasma or serum samples selected for anti-HSV-1 or anti-HSV-2 hyperimmune globulin. [Section 72] 72. The method of claim 71, wherein the one or more blood, plasma or serum samples are human blood, plasma or serum samples. [Section 73] 73. The method of any one of claims 71 or 72, wherein the blood, plasma or serum sample is obtained from a donor immunized with the composition of any one of claims 1 to 10 and 24 to 26, or with a multimeric protein of HSV-1 or HSV-2 involved in mediating binding, fusion and entry of HSV-1 or HSV-2 into the host, such as trimeric gH / gL or trimeric gB. [Section 74] Item 74. The method according to any one of Items 71 to 73, wherein the subject is at risk of developing encephalitis caused by HSV-1 or HSV-2 infection, or the subject is a pregnant woman with active HSV-2 or HSV-1 infection and / or herpes simplex virus (HSV)-associated encephalitis. [Section 75] Prior to administering the anti-HSV-1 or HSV-2 hyperimmune globulin to the subject, administering to one or more human donor subjects an immunogenic composition comprising at least two of an HSV-1 or HSV-2 glycoprotein D (gD) polypeptide, an HSV-1 or HSV-2 gH / gL heterodimer comprising an HSV-1 or HSV-2 gH polypeptide and an HSV-1 or HSV-2 gL polypeptide, and an HSV-1 or HSV-2 gB polypeptide in an amount sufficient to generate anti-HSV-1 or HSV-2 high titer immunoglobulins; collecting said anti-HSV-1 and / or anti-HSV-2 hyperimmune globulin from said one or more human donor subjects; Item 75. The method according to any one of items 71 to 74, further comprising: [Section 76] 76. The method of claim 75, wherein the HSV-1 or HSV-2 gB polypeptide is a monomer, dimer, or trimer, and the HSV-1 or HSV-2 gH / gL heterodimer is a monomer, dimer, trimer, or tetramer. [Section 77] A method for passively transferring immunity to Varicella-Zoster Virus (VZV), comprising administering immune cells or high titer anti-VZV immunoglobulin to a subject in need thereof, wherein the immune cells or high titer anti-VZV immunoglobulin are obtained from one or more blood, plasma or serum samples selected for the high titer anti-VZV immunoglobulin. [Section 78] 78. The method of claim 77, wherein the one or more blood, plasma or serum samples are human blood, plasma or serum samples. [Section 79] The method according to claim 77 or 78, wherein the blood, plasma or serum sample is obtained from a donor subject immunized with the composition according to any one of claims 1 to 10 and 27 to 29, or with a multimeric VZV protein involved in mediating VZV binding, fusion and entry into host cells, such as trimeric gH / gL or trimeric gB. [Section 80] 80. The method of any one of paragraphs 77 to 79, wherein the subject is at risk of developing herpes zoster (shingles) or chickenpox (varicella). [Section 81] Prior to administering the high titer anti-VZV immunoglobulin to the subject, administering to one or more human donor subjects an immunogenic composition comprising at least two of a VZV gH / gL heterodimer comprising a VZV gH polypeptide and a VZV gL polypeptide, a VZV gB polypeptide, a VZV glycoprotein C (gC) polypeptide, a VZV glycoprotein E (gE) polypeptide, and a VZV glycoprotein I (gI) polypeptide, in an amount sufficient to generate high titer anti-VZV immunoglobulin; collecting said high titer anti-VZV immunoglobulin from said one or more human donor subjects; Item 81. The method according to any one of items 77 to 80, further comprising: [Section 82] 82. The method of claim 81, wherein the VZV gB polypeptide is a monomer, dimer, or trimer, and the VZV gH / gL heterodimer is a monomer, dimer, trimer, or tetramer. [Section 83] A method for passively transferring immunity to human herpesvirus 6 (HHV-6) or human herpesvirus 7 (HHV-7), comprising administering immune cells or anti-HHV-6 or anti-HHV-7 hyperimmune globulin to a subject in need thereof, wherein the immune cells or anti-HHV-6 or anti-HHV-7 hyperimmune globulin are obtained from one or more blood, plasma or serum samples selected for the anti-HHV-6 or anti-HHV-7 hyperimmune globulin. [Section 84] 84. The method of claim 83, wherein the one or more blood, plasma or serum samples are human blood, plasma or serum samples. [Section 85] The method according to item 83 or 84, wherein the blood, plasma or serum sample is obtained from a donor subject immunized with the composition according to any one of items 1 to 10 and items 30 to 31, or with a multimeric protein of HHV-6 or HHV-7 involved in mediating binding, fusion and entry of HHV-6 or HHV-7 into host cells, such as trimeric gH / gL or trimeric gB. [Section 86] Prior to the step of administering the anti-HHV-6 or anti-HHV-7 hyperimmune globulin to the subject, administering to one or more human donor subjects an immunogenic composition comprising an HHV-6 or HHV-7 gH / gL heterodimer, comprising an HHV-6 or HHV-7 gH polypeptide and an HHV-6 or HHV-7 gL polypeptide, and an HHV-6 or HHV-7 gB polypeptide, in an amount sufficient to generate anti-HHV-6 or anti-HHV-7 high titer immunoglobulin; collecting said anti-HHV-6 or anti-HHV-7 hyperimmune globulin from said one or more human donor subjects; 86. The method according to any one of items 83 to 85, further comprising: [Section 87] 87. The method of claim 86, wherein the HHV-6 or HHV-7 gB polypeptide is a monomer, dimer, or trimer, and the HHV-6 or HHV-7 gH / gL heterodimer is a monomer, dimer, trimer, or tetramer. [Section 88] A method for passively transferring immunity to Kaposi's sarcoma herpesvirus (KSHV), comprising administering immune cells or high titer anti-KSHV immunoglobulin to a subject in need thereof, wherein the immune cells or high titer anti-KSHV immunoglobulin are obtained from one or more blood, plasma or serum samples selected for the high titer anti-KSHV immunoglobulin. [Section 89] 89. The method of claim 88, wherein the one or more blood, plasma or serum samples are human blood, plasma or serum samples. [Section 90] 89. The method of claim 88 or 89, wherein the blood, plasma or serum sample is obtained from a donor immunized with the composition of any one of claims 1 to 10 and 32 to 34, or with a multimeric KSHV protein involved in mediating KSHV binding, fusion and entry into host cells, such as trimeric gH / gL or trimeric gB. [Section 91] 91. The method of any one of claims 88 to 90, wherein the subject is at risk of developing KSHV-associated Kaposi's sarcoma, primary effusion lymphoma, multicentric Castleman disease, KSHV-associated inflammatory cytokine syndrome, or KSHV immune reconstitution inflammatory syndrome. [Section 92] Prior to administering the high titer anti-KSHV immunoglobulin to the subject, administering to one or more human donor subjects an immunogenic composition comprising at least two of a KSHV gH / gL heterodimer comprising a KSHV gH polypeptide and a KSHV gL polypeptide, a KSHV gB polypeptide, a KSHV gM polypeptide, a KSHV gN polypeptide, a KSHV ORF68 polypeptide, and a KSHV K8.1 polypeptide in an amount sufficient to generate high titer anti-KSHV immunoglobulin; collecting said high titer anti-KSHV immunoglobulin from said one or more human donor subjects; Item 88 to 91, further comprising the method of any one of items 88 to 91. [Section 93] 93. The method of paragraph 92, wherein the KSHV gB polypeptide is a monomer, dimer, or trimer and the gH / gL heterodimer is a monomer, dimer, trimer, or tetramer. EXAMPLES

[0254] 1. Epstein-Barr virus (EBV) Example 1.1 - Production of EBV gH and EBV gL polypeptides For recombinant production of EBV gH and gL polypeptides, the coding sequences of EBV gH and gL were downloaded from the NCBI website from reference sequence NC_009334.1, which includes EBV gH nucleotides 129454-131574 and EBV gL nucleotides 98500-98913. The gL sequence encoding amino acids 23-137 was used, replacing the signal peptide at amino acids 1-22 with the IgGκ leader sequence. The gH sequence encoding amino acids corresponding to amino acids 19-678 was linked to the 3' end of the gL sequence, separated by a 15 amino acid linker (Gly4Ser)3 (SEQ ID NO:3) sequence (see representative schematic in FIG. 1). The Foldon trimerization domain coding sequence from T4 phage fibritin (e.g., U.S. Pat. No. 6,911,205; U.S. Pat. No. 8,147,843; and WO 01 / 19958) was ligated to the 3' end of gH followed by a His6 (SEQ ID NO: 49) coding sequence. DNA encoding trimeric gH / gL was synthesized and cloned into vector pOptiVEV (Invitrogen, Carlsbad, Calif., USA) and the sequence verified by sequencing. A monomeric EBV gH / gL construct was generated by PCR amplification of EBV gH / gL without the Foldon trimerization coding sequence and cloned into pOptiVEV. The sequence was verified by sequencing.

[0255] Chinese hamster ovary (CHO) cells (strain DG44, Invitrogen, Carlsbad, CA, USA) were transfected with the resulting pOptiVEV-gH / gL constructs and positive cells were selected with gradually increasing concentrations of methotrexate (MTX) up to 4 μM. Selected CHO cells were loaded into "Fibercell" cartridges (FiberCell Systems, Frederick, MD, USA) for protein production. The supernatant was concentrated and purified using cobalt affinity purification (Thermo Fisher Scientific, Waltham, MA, USA). Size-exclusion chromatography was performed using a Sephadex™ G200 column or a Superose™ 6 Increase 10 / 300 GL column (GE Healthcare, Little Chalfont, UK). The recombinant protein was further purified by

[0256] Anti-His6 (SEQ ID NO: 49) mAb or anti-EBV gH / gL mAb (clone E1D1, a gift from Dr. LM Hutt-Fletcher, Louisiana State University Health Sciences Center, Shreveport, LA, USA) were used under reducing conditions to disrupt native oligomers. Western blot analysis of the trimeric gH / gL polypeptide using ELISA kits revealed a molecular weight (MW) band of approximately 90 kilodaltons (kDa), consistent with the predicted size of monomeric gH / gL (Figure 2A). Under non-reducing conditions, a MW band of approximately 270 kDa was observed, consistent with the predicted size of trimeric gH / gL (Figure 2A).

[0257] Example 1.2 - Production of EBV gB Polypeptide To recombinantly produce EBV gB polypeptide, the coding sequence of EBV gB corresponding to reference sequence NC_009334.1, nucleotides 157775-160348 was downloaded from the NCBI website. The sequence encoding the extracellular domain of EBV gB (amino acids 23-732 of wild-type EBV) was used to design a construct for trimeric gB expression. The signal peptide corresponding to amino acids 1-22 was replaced by an IgGκ leader sequence, and the coding sequence for the furin cleavage site between amino acids 427 (L) and 434 (A) (RRRRD) (SEQ ID NO: 50) was replaced by a 15 amino acid (Gly4Ser)3 (SEQ ID NO: 3) linker sequence (Figure 1). A His6 (SEQ ID NO: 49) sequence was ligated to the 3' end for protein purification. All steps below were as described above for EBV gH / gL.

[0258] Western blot analysis under completely reducing conditions using anti-His6 (SEQ ID NO: 49) mAb or anti-gB mAb (Virusys Corp., Taneytown, MD, USA) demonstrated that E We demonstrated that the EBV gB protein was of the predicted size of the monomeric form (approximately 80 kDa) (Figure 2B). Under modified non-reducing conditions that allow detection of the native form of the EBV gB protein, a homogenous band with the predicted size of trimeric EBV gB (approximately 240 kDa) was observed (Figure 2B).

[0259] Example 1.3 - Production of EBV gp350 Polypeptides EBV gp350 polypeptide was expressed as previously described (see Cui et al., Vaccine, 31:3039-45, 2013; which is hereby incorporated by reference in its entirety). (See also US Pat. No. 5,399,993, which is incorporated herein by reference). Briefly, a monomeric gp350 construct of EBV was generated by PCR amplification of gp350 cDNA of the B95-8 strain. The sequence encoding amino acids 1-470 was cloned with an IgGκ leader sequence added to the 5' end and a His6 (SEQ ID NO: 49) coding sequence added to the 3' end. A tetrameric gp350 construct was generated by ligation of a second gp350 fragment (1-470) to the 3' end of the monomeric gp350 construct (without His6 (SEQ ID NO: 49)). The second gp350 fragment has a (Gly4Ser)3 (SEQ ID NO: 3) linker at the 5' end for homodimerization, a leucine zipper sequence at the 3' end followed by a His6 (SEQ ID NO: 49) sequence for protein purification (Figure 1). Monomeric and tetrameric gp350 DNA were cloned into pOptiVEV and their sequences were verified by sequencing. All steps below were as described above for EBV gH / gL.

[0260] Anti-gp350 mAb clone 2L10 (Merck Millipore, Billerica, MA, USA), 72A1 (ATCC, Manassas, VA, USA) or anti-His6 (SEQ ID NO: 1) were used. (Lane #49) Western blot analysis using the mAb under denaturing (reducing) conditions revealed a single ≈100 kDa band corresponding to monomeric gp350 and a single ≈200 kDa band corresponding to a gp350 dimer resulting from dissociation of two gp350 dimers to form tetrameric gp350 (Figure 2C). Under native (non-reducing) conditions, a single ≈100 kDa band corresponding to monomeric gp350 was revealed, and a single ≈400 kDa band corresponding to tetrameric gp350 was observed (Figure 2C).

[0261] Example 1.4 - Induction of EBV immune responses in rabbits The resulting EBV polypeptides were used in vaccine preparations to induce immune responses in rabbits. In this study and the examples that follow, the level of immune response was determined by the level of EBV polypeptide-specific antibodies found in the serum. In this study, groups of five male New Zealand White rabbits, aged 12-15 weeks, were immunized subcutaneously with 25 μg of each EBV antigen, including tetrameric EBV gp350, trimeric EBV gH / gL, or trimeric EBV gB, versus monomeric EBV gp350 or monomeric EBV gH / gL. The antigen was adsorbed onto aluminum hydroxide (alum; 0.25 μg of alum per mg of protein) and mixed prior to injection with 50 μg of a 12-mer phosphorothioate-modified CpG oligodeoxynucleotide (ODN) (hereinafter ODN 2007, TCGTCGTTGTCGTTTTGTCGTT (SEQ ID NO: 51)) optimized for use in rabbits (see Ioannou et al., Vaccine, 21:4368-72, 2003). The activity of ODN 2007 was confirmed by its ability to stimulate IgM secretion when added to rabbit splenocytes (ibid.). Rabbits immunized with alum and CpG-ODN alone served as negative controls. Rabbits were immunized on days 0, 21, and 42. Serum samples were taken before the first immunization and 10 days after each immunization.

[0262] IC in cultures of Raji B lymphoma cells and green fluorescent protein (GFP)-tagged EBV 50 Sera were obtained 10 days after the last immunization for measurement of neutralization titers. IC 50 Values ​​represent the reciprocal of serum titers resulting in 50% EBV neutralization. EBV infection was measured by flow cytometry. As shown in Figure 3, tetrameric gp350 and trimeric gH / gL were significantly more potent than their monomeric counterparts. * p<0.05) higher IC 50 Of note, the gH / gL (IC 50 =506)>gB(IC 50 =89)>gp350(IC 50 = 22) and IC between multimeric proteins 50 A significant difference in potency (p<0.05) was also observed.

[0263] Thus, as shown in Figure 3, the five EBV polypeptides each induced an increased IgG response after the first boost, including monomeric gp350 (Figure 3, left panel, open circles) and monomeric gH / gL (Figure 3, center panel, open circles). A further significant increase in serum IgG titers followed after the second boost. Tetrameric EBV gp350 (Figure 3, left panel, filled circles) induced more than 20-fold higher serum gp350-specific IgG titers after the first and second boosts, compared with monomeric EBV gp350 (Figure 3, left panel, open circles). Trimeric EBV gH / gL (FIG. 3, center panel, filled circles) induced a >30-fold and >90-fold increase in serum gH / gL-specific IgG titers after primary immunization and the first boost, respectively, with titers comparable by a second boost. These data are consistent with previous studies performed in mice using tetrameric and monomeric gp350, which showed that multimerization of tetrameric fusion EBV gp350 polypeptides induced a significant increase in immunogenicity (Cui et al., Vaccine, 31:3039-45, 2013).

[0264] Example 1.5 - EBV antibody titers induced by monomeric gH / gL, trimeric gH / gL and trimeric gB compared to titers induced by monomeric and tetrameric gp350 Determination of serum in vitro EBV neutralization titers was performed using Raji cells (EBV-positive human Burkitt's lymphoma cell line) as described (Sashihara et al., Virology, 391:249-56, 2009). Briefly, GFP-EBV (B95-8 / F) was generated by transfecting 293 / 2089 cells with plasmids p509 and p2670 expressing EBV BZLF1 and EBV BALF4, respectively (kindly provided by Dr. Jeffrey I. Cohen, NIH, Bethesda, MD, USA) (Non-Patent Document 4, and Delecluse et al., Proc. Natl. Acad. Sci. USA, 95:8245-50, 1998). Serial dilutions of serum were mixed with GFP-EBV in 96-well plates for 2 h, followed by addition of Raji cells for an additional 1 h. Cells were then washed and re-cultured in medium alone for 3 days, fixed with paraformaldehyde, and analyzed by flow cytometry for GFP+ Raji cells. A 50% infectivity (IC 50 ) Inhibited serum dilution The percentiles were calculated by nonlinear regression analysis using Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA). b(72A1) was used as a positive control. Pre-immune serum and serum from rabbits immunized with alum+CpG-ODN alone served as negative controls. For determination of serum neutralization titers using peripheral blood naive human B cells, naive human B cells isolated from peripheral blood of healthy donors were incubated with GFP-EBV and treated with 100 ng / ml IL-4 (BioLegend, San Diego, CA, USA) and 1 μg / ml CD40 antibody. in RPMI 1640 medium containing 100% lysine (R&D Systems, Minneapolis, MN, USA). Cultured.

[0265] As shown in Figure 4A, tetrameric EBV gp350 had a significantly higher IC than monomeric EBV gp350. 50 The titer (effective dilution of antibody that inhibited infectivity by 50%) was induced (IC 50 , 22 vs. less than 5, respectively). Of note, trimeric gH / gL had a significantly higher IC than monomeric gH / gL. 50 Titer (IC 50 , 506 vs. 107, respectively), i.e., tetrameric gp350. Similarly, trimeric EBV gB induced significantly higher titer levels than those induced by tetrameric gp350 (IC 50 22) Significantly higher IC 50 Titer (IC 50 89) and induce monomeric gH / gL (IC 50 107) Compared with monomeric gp350 previously tested in phase II clinical trials, trimeric gH / gL, monomeric gH / gL, trimeric gB, and tetrameric gp350 showed 100-fold, 20-fold, 18-fold, and 4-fold higher IC 50 The IC titers were slightly higher for monomeric and tetrameric gp350 compared to those calculated using Raji cells (Figure 4A). 50Similar data were obtained from pooled sera from each group shown in Figure 4A, utilizing GFP-EBV and naive peripheral blood human B cells from healthy donors for determination of EBV neutralization titers (Figure 4B), except that EBV gH / gL and EBV gB proteins, like EBV gp350, elicit antibodies that block EBV entry into Raji Burkitt lymphoma and naive peripheral human B cells in rabbits. However, EBV gH / gL and EBV gB proteins appear to be significantly more potent on a weight basis than EBV gp350.

[0266] Example 1.6 - Immunization of rabbits with EBV trimeric gB and monomeric gH / gL New Zealand white rabbits aged 12 to 15 weeks were adsorbed to aluminum hydroxide (alum; 0.25 μg alum per mg protein) and mixed with 100 μg of 12-mer phosphorothioate-modified CpG-ODN (TCATAACGTTCC (SEQ ID NO: 52)) optimized for rabbits (Ioannou et al., Vaccine, 21:4368-72, 2003). Rabbits were immunized subcutaneously with 25 μg each of a combination of trimeric gB and monomeric gH / gL of EBV. Rabbits were immunized on days 0, 21, and 42, and serum samples were taken before the first immunization and 10 days after each immunization. An EBV neutralization assay based on flow cytometric analysis of GFP-labeled EBV entry into Raji Burkitt's lymphoma B cells was used to determine serum EBV neutralizing titers (IC) that inhibited infectivity of Raji B cells by 50%. 50 ) was measured. Administration of both EBV trimeric gB and monomeric gH / gL resulted in synergistic results compared to administration of individual EBV proteins. More specifically, on day 52, rabbits immunized with EBV trimeric gB and monomeric gH / gL demonstrated 16- and 14-fold higher EBV neutralizing activity compared to rabbits immunized with EBV trimeric gB or monomeric gH / gL alone, respectively (Figure 5).

[0267] Example 1.7 - EBV neutralization in vitro by combinations of antisera Various combinations of sera obtained from rabbits immunized with trimeric EBV gB, monomeric EBV gH / gL, or monomeric EBV gp350 were incubated in vitro with Raji cells. The serum was analyzed for EBV neutralization titers in vitro. Trimeric gB + monomeric gp350 serum, monomeric gH / gL + monomeric gp350 serum, and trimeric gB + monomeric gH / gL + monomeric gp350 serum all showed a more than two-fold increase in EBV neutralizing activity compared with the sum of the neutralizing activities of the individual protein immune sera, clearly demonstrating synergy on EBV neutralizing activity (Fig. 6B ).

[0268] Various combinations of sera from rabbits immunized with EBV trimer gB, trimer gH / gL, or tetramer gp350 were also analyzed for in vitro EBV neutralization titers using Raji cells. Trimer gB+trimer gH / gL sera, trimer gH / gL+tetramer gp350 sera, and trimer gB+trimer gH / gL+tetramer gp350 sera showed EBV neutralization activity comparable to the sum of the neutralization activities of the individual protein immune sera (Figure 6B). Trimer gB+tetramer gp350 sera showed more than a two-fold increase in EBV neutralization activity compared to the sum of the neutralization activities of the individual protein immune sera, demonstrating synergy (Figure 6A).

[0269] The synergistic results obtained when combining certain EBV proteins were unexpected, as were the additive results obtained when combining other EBV proteins, considering the possibility of attenuated antibody responses due to vaccines or immune interference.

[0270] Example 1.8 - Passive transfer of immunity against EBV in NOG mice In this study, mice were challenged with live EBV to determine whether antisera derived from rabbits exposed to EBV polypeptides as above, i.e., by passive immune transfer model, could protect mice from EBV infection. null The NOG (NOG) mouse is an art-recognized humanized mouse model of EBV infection that mimics key aspects of EBV infection in humans (Yajima et al., J. Infect. Dis., 198:673-82, 2008). NOG mice are immunodeficient and lack mature T cells, B cells, and natural killer cells. The immune system of NOG mice can be reconstituted with a functional human immune system by transplanting hematopoietic stem cells (HSCs) derived from human umbilical cord blood to generate humanized NOG (hu-NOG) mice (Yajima et al., J. Infect. Dis., 198:673-82, 2008). Approximately 1×10 3 T.D. 50 Inoculation of mice with (50% transforming dose) EBV induces B cell lymphoproliferation with histopathological findings and latent EBV gene expression similar to that observed in immunocompromised humans, and death up to 10 weeks after infection, making it a useful model for EBV-induced PTLD in humans (Dittmer et al., Curr. Opin. Virol., 14:145-50, 2015).

[0271] Hu-NOG mice are still not suitable for direct vaccination studies due to the lack of induction of specific human IgG responses to protein antigens (Seung et al., J. Infect. Dis., 208 Suppl 2:S155-9, 2013), and thus are not suitable for determining the protective role of EBV-specific antibodies. In this regard, passive immunization studies are needed to determine whether EBV-specific immunization can be achieved. In this regard, an early study reported that 85% of SCID mice injected intraperitoneally with peripheral blood mononuclear cells (PBMCs) from EBV-seropositive healthy donors developed B-cell lymphomas over a period of 150 days. However, tumor formation was prevented by weekly treatment with two different commercially available IVIg preparations (not specifically selected for high EBV neutralizing activity) or by purified IgG from EBV-seronegative but not seronegative donors (Abedi et al., Int. J. Cancer, 71:624-9, 1997).

[0272] In this study, hu-NOG mice were generated by intravenous injection of human CD34(+) HSCs isolated from umbilical cord blood (approximately 1 × 10 4 ~1.2×10 5 After reconstitution of the human blood-immune system, four groups (n=4) of hu-NOG mice were administered 500 EBV-derived IgG1-derived EBV gp350, EBV-trimeric gH / gL, EBV-trimeric gB, or control (adjuvant (alum + CpG-ODN) alone) derived from rabbits immunized with EBV gp350, EBV-trimeric gH / gL, EBV-trimeric gB, or control (adjuvant (alum + CpG-ODN) alone). An intraperitoneal injection of 300 μl of pooled serum was performed on day 2. Two hours after intraperitoneal injection of rabbit serum, approximately 1 × 10 mice were injected with the serum, a dose that induces B cell lymphoproliferation and lethality within about 10 weeks or within about 10 weeks. 3 T.D. 50 hu-NOG mice were infected intravenously with EBV (AKATA Burkitt's lymphoma cell line) (Yajima et al., J. Infect. Dis., 198:673-682, 2008). .

[0273] 75 days after EBV infection, all three hu-NOG mice that received serum from rabbits injected with alum+CpG-ODN died, whereas all three mice that received trimeric gB-specific pooled antisera survived 132 days after EBV infection (Figure 7A). One hu-NOG mouse that received tetrameric gp350-specific pooled antisera survived 119 days, and one hu-NOG mouse that received trimeric gH / gL-specific pooled antisera survived 132 days (Figure 7A). Compared to hu-NOG mice that received the control (alum+CpG-ODN serum), the copy numbers of EBV from multiple organs of mice that received trimeric gH / gL-specific pooled antisera or tetrameric gp350-specific pooled antisera were significantly lower in multiple organs than serum from rabbits injected with alum+CpG-ODN alone (Figure 7B). The effect of gB-specific pooled antisera on EBV organ involvement was not reported because the experiment was ongoing. Hu-NOG mice receiving pooled antisera specific for gB, gH / gL, or gp350 also showed significantly lower EBV DNA blood levels compared to adjuvant controls, but hu-NOG mice receiving trimeric gB-specific pooled antisera had higher EBV loads in peripheral blood compared to mice receiving tetrameric gp350-specific pooled antisera or trimeric gH / gL-specific pooled antisera (Figure 7C).

[0274] 2. Human cytomegalovirus (HCMV) Example 2.1 - Production of trimeric HCMV gB The above results with EBV fusion / cell entry proteins showed unexpectedly high levels of antibody induction when EBV polypeptides were combined. Based on these novel findings, the inventors expected to obtain similar results when combining fusion / cell entry proteins from other HHV family members, such as HCMV. To this end, similar studies were planned to show that the observations made in EBV studies can be extended to other HHV family members, such as HCMV.

[0275] For HCMV, the coding sequence of HCMV gB was obtained from the NCBI website reference sequence NC_006273.2, Merlin strain, nucleotides 82066-84789. The DNA sequence encoding amino acids 23-750 (corresponding to the extracellular domain of gB) of HCMV gB was used, and the signal peptide (corresponding to amino acids 1-22) was replaced with an IgGκ leader sequence. To generate the trimeric form of the gB polypeptide, the cleavage site coding sequence, RTKRS (SEQ ID NO:53) between amino acids 456 (N) and 462 (T), was replaced with a 15 amino acid (Gly4Ser)3 (SEQ ID NO:3) linker sequence (Figure 8A). A His6 (SEQ ID NO:49) sequence was added to the 3' end for protein purification. DNA encoding the gB protein was synthesized and cloned into pOptiVEV (Invitrogen, Carlsbad, CA, USA), and the sequence was verified by sequencing. CHO cells (strain DG44; Invitrogen, Carlsbad, CA, USA) were stably transfected with pOptiVEC-gB, and positive cells were selected with increasing concentrations of methotrexate up to 4 μM. Supernatants were concentrated for affinity purification using a cobalt column (Thermo Fisher Scientific, Waltham, MA, USA).

[0276] The purified proteins were analyzed by electrophoresis on 3%-8% NuPAGE Tris Acetate Mini-Gels under reducing conditions. Purified HCMV gB was diluted with 50 mM The samples were boiled for 10 min in lithium dodecyl sulfate sample loading buffer containing 100 μl of DTT and blotted with anti-gB monoclonal antibody 2F12 (Virusys Corp., Taneytown, MD, USA) or LS-C64457 (LifeSpan BioSciences, Inc., Seattle, WA, USA). Both antibodies showed a 120 kDa band corresponding to the monomer. Purified HCMV gB was also analyzed by PAGE under modified nonreducing conditions (proteins were mixed with lithium dodecyl sulfate sample buffer without DTT and resolved on a 3%-8% PAGE in native running buffer) and blotted with anti-gB monoclonal antibody LS-C64457, which showed a band with a molecular mass of approximately 360 kDa, consistent with trimeric gB (Fig. 9B).

[0277] Example 2.2 - Production of monomeric and trimeric HCMV gH / gL polypeptides Similarly, the coding sequences of HCMV gH and gL were obtained from the NCBI website reference sequence NC_006273.2, strain Merlin, gH nucleotides 109224 to 111452, gL nucleotides 165022 to 165858. Constructs for expression of trimeric HCMV gH / gL were prepared using MacVector (MacVector, Inc., Apex, NC, USA), which was used to express trimeric EBV gH / gL. The gH / gL constructs were synthesized according to the design. The gL sequence encoding amino acids 31-278 was used, and the signal peptide corresponding to amino acids 1-30 was replaced with an IgGκ leader sequence. The gH sequence encoding amino acids 24-718 was linked to the 3' end of gL and separated by a 15 amino acid linker (Gly4Ser)3 (SEQ ID NO:3) sequence. The Foldon trimerization domain coding sequence from T4 phage fibritin was linked to the 3' end of gH, followed by a His6 (SEQ ID NO:49) coding sequence for protein purification. DNA encoding trimeric gH / gL was synthesized, cloned into pOptiVEV (Invitrogen, Carlsbad, CA, USA), and sequence verified by sequencing. Monomeric HCMV gH / gL constructs were generated by PCR amplification of trimeric HCMV gH / gL without the Foldon trimerization domain coding sequence, cloned into pOptiVEV, and sequence verified by sequencing.

[0278] The resulting pOptiVEC-gH / gL construct was stably transfected into Chinese hamster ovary (CHO) cells (strain DG44) (Invitrogen) using Free-style Max reagent (Invitrogen, Carlsbad, CA, USA) and positive transformants were selected using gradually increasing concentrations of methotrexate up to 4 μM. Supernatants were concentrated and purified using cobalt affinity purification (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed by Western blot using both anti-His6 (SEQ ID NO: 49) and anti-HCMV gH / gL antibodies (Santa Cruz Biotech, Dallas, TX, USA). Under reducing conditions, the Western blot showed monomeric gH / gL as a band at approximately 110 kDa (Figure 9C), and under non-reducing conditions, trimeric gH / gL appeared as a band at approximately 330 kDa (Figure 9D).

[0279] Example 2.3 - Induction of HCMV IgG by trimeric gB and monomeric gH / gL Having generated the desired HCMV polypeptide constructs, a comparative study was performed to determine whether multimeric polypeptides and / or combinations of various polypeptides would generate a substantially greater immune response than monomeric polypeptides. Thus, seven groups of five 12-15 week old male New Zealand White rabbits were immunized subcutaneously with 25 μg of a single HCMV envelope protein or a combination of HCMV envelope proteins (25 μg of each protein in the combination). 25 μg of each protein was adsorbed onto aluminum hydroxide (alum; 0.25 μg alum per mg protein) and mixed with 25 μg of CpG-ODN (ODN 2007 with the sequence TCGTCGTTGTCGTTTTGTCGTT (SEQ ID NO: 51)) with known activity in rabbits. The HCMV proteins / combinations used were monomeric gH / gL, monomeric UL128 / UL130 / UL1 The immunizations were: 31A, monomeric gB (Sino gB), trimeric gB, monomeric gH / gL + monomeric UL128 / UL130 / UL131A, trimeric gB + monomeric gH / gL, or trimeric gB + monomeric gH / gL + monomeric UL128 / UL130 / UL131A. Rabbits were immunized on days 0, 21, and 42, and serum samples were collected before the first immunization and on days 10, 31, 52, and 72 after immunization. Serum titers of antigen-specific IgG against live HCMV were determined using fibroblast (cell line MRC-5, ATCC, Manassas, VA, USA) and epithelial cells (cell line ARPE-19, ATCC, Manassas, VA, USA). Trimeric HCMV gB and monomeric HCMV gH / gL recombinant proteins were incubated together for 30 min at room temperature and were found to induce high titers of protein-specific IGg (FIG. 11).

[0280] HCMV neutralization assay. Pooled day 52 and day 72 sera from five rabbits from each cohort immunized with single or combinations of HCMV envelope proteins were heat inactivated at 56°C for 30 min to eliminate complement activity or were not heat treated. Serum HCMV neutralizing antibody titers were determined using an ELISpot assay. Each serum sample was prepared in quadruplicate 1:2 serial dilutions with culture medium. Each dilution was mixed with an equal volume of culture medium containing HCMV strain AD169WT131 and incubated at 37°C for 4 h, then added to wells of a 96-well plate containing monolayers of ARPE-19 (epithelial cell line, ATCC, Manassas, VA, USA) or MRC-5 (fibroblast cell line, ATCC, Manassas, VA, USA) and cultured overnight at 37°C with 5% CO2. Cells were fixed in absolute ethanol, rehydrated, blocked with 5% normal horse serum in PBS, and then incubated with anti-IE1 monoclonal antibody MAB810 (Merck Millipore, Burlington, MA, USA), goat anti-mouse secondary antibody (Merck Millipore, Burlington, MA, USA), and 10% PBS. Jackson ImmunoResearch Labs, West Grove, 1 hour each, and VEC TASTAIN ABC reagent (Vector Labs, Burlingame, CA, USA) and Both were incubated for 30 min. Between each step, the plates were washed three times with 0.05% Tween® 20 in PBS and TrueBlue (Sigma-Aldrich, St. Louis, MO, USA) was added for color development. CTL-ImmunoSpot™ S6 Microplates Plates were scanned and analyzed using an ImmunoSpot Analyzer (Cellular Technology Limited, Cleveland, OH, USA). The 50% inhibitory concentration (IC5 IC values ​​and standard errors of the means were plotted against the log serum concentration of the mean triplicates for each serum dilution using GraphPad Prism 6 software, and a four-parameter equation of best fit of the data was calculated to determine IC 50 Neutralization titers were calculated by interpolating the serum dilution rate to the midpoint of the curve.

[0281] Figure 12A shows the HCMV neutralizing activity assayed using ARPE-19 cells when rabbit immune serum was not heat inactivated. Immunization of rabbits with monomeric UL128 / UL130 / UL131A induced little HCMV neutralizing activity, with an IC of less than 10. 50 Immunization with monomeric gH / gL induced low levels of complement-dependent HCMV neutralizing activity (IC of 190.9). 50 Immunization of rabbits with the combination of monomeric gH / gL + monomeric UL128 / UL130 / UL131A induced a complement-dependent HCMV neutralizing activity (IC of 676.9) that was three-fold higher than the sum of HCMV neutralization induced by monomeric gH / gL or monomeric UL128 / UL130 / UL131A alone. 50 ) (Fig. 12A). Immunization of rabbits with monomeric gB (Sino gB) induced moderate complement-dependent HCMV neutralizing activity (IC of 528.0). 50 ), and trimeric gB induced a higher concentration of monomeric gB (IC of 2168.8). 50) (Figure 12A). Immunization with the combination of trimeric gB and monomeric gH / gL induced a two-fold higher complement-dependent HCMV neutralization activity (IC of 4299.2) than the sum of HCMV neutralization induced by trimeric gB and monomeric gH / gL individually. 50 ) and demonstrated a synergistic effect (Figure 12A). Immunization of rabbits with the UL131A combination induced a complement-dependent HCMV neutralizing activity (IC of 10910.8) that was 5-fold higher than the sum of HCMV neutralization induced by trimeric gB, monomeric gH / gL, and monomeric UL128 / UL130 / UL131A individually. 50 ) and demonstrated synergistic effects (Figure 12A). The complement-dependent HCMV neutralizing activity induced by immunization with a combination of trimeric gB, monomeric gH / gL, and monomeric UL128 / UL130 / UL131A was 20-fold higher than that of monomeric gB (Sino gB), which demonstrated 50% efficacy in preventing HCMV infection in a phase II clinical trial.

[0282] HCMV neutralization activity assayed using the fibroblast cell line MRC-5 when rabbit immune serum was heat inactivated at 56°C for 30 min to eliminate complement activity is shown in Figure 12B. Immunization of rabbits with monomeric gB (Sino gB) induced low levels of complement-independent HCMV neutralization activity (IC of 103.5). 50 ), and trimeric gB induced 20-fold higher complement-independent HCMV neutralizing activity compared to monomeric gB (IC of 2185.2). 50 , Fig. 12B). Immunization of rabbits with monomeric gH / gL also induced a low level of complement-independent HCMV neutralizing activity (IC of 167.7). 50 In contrast, immunization with a combination of trimeric gB and monomeric gH / gL induced a complement-independent HCMV neutralizing activity (IC of 12299.4) that was 5-fold higher than the sum of the HCMV neutralizing activities induced by trimeric gB and monomeric gH / gL individually. 50), demonstrating a synergistic effect (Figure 12B). The complement-independent HCMV neutralizing activity induced by immunization with a combination of trimeric gB and monomeric gH / gL was more than 100-fold higher than that of monomeric gB (Sino gB), which demonstrated 50% efficacy in preventing HCMV infection in a phase II clinical trial.

[0283] Example 2.4 - In vitro neutralization assay using HCMV gB and gH / gL antisera Serum HCMV neutralizing antibody titers were determined using an ELISpot assay. Serum samples were pooled and then split by serial 1:2 dilutions in triplicate with culture medium. Each dilution was diluted with 200 pfu of HCMV strain AD169. WT131 The cells were mixed with an equal volume of culture medium containing 0.05% Tween™ 20 in PBS, incubated at 37°C for 1 h, then added to wells of a 96-well plate containing MRC-5 monolayers and cultured at 37°C with 5% CO2. The cells were fixed with absolute ethanol, rehydrated, blocked with 1% BSA in PBS, and incubated with anti-IE1 monoclonal antibody MAB810 (Millipore), biotin-labeled goat anti-mouse secondary antibody, and ABC reagent (Vector Laboratories) for 1 h each. Between each step, the plates were washed three times with 0.05% Tween™ 20 in PBS, and TrueBlue was added for color development. The plates were scanned using a CTL-ImmunoSpot™ S6 Micro Analyzer (Cellular Technology Limited, Cleveland, OH). The 50% inhibitory concentration (IC 50 ) values ​​and standard errors of the means were plotted against the log serum concentration of the mean triplicates for each serum dilution using GraphPad Prism7 software, and a best-fit four-parameter equation for the data was calculated to determine the IC 50 Neutralization titers were calculated by interpolating the serum dilution rate to the midpoint of the curve.

[0284] In vitro HCMV neutralization results obtained using pooled immune sera from rabbits immunized with monomeric HCMV gB, trimeric HCMV gB, monomeric HCMV gH / gL, and their in vitro combinations are presented in Figures 15-20. Multimerizing HCMV polypeptides significantly enhanced the neutralizing activity of antibodies generated against the multimerized polypeptides compared to the monomeric forms of the polypeptides. For example, the IC of trimeric HCMV gB was 1.2-fold higher than that of the monomelic form. 50 IC of monomeric HCMV gB compared with 2283 50 The neutralizing activity of HCMV gB- and gH / gL-immune sera was 91.94 (Figures 15 and 16). Combining HCMV gB- and gH / gL-immune sera unexpectedly induced a higher HCMV neutralizing activity than the sum of the neutralizing activities induced by each protein individually, demonstrating synergy. For example, in vitro neutralization of monomeric HCMV gB- and gH / gL-immune sera Combination IC 50 IC of 91.94 and 169.6 for each individual protein 50 (Figures 15 and 17), and 836.4 (Figure 18). Similarly, the IC 50 The IC of 2283 and 169.6 50 (FIG. 16 and FIG. 17, respectively, for each protein individually) compared to 3093 (FIG. 19). These synergistic results are summarized in FIG.

[0285] Thus, as in the case of EBV, these comparative studies demonstrate that combining HCMV fusion / cell entry proteins (e.g., gB and gH / gL) unexpectedly enhances HCMV neutralizing activity in vivo. Immunization of rabbits with a combination of HCMV trimeric or monomeric gB and monomeric gH / gL elicited significantly higher HCMV neutralizing activity than the sum of the individual proteins, demonstrating unexpected synergy.

[0286] Example 2.5 - Production of monomeric and trimeric UL128 / 130 / 131 polypeptides of HCMV In an effort to further characterize the potential for generating enhanced antibody titers by administering antigen compositions containing HHV polypeptides, the HCMV proteins UL128, UL130, and UL131 were recombinantly produced. Briefly, the coding sequence of HCMV UL128 was obtained from the NCBI website reference sequence GQ121041.1, Towne strain, nucleotides 175653-176410. Also, the coding sequences of HCMV UL130 and UL131A were obtained from the NCBI website reference sequence NC_006273.2, Merlin strain, nucleotides 177649-177802 linked to UL130 nucleotides 176984-177628 and UL131A nucleotides 177911-178146. UL128 from the Towne strain was used because UL128 from the Merlin strain was mutated and non-functional. A construct for expression of the trimer UL128-UL130-UL131A was designed using MacVector. The UL128 sequence encoding amino acids 28-171, the UL130 sequence encoding amino acids 26-214, and the UL131A sequence encoding amino acids 19-129 were linked by a 15 amino acid linker (Gly4Ser)3 (SEQ ID NO: 3) between each coding sequence (Figure 10). The Foldon trimerization domain coding sequence from T4 phage fibritin was linked to the 3' end of UL131A, followed by a His6 (SEQ ID NO: 49) coding sequence, and an IgGκ leader sequence was placed 5' to the UL128 sequence for secretion of the recombinant protein (Figure 10). DNA encoding the trimer UL128-UL130-UL131A was synthesized and cloned into pOptiVEV (Invitrogen, Carlsbad, CA, USA) and the sequence was verified. A monomeric UL128-UL130-UL131A construct was generated by PCR amplification of trimeric UL128-UL130-UL131A without the Foldon trimerization domain coding sequence, cloned into pOptiVEV and sequence verified.

[0287] CHO cells (DG44 line, Invitrogen, Thermo Scientific, Carlsbad, CA, USA) Thermo Fisher Scientific) was stably transfected with the resulting pOptiVEC-UL128-UL130-UL131A construct using Free-style Max reagent (Invitrogen, Carlsbad, CA) and positive transfectants were selected with gradually increasing concentrations of methotrexate up to 4 μM. Supernatants were concentrated and purified using cobalt affinity purification (Thermo Fisher Scientific, Waltham, MA, USA). Western blot analysis of supernatants from CHO cells transfected with the monomeric UL128-UL130-UL131A construct using anti-His6 (SEQ ID NO: 49) and anti-UL128 antibodies showed a band of approximately 57 kDa consistent with monomeric UL128 / UL130 / UL131A (Figure 9E).

[0288] Example 2.6 - Production of the pentameric gH / gL / UL128 / 130 / 131 complex of HCMV The coding sequences of gH, gL, UL128, UL130 and UL131A of HCMV were obtained from the NCBI website. A construct for expression of the pentameric complex gH / gL / UL128 / UL130 / UL131A was designed using MacVector and is shown in FIG. 13. The construct contains the gL sequence encoding amino acids 31-278, the gH sequence encoding amino acids 24-718, and the signal peptide of both sequences replaced by the IgGκ leader sequence. An EV71 internal ribosome entry site (IRES) sequence was inserted between the gH and gL sequences, and a His6 (SEQ ID NO: 49) coding sequence was added to the 3' end of gH for protein purification. Additionally, the signal peptides of UL128, UL130 and UL131A were replaced with an IgGκ leader sequence, and the UL128 sequence encoding amino acids 28-171, the UL130 sequence encoding amino acids 26-214, and the UL131A sequence encoding amino acids 19-129 were linked together by insertion of an EV71 IRES sequence between each. UL128, UL130 and UL131A were driven by a second CMV promoter located at the 5' end of UL128 and the 3' end of the gH-His6 (SEQ ID NO: 49) coding sequence. HCMV gL and gH were driven by a first CMV promoter derived from the vector pOptiVEC.

[0289] DNA encoding the pentameric complex gH / gL / UL128 / UL130 / UL131A was synthesized, cloned into pOptiVEV (Invitrogen) and verified. CHO cells (strain DG44; Invitrogen) can be transfected with pOptiVEC-gH / gL / UL128 / UL130 / UL131A and positive transformants can be selected in gradually increasing concentrations of methotrexate up to 4 μM using the procedure already outlined above for similar constructs.

[0290] Example 2.7 - Production of HCMV gH / gL / gO Complex As with the other HCMV constructs discussed above, the coding sequences for HCMV gH, gL were obtained from the NCBI website, and the coding sequence for HCMV gO was obtained from the NCBI website reference sequence NC_006273.2, Merlin strain, gO nucleotides 107430-108848. Constructs for gH / gL / gO complex expression were designed using MacVector, including the gL sequence encoding amino acids 31-278 and the gH sequence encoding amino acids 24-718, as shown in FIG. 14. The signal peptides of both sequences were replaced with IgGκ leader sequences. An EV71 internal ribosome entry site (IRES) sequence was inserted between the gH and gL sequences, and a His6 (SEQ ID NO:49) coding sequence was attached to the 3' end of gH for protein purification. In addition, the signal peptide of gO was replaced with an IgGκ leader sequence, and the gO sequence encoding amino acids 31-466 was driven by a second CMV promoter located at the 5' end of gO and the 3' end of the gH-His6 (SEQ ID NO: 49) coding sequence. HCMV gH and gL were driven by the first CMV promoter derived from the vector pOptiVEC.

[0291] DNA encoding the gH / gL / gO complex was synthesized and cloned into pOptiVEV as previously described. Stable CHO transformants are purified and analyzed by size-exclusion chromatography and multi-angle light scattering (SEC-MALS).

[0292] Example 2.8 - Immunization of mice with HCMV trimeric and monomeric gB Six groups of 7- to 10-week-old Balb / c mice (n=5) were immunized with 1 μg, 5 μg, or 25 μg of HCMV trimer gB or 1 μg, 5 μg, or 25 μg of HCMV monomeric gB (Sino gB, Sino Biological Inc., China) by the intraperitoneal (ip) route. The antigens were adsorbed onto aluminum hydroxide (alum; 0.25 μg alum per mg protein) and delivered to mice using 30-mer phosphorothioate-modified CpG-ODN (AAAAAAAAAAAAAAACGTTAAAAAAAAAAAA) optimized for mouse. A (SEQ ID NO: 54)) was mixed with 25 μg of Alum+CpG-ODN. Mice immunized with Alum+CpG-ODN alone served as negative controls. Mice were immunized on days 0, 21, and 42, and serum samples were taken before the first immunization, 10 days after each immunization, and on day 63. Individual mouse serum samples were analyzed for gB-specific IgG titers by ELISA, and in vitro neutralizing activity using fibroblasts (MRC-5) and epithelial cells (ARPE-19).

[0293] HCMV neutralization assay. Sera from mice immunized with monomeric or trimeric gB were heat inactivated at 56°C for 30 min to eliminate complement activity or were not heat treated. Serum HCMV neutralizing antibody titers were determined using an ELISpot assay. Each serum sample was prepared in triplicate 1:2 serial dilutions with culture medium. Each dilution was mixed with an equal volume of culture medium containing HCMV strain AD169WT131 and incubated at 37°C for 4 h, then added to wells of a 96-well plate containing a monolayer of MRC-5 (fibroblast cell line, ATCC, Manassas, VA, USA) and cultured overnight at 37°C in 5% CO2. Cells were fixed with absolute ethanol, rehydrated, blocked with 5% normal horse serum in PBS, and then incubated with anti-IE1 monoclonal antibody MAB810 (Merck Millipore, Burlington, MA, USA), goat anti-mouse secondary antibody (Jackson ImmunoResearch Labs, West Grove, PA, USA) for 1 h each, and with VECTASTAIN ABC reagent (Vector Labs, Burlingame, CA, USA) for 30 min. Between each step, the plates were washed three times with 0.1% Tween 20 in PBS and TrueBlue (Sigma-Aldrich, St. Louis, MO, USA) was added for color development. The CTL-ImmunoSpot™ S6 Micro Analyzer (ImmunoSpot, Cellular Technology Limited, Cleveland, OH, USA) was used. The plates were scanned and analyzed. The 50% inhibitory concentration (IC 50 ) values ​​and standard errors of the means were plotted against the log serum concentration of the mean triplicates for each serum dilution using GraphPad Prism 6 software, and a best-fit four-parameter equation for the data was calculated to determine the IC 50 Neutralization titers were calculated by interpolating the serum dilution rate to the midpoint of the curve.

[0294] Monomeric and trimeric HCMV gB were directly compared side-by-side for induction of total serum titers of antigen-specific IgG. As shown in FIG. 21A, each group of HCMV proteins induced an increased serum IgG response after the first boost, and an even more significant increase in serum IgG titers after the second boost. Trimeric HCMV gB induced 5- to 11-fold higher serum titers of gB-specific antibody IgG titers compared to monomeric HCMV gB after the first and second immunizations, with larger differences observed at lower doses. The difference in HCMV gB-specific IgG titers induced by trimeric and monomeric HCMV gB decreased after the third immunization, with smaller differences observed at higher doses. 5 μg of trimeric HCMV gB induced the optimal antigen-specific IgG response. 25 μg of trimeric HCMV gB induced slightly higher gB-specific IgG titers, which were not significantly different from those of 5 μg of HCMV trimeric gB.

[0295] By using the MRC-5 fibroblast cell line, immune sera from mice immunized with trimeric HCMV gB that had been heat-inactivated at 56°C for 30 min (to eliminate complement activity) demonstrated 50-fold higher HCMV neutralizing activity against HCMV strain AD169wt131 compared to the HCMV neutralizing activity of immune sera from mice immunized with monomeric HCMV gB (Figure 21B). Non-heat-inactivated sera from mice immunized with monomeric HCMV gB (Figure 21C) demonstrated 6-fold higher HCMV neutralizing activity compared to heat-inactivated sera (Figure 21B), whereas non-heat-inactivated sera from mice immunized with trimeric gB demonstrated 2- to 3-fold higher HCMV neutralizing activity compared to heat-inactivated sera. Without heat inactivation, the HCMV neutralizing activity against HCMV strain AD169wt131 induced by trimeric HCMV gB was 20-fold higher than that of monomeric HCMV gB and 10-fold higher than that of monomeric HCMV gB. These results suggest that trimeric HCMV gB induces a more complement-dependent response (Figure 21C). CytoGam™ (CSL Behring, King of Prussia, PA, USA), a commercially available cytomegalovirus CMV-IgIV immunoglobulin containing high titer HCMV neutralizing antibodies derived from the plasma of HCMV-seropositive healthy donors, inhibits trimeric gB. CytoGam™ demonstrated much lower HCMV neutralizing activity against HCMV strain AD169wt131 compared to sera from mice immunized with trimeric HCMV gB. Using the MRC-5 cell line, 10 mg / ml of CytoGam™ demonstrated approximately 30-fold lower complement-independent HCMV neutralizing activity than sera from mice immunized with trimeric HCMV gB. Heat inactivation had no effect on CytoGam™, rendering it much less complement-dependent HCMV neutralizing activity compared to non-heat inactivated sera from mice immunized with trimeric HCMV gB or monomeric HCMV gB.

Claims

1. The following human herpesvirus polypeptides: a human herpesvirus glycoprotein B (gB) polypeptide comprising the extracellular domain of gB; glycoprotein L (gL) polypeptide, and a human herpesvirus glycoprotein H (gH) polypeptide comprising the extracellular domain of gH; Including, the gH polypeptide and the gL polypeptide are fusion proteins of gH and gL, The composition is free of virus-like particles, the furin cleavage site of the gB polypeptide is replaced with a linker sequence; the human herpesvirus polypeptide is an Epstein-Barr virus (EBV) polypeptide, the gB polypeptide is a trimer, and the fusion protein of gH and gL is a monomer; Vaccine compositions.

2. The vaccine composition of claim 1 , which does not contain an EBV glycoprotein 350 (gp350) polypeptide.

3. The vaccine composition of claim 1 or 2, wherein the gB polypeptide and the gH polypeptide comprise an intracellular domain.

4. A vaccine composition according to any one of claims 1 to 3, comprising a pharma- ceutically acceptable excipient and / or adjuvant.

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