SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 30, 2019, is named 043214-080293WOPT_SL.txt and is 83,579 bytes in size.

TECHNICAL FIELD

The present invention is primarily concerned with vaccines that can be used to immunize subjects against Herpes Simplex Virus type 2 (HSV-2) infections associated with chronic genital ulcers. The vaccines use a replication defective HSV-2 virus that has been engineered to express high levels of both HSV-2 glycoprotein B antigen (gB2) and HSV-2 glycoprotein D antigen (gD2).

BACKGROUND

Herpes Simplex Viruses (HSV) and HSV Infections

Herpes simplex virus 2 (HSV-2) is the primary cause of genital ulcer disease. It can cause both an acute, productive infection and a long-term latent infection characterized by unpredictable periodic recurrences (Whitley, et al.,Clin. Infect. Dis.26:541-553; see also 554-555 (1998)). Apart from causing lifelong recurrent genital ulcers, HSV infections are a major concern in AIDS patients. It has been documented that genital HSV-2 infection triples the risk for sexually acquiring HIV infection (Freeman, et al,Aids20:73-83 (2006)), and in Africa, this increase in risk may contribute to 25-35% of incident HIV infections (Abu-Raddad, et al.,PLoS ONE3:e2230 (2008)).

Although the severity and duration of most symptomatic HSV primary infections can be reduced by oral or intravenous treatment with acyclovir, valacyclovir, or famciclovir, antiviral therapy neither prevents the establishment of latent infection from primary infection nor reduces subsequent recurrences (Whitley, et al.,Clin. Infect. Dis.26:541-553; see also 554-555 (1998)). The continued spread of genital herpes in the United States over the past decades (Fleming, et al.,N. Engl. J. Med.337:1105-11 (1997); Bradley, et al.,J Infect. Dis.209(3):325-333 (2014)) and the increasing incidence of HSV resistant to current antiviral medications suggest that there is a need for safe and efficacious vaccines against HSV infections (Koelle, et al.,Annu. Rev. Med.59:381-395 (2008); Stanberry,Herpes11(Suppl 3):161A-169A (2004)). In addition, the finding that HSV suppressive therapy leads to a significant reduction in levels of HIV in the genital mucosa and plasma of women infected with both HSV-2 and HIV (Nagot, et al.,N. Engl J. Med.356:790-9 (2007)) suggests that an effective HSV vaccine may also have major implications in control of HIV infection (Abu-Raddad, et al.,PLoS ONE3:e2230 (2008); Koelle, et al.,Annu. Rev. Med.59:381-395 (2008)).

HSV-2 Glycoproteins D and B

HSV glycoproteins gD2 and gB2 are abundantly expressed on the surface of infected cells (Glorioso, et al.,J. Virol.50:805-12 (1984)) and constitute the major glycoproteins on the viral envelope (Handler, et al.,J. Virol.70:6067-70 (1996)). They are essential for virus entry into cells and are major targets for neutralizing antibodies against HSV (Sim, et al.,J. Gen. Virol.19:217-33 (1973); Para, et al.,J. Virol.55:483-8 (1985); Cohen, et al.,J. Virol.49:102-8 (1984); Minson, et al.,J. Gen. Virol.67(Pt 6):1001-13 (1986); Sanchez-Pescador, et al.,J. Infect. Dis.166:623-7 (1992); Cheshenko, et al.,J. Gen. Virol.83:2247-55 (2002)). gD2 and gB2 are also the dominant viral targets for both CD4+and CD8+T cells in humans, and in murine models of HSV infection (Mikloska, et al.,J. Gen. Virol.79:353-61 (1998); Chentoufi, et al.,J. Virol.82:11792-802 (2008); Zarling, et al.,J. Immunol.136:4669-73 (1986); Johnson, et al.,J. Immunol.145:702-10 (1990); Wallace, et al.,J. Virol.73:7619-26 (1999); Kim, et al.,J. Immunol.181:6604-15 (2008); Sheridan, et al.,J. Virol.83:2237-45 (2009); Koelle, et al.,J. Clin. Invest.91:961-8 (1993); Koelle, et al.,Virol.68:2803-10 (1994); Tigges, et al.,J. Virol.66:1622-34 (1992); Hosken, et al.,J. Virol.80:5509-15 (2006)). Based on their biological importance, gD and gB are prime candidates for HSV subunit vaccine development (Stanberry,Herpes11(Suppl 3):161A-169A (2004)).

In a phase 3 clinical trial, Stanberry et al. showed that vaccination with recombinant HSV-2 gD (gD2) in combination with adjuvant AS04 (gD2/AS04) provided 73-74% efficacy in protecting against the development of genital herpes in HSV-seronegative women. However, no significant protection was observed in men or HSV-1 seropositive subjects (Stanberry, et al.,N. Engl. J. Med.347:1652-61 (2002)). In more recent phase 3 clinical trials, no protection against HSV-2 disease or infection was observed (Belshe, et al.,N. Eng. J. Med.366:34-43 (2012)). Notably, the gD2/AS04 vaccine elicits little or no CD8+T cell response (Belshe, et al.,N. Eng. J. Med.366:34-43 (2012); Koelle, et al.,Annu. Rev. Med.59:381-395 (2008)) and this response is crucial in controlling primary and recurrent HSV infection as well as preventing reactivation of HSV from latently infected neurons (Koelle, et al.,J. Clin. Invest.101:1500-8 (1998); van Lint, et al.,J. Immuol.172:392-7 (2004); Wakim, et al.,Immunol. Cell. Biol.86:666-75 (2008); Liu, et al.,J. Exp. Med.191:1459-66 (2000); Zhu, et al.,J. Exp. Med.204:595-603 (2007); Knickelbein, et al.,Science322:268-71 (2008); Zhang, et al.,Mucosal Immunol.2:129-43 (2009); Milligan, et al.,J. Immunol.160:6093-100 (1998); Schiffer, et al.,Proc. Natl. Acad. Sci. USA107:18973-8 (2010)). These studies highlight the need to develop HSV vaccine candidates capable of eliciting broader and more robust humoral as well as CD4+and CD8+T-cell responses to gD2 and other HSV antigens (Jones, et al.,Herpes11:12-7 (2004); Koelle, et al.,Annu. Rev. Med.59:381-395 (2008); Cohen,J., Science330:304 (2010)).

Viral Vaccines

It is well documented that live viral vaccines capable of de novo synthesis of immunogens in the host induce a broader and more durable immune response than vaccines consisting of only peptides or proteins. Various forms of replication-defective HSV and neuro-attenuated, replication-competent mutants have been developed and tested as potential vaccines against HSV infection (U.S. Pat. No. 7,223,411; Dudek, et al.,Virology344:230-9 (2006)). However, because both replication-defective viruses and neuro-attenuated mutants can co-replicate with wild-type virus or become replication-competent in the context of wild-type virus, their use as a vaccine in humans poses a safety concern, particularly in individuals who harbor latent HSV infection (Koelle, et al.,Curr. Eye Res.30:929-42 (2005)). The observation that replication-defective HSV-1 mutants can reactivate the latent HSV-1 immediate-early promoter in the rodent brain has raised additional safety concerns about the possibility of such recombinants triggering outbreaks of productive viral infections in latently infected individuals (Starr, et al.,Gene Ther.3:615-23 (1996)).

To minimize the risk of activating latent wild-type infections, an HSV-2 vaccine that expresses a dominant-negative HSV replication initiating polypeptide, UL9 mutant (UL9-C535C), was generated (U.S. Pat. No. 8,809,047; Akhramayeva et al, J. Virol. 85(10): 5036-5047 (2011); Zhang et al, PLoS ONE 9(6): e101373. doi: 10.1371). This replication defective HSV-2 recombinant vaccine, also referred to as CJ2-gD2, encodes 2 copies of the HSV-2 gD2, and is more effective than the gD2 subunit vaccines. Immunization with CJ2-gD2 elicits effective HSV-2 specific neutralizing antibody and T-cell response, as well as inhibits wild-type latent infections.

While significant progress has been made with respect to potential HSV-2 vaccination for clinical use, there continues to be a need in the art for vaccines that can achieve a robust immune response, that minimize the potential of activation of latent wild-type virus, and that are easy to produce.

SUMMARY OF THE INVENTION

In general, the present invention makes use of tetracycline gene-switch technology (T-REx, Invitrogen/ThermoFisher Scientific) (Yao, et al.,Hum. Gene Ther.9:1939-50 (1998)) and uses deletion of essential genes or a dominant-negative mutant form of the HSV-1 or HSV-2 UL9 polypeptide, e.g., UL9-C535C to make the virus replication-defective and safe for immunization against HSV-2.

More specifically, one aspect of the present invention described herein provides a replication-defective, Herpes simplex virus-2 (HSV-2) recombinant viruses that lacks the sequences encoding functional ICP0 protein and gG2 protein and which comprise within the HSV-2 genome: (a) a first coding sequence, wherein said first coding sequence encodes HSV-2 glycoprotein B (gB2); wherein said first coding sequence is operably linked to a first immediate-early promoter, (b) a second coding sequence, wherein said second coding sequence encodes HSV-2 glycoprotein D (gD2) and is operably linked to a second immediate-early promoter, and (c) a third coding sequence, wherein said third coding sequence encodes HSV-2 glycoprotein D (gD2) and is operably linked to a third immediate-early promoter.

In some embodiments, said first promoter is an HSV-1 or HSV-2 immediate early promoter that is optionally operably linked to a first tetracycline operator (tet-O) sequence; said second promoter is an HSV-1 or HSV-2 immediate early promoter that is optionally operably linked to a second tetracycline operator (tet-O) sequence; and said third promoter is an HSV-1 or HSV-2 immediate early promoter that is optionally operably linked to a third tetracycline operator (tet-O) sequence. In one embodiment, at least one promoter is operably linked to a tetracycline operator (tet-O) sequence.

In another aspect of the invention described herein is a replication-defective HSV-2 recombinant virus, comprising within its genome (a) a first coding sequence, wherein said first coding sequence encodes HSV-2 glycoprotein B (gB2), wherein said first coding sequence is operably linked to a first promoter, and said first promoter is an HSV-1 or HSV-2 immediate early promoter that is operably linked to a first tetracycline operator (tet-O) sequence; (b) a second coding sequence, wherein said second coding sequence encodes HSV-2 glycoprotein D (gD2) and is operably linked to a second immediate early promoter, wherein said second promoter is operably linked to a second tet-O sequence; and (c) a third coding sequence, wherein said third coding sequence encodes HSV-2 glycoprotein D (gD2) and is operably linked to a third immediate early promoter, wherein said third promoter is operably linked to a third tet-O sequence. In one embodiment, the second promoter and third promoter are a HSV-1 or HSV-2 immediate early promoter.

In one aspect of the invention, the genome of the replication defective recombinant virus does not encode functional ICP0 and functional gG2. In certain embodiments, the genome of the replication defective recombinant virus does not encode functional ICP0 and functional gG2, and further does not encode functional UL19 (VP5 protein).

In still another aspect of the invention described herein is a replication-defective HSV-2 recombinant virus, comprising within its genome: (a) a first coding sequence, wherein said first coding sequence encodes HSV-2 glycoprotein B (gB2), wherein said first coding sequence is operably linked to a first promoter, and said first promoter is an HSV-1 or HSV-2 immediate early promoter that is operably linked to a first tetracycline operator (tet-O) sequence; (b) a second coding sequence, wherein said second coding sequence encodes HSV-2 glycoprotein D (gD2) and is operably linked to a second HSV-1 or HSV-2 immediate early promoter, wherein said second promoter is operably linked to a second tet-O sequence; and (c) a third coding sequence, wherein said third coding sequence encodes HSV-2 glycoprotein D (gD2) and is operably linked to a third HSV-1 or HSV-2 immediate early promoter, wherein said third promoter is operably linked to a third tet-O sequence.

In certain preferred embodiments, a non-essential gene encoding HSV-2 glycoprotein G (gG2), has been disrupted by the insertion of an HSV-2 glycoprotein B (gB2) sequence. In one embodiment, the first coding sequence is located at the gG2 locus of the HSV-2 genome and gG2 is not expressed. This preferred embodiment is counter-intuitive, as insertion at this location removes expression of the gG2 antigen, and thus this antigen can no longer contribute to overall immunogenicity of the vector. However, we have demonstrated that the recombinant viruses of the invention provide surprisingly strong and protective immune responses even in the absence of gG2 antigen. One advantage of this is that, since the loss of gG2 does not prevent HSV-2 viral replication (Liljeqvist, et al.,J. Virol.73:9796-802 (1999); Harland, et al.,J. Gen. Virol.69(Pt 1):113-24 (1988)), one can produce gG2 deletion mutants in cells that do not need to be engineered to produce compensatory gG2. An additional advantage is that assays, e.g., by PCR or serological tests, can be performed to determine whether gG2 is being expressed in the vaccinated host in order to differentiate between infection with wild type HSV-2 and with the vaccine vector. This ability may be important in serological differentiation for breakthrough HSV-2 infections in clinical settings.

In some embodiments, a dominant-negative mutant form of the HSV-1 or HSV-2 UL9 polypeptide, e.g., UL9-C535C, is used to develop a safe recombinant viral vaccine against HSV-2 infection. A reference providing guidance on how to make and use HSV-2 vaccines based on this technology is provided by U.S. Pat. No. 8,809,047 (incorporated herein by reference in its entirety).

In certain embodiments of various aspects, the recombinant virus is a dominant-negative virus that further comprises a fourth coding sequence, wherein said fourth coding sequence encodes a dominant negative mutant HSV-1 or HSV-2 UL9 protein, and is operably linked to a fourth promoter, wherein said fourth promoter is operably linked to a fourth tet-O sequence.

In certain embodiments of various aspects, the recombinant dominant-negative virus further comprises a fifth coding sequence, wherein said fifth coding sequence encodes a dominant negative mutant HSV-1 or HSV-2 UL9 protein, and is operably linked to a fifth promoter, wherein said fifth promoter is operably linked to a fifth tet-O sequence.

In one aspect of any of the embodiments, described herein is a replication-defective Herpes simplex virus 2 (HSV-2) recombinant virus, comprising within its genome: a) a first coding sequence, wherein said first coding sequence encodes HSV-2 glycoprotein B (gB2), wherein said first coding sequence is operably linked to a first immediate-early promoter; b) a second coding sequence, wherein said second coding sequence encodes HSV-2 glycoprotein D (gD2), and is operably linked to a second immediate-early promoter; c) a third coding sequence, wherein said third coding sequence encodes HSV-2 glycoprotein D (gD2); and is operably linked to a third immediate-early promoter; and wherein said genome does not comprise a sequence encoding a functional ICP0 protein, and does not comprise a sequence encoding functional HSV-2 gG2 protein.

In one aspect of any of the embodiments, described herein is a replication-defective Herpes simplex virus 2 (HSV-2) recombinant virus, comprising within its genome: a) a first coding sequence, wherein said first coding sequence encodes HSV-2 glycoprotein B (gB2), wherein said first coding sequence is operably linked to a first immediate-early promoter, and said first promoter is an HSV-1 or HSV-2 immediate early promoter that is operably linked to a first tetracycline operator (tet-O) sequence; b) a second coding sequence, wherein said second coding sequence encodes HSV-2 glycoprotein D (gD2) and is operably linked to a second immediate-early promoter, wherein said second promoter is operably linked to a second tet-O sequence; c) a third coding sequence, wherein said third coding sequence encodes HSV-2 glycoprotein D (gD2) and is operably linked to a third immediate-early promoter, wherein said third promoter is operably linked to a third tet-O sequence; and wherein said genome does not comprise a sequence encoding a functional ICP0 protein, and does not comprise a sequence encoding functional HSV-2 gG2 protein.

In some embodiments of any of the aspects, said second promoter and third promoter are a HSV-1 or HSV-2 immediate early promoter operably linked to a tetracycline operator (tet-O) sequence.

In some embodiments of any of the aspects, the first coding sequence is located at the gG2 locus of the HSV-2 genome.

In some embodiments of any of the aspects, said genome further does not comprise a sequence encoding a functional UL19 (VP5) protein.

In some embodiments of any of the aspects, the recombinant virus further comprises a fourth coding sequence, wherein said fourth coding sequence encodes a dominant negative mutant HSV-1 or HSV-2 UL9 protein, and is operably linked to a fourth promoter, wherein said fourth promoter is operably linked to a fourth tet-O sequence.

In some embodiments of any of the aspects, the recombinant virus further comprises a fifth coding sequence, wherein said fifth coding sequence encodes a dominant negative mutant HSV-1 or HSV-2 UL9 protein, and is operably linked to a fifth promoter, wherein said fifth promoter is operably linked to a fifth tet-O sequence.

In some embodiments of any of the aspects, said fourth sequence encodes UL9-C535C.

In some embodiments of any of the aspects, the recombinant virus comprises said fifth sequence, wherein said fifth sequence encodes UL9-C535C.

In some embodiments of any of the aspects, each of said first, second and third promoters are HSV-1 or HSV-2 immediate early promoters.

In some embodiments of any of the aspects, each of said first, second and third promoters are selected from the group consisting of an ICP0 promoter, an ICP27 promoter, and an ICP4 promoter.

In some embodiments of any of the aspects, the first promoter is a HSV-1 or HSV-2 ICP0 promoter.

In some embodiments of any of the aspects, the first promoter is a modified HSV-1 or HSV-2 ICP0 promoter comprising a human cytomegalovirus (hCMV) TATA element.

In some embodiments of any of the aspects, the first promoter comprises SEQ ID NO: 08.

In some embodiments of any of the aspects, the fourth and fifth promoters are hCMV immediate-early promoters.

In some embodiments of any of the aspects, said first sequence is a codon optimized sequence.

In one aspect of any of the embodiments, described herein is a replication-defective Herpes simplex virus 2 (HSV-2) recombinant virus, comprising within its genome: a) a first coding sequence, wherein said first coding sequence encodes HSV-2 glycoprotein B (gB2), wherein said first coding sequence is operably linked to a first immediate-early promoter, and said first promoter is an HSV-1 or HSV-2 immediate early promoter that is operably linked to a first tetracycline operator (tet-O) sequence; b) a second coding sequence, wherein said second coding sequence encodes HSV-2 glycoprotein D (gD2) and is operably linked to a second HSV-1 or HSV-2 immediate-early promoter, wherein said second promoter is operably linked to a second tet-O sequence; c) a third coding sequence, wherein said third coding sequence encodes HSV-2 glycoprotein D (gD2) and is operably linked to a third HSV-1 or HSV-2 immediate-early promoter, wherein said third promoter is operably linked to a third tet-O sequence; and wherein said genome does not comprise a sequence encoding a functional ICP0 protein, and does not comprise a sequence encoding functional HSV-2 gG2 protein.

In one aspect of any of the embodiments, described herein is a replication-defective HSV-2 recombinant virus, comprising within its genome: a) a first coding sequence, comprising a codon optimized HSV-2 gB2 sequence operably linked to a first promoter, wherein said first promoter is an HSV-1 or HSV-2 ICP0 or ICP4 promoter that is operably linked to a first tet-O sequence; b) a second coding sequence, comprising a codon-optimized HSV gD2 sequence operably linked to a second promoter, wherein said second promoter is an HSV-1 ICP4 promoter that is operably linked to a second tet-O sequence and wherein the second coding sequence operably linked to the second promoter is located at the UL26/UL27 intergenic region; c) a third coding sequence, comprising a codon optimized HSV gD2 sequence operably linked to a third promoter, wherein said third promoter is an HSV-1 ICP27 promoter that is operably linked to a third tet-O sequence, wherein the third coding sequence operably linked to the third promoter displaces the UL19 gene; and wherein said genome does not comprise a sequence encoding a functional ICP0 protein, and does not comprise a sequence encoding functional HSV-2 gG2 protein.

In one aspect of any of the embodiments, described herein is a replication-defective HSV-2 recombinant virus, comprising within its genome: a) a first coding sequence, comprising a codon optimized HSV-2 gB2 sequence operably linked to a first promoter, wherein said first promoter is an HSV-1 or HSV-2 ICP0 or ICP4 promoter that is operably linked to a first tet-O sequence; b) a second coding sequence, comprising an HSV-2 gD2 sequence operably linked to a second promoter, wherein said second promoter is an HSV-1 or HSV-2 ICP0, ICP4, or ICP27 promoter that is operably linked to a second tet-O sequence; c) a third coding sequence, comprising an HSV-2 gD2 sequence operably linked to a third promoter, wherein said third promoter is an HSV-1 or HSV-2 ICP0, ICP4, or ICP27 promoter that is operably linked to a third tet-O sequence; and wherein said genome does not comprise a sequence encoding a functional ICP0 protein, and does not comprise a sequence encoding HSV-2 gG2 protein.

In some embodiments of any of the aspects, the first coding sequence is located at the gG2 locus of the HSV-2 genome.

In some embodiments of any of the aspects, said genome does not comprise a sequence encoding a functional UL19 (VP5) protein.

In some embodiments of any of the aspects, the replication-defective HSV-2 recombinant virus further comprises i) a fourth coding sequence, encoding a dominant negative UL9-C535C protein, and operably linked to a third promoter, wherein said third promoter is an hCMV immediate early promoter that is operably linked to a third tet-O sequence; and ii) a fifth sequence, encoding a dominant negative UL9-C535C protein, operably linked to a fifth promoter, wherein said fifth promoter is an hCMV immediate early promoter that is operably linked to a fifth tet-O sequence.

In some embodiments of any of the aspects, the first promoter is an HSV-1 or HSV-2 ICP0 promoter.

In some embodiments of any of the aspects, the first promoter is a modified HSV-1 or HSV-2 ICP0 promoter comprising a human cytomegalovirus (hCMV) TATA element.

In some embodiments of any of the aspects, the first promoter comprises SEQ ID NO: 08.

In some embodiments of any of the aspects, at least one of the second and third promoters is a HSV-1 or HSV-2 ICP27 promoter operably linked to a tet-O sequence.

In some embodiments of any of the aspects, at least one of the second and third promoters is a HSV-1 or HSV-2 ICP4 promoter operably linked to a tet-O sequence.

In some embodiments of any of the aspects, each of the second and third promoters are the same, and wherein said same promoter is an HSV-1 or HSV-2 promoter selected from the group consisting of: an ICP4 promoter, an ICP27 promoter.

In some embodiments of any of the aspects, each of the second and third promoters are different, and wherein one of these promoters is an HSV-1 or HSV-2 ICP4 promoter, and wherein the other of these promoters is an HSV-1 or HSV-2 ICP27 promoter.

In one aspect of any of the embodiments, described herein is a replication defective HSV recombinant virus, comprising a modified HSV-1 or HSV-2 ICP0 promoter comprising a human cytomegalovirus (hCMV) TATA element, wherein said modified promoter is operably linked to a transgene.

In some embodiments of any of the aspects, the transgene encodes HSV-2 glycoprotein B (gB).

In some embodiments of any of the aspects, said modified promoter comprises SEQ ID NO: 08.

In one aspect of any of the embodiments, described herein is a vaccine comprising a recombinant virus described herein in unit dose form.

In one aspect of any of the embodiments, described herein is a method of immunizing a subject against HSV-1 or HSV-2 infection, comprising administering to said subject a vaccine described herein.

In some embodiments of any of the aspects, said subject is seropositive for HSV-1.

In some embodiments of any of the aspects, said subject is seropositive for HSV-2.

In some embodiments of any of the aspects, said subject is seronegative for HSV-1 and HSV-2.

In one aspect of any of the embodiments, described herein is a method for producing a virus described herein, the method comprising; a) infecting complementing cells with the virus, wherein the complementing cells express a functional gene product or products that are needed for replication of the virus and for which sequences encoding such are lacking from the virus genome; b) culturing the complementing cells such that the virus replicates; and c) harvesting said replicated virus from the complementing cells.

In some embodiments of any of the aspects, the complementary cells further express TetR.

In some embodiments of any of the aspects, the complementary cells express ICP0 functional gene product.

In some embodiments of any of the aspects, the complementary cells express UL19 (VP5) functional gene product.

In one aspect of any of the embodiments, described herein is a composition comprising a vaccine comprising a recombinant virus described herein for use in unit dose form in the treatment of an infection with HSV-1 or HSV-2.

In one aspect of any of the embodiments, described herein is a composition comprising a virus for use in the treatment of an infection with HSV-1 or HSV-2, the composition comprising; a) infecting complementing cells with the virus, wherein the complementing cells express a functional gene product or products that are needed for replication of the virus and for which sequences encoding such are lacking from the virus genome; b) culturing the complementing cells such that the virus replicates; and c) harvesting said replicated virus from the complementing cells.

Definitions

As described above, the genome of the HSV-2 virus according to the invention has a first coding sequence which codes for gB2 and which is operably linked to a first promoter (e.g. an HSV-1 or HSV-2 immediate early promoter) that is under the control of (operably linked to) a first tetracycline operator (tet-O) nucleotide segment. The genome also includes two sequences encoding HSV-2 gD (gD2), a “second coding sequence” and a “third coding sequence”. In one embodiment, at least one sequence encoding HSV-2 gD2 is operably linked to a promoter under control of a tet-O nucleotide segment. In one embodiment, the second coding sequence is operably linked to a second promoter under the control of a second tet-O nucleotide segment and the third coding sequence is operably linked to a third promoter under the control of a third tet-O nucleotide segment.

In one embodiment of various aspects, in order to control viral replication, the genome comprises a fourth coding sequence and that codes for a dominant negative mutant HSV-1 or HSV-2 UL9 protein. In some embodiment, there is also a fifth coding sequence that codes for a dominant negative mutant HSV-1 or HSV-2 UL9 protein. In some embodiments, the dominant negative mutant of the fourth and/or fifth sequence is UL9-C535C. When this sequence is expressed, the mutant protein produced acts in trans to inhibit the replication of HSV-2. The fourth coding sequence is operably linked to a fourth promoter, and this is operably linked to a fourth tet-O sequence. A second copy of a dominant negative mutant HSV-1 or HSV-2 UL9 protein (a “fifth coding sequence,” e.g., UL9-C535C) may also be present and is operably linked to a fifth promoter under the control of a fifth tet-O nucleotide segment.

In certain embodiments of various aspects, in order to control viral replication, the genome of the HSV-2 recombinant virus lacks the sequence encoding the essential UL19 (VP5) protein. In such embodiments, it is not necessary to have a dominant negative UL9 protein for replication incompetency.

Each of the tet operators allows transcription to proceed from its associated promoter when free of tet repressor (tetR) but represses transcription when bound by repressor.

In addition, the genome of the virus is characterized by the absence of a sequence encoding a functional ICP0 protein.

In order to enhance its antigenicity, the genome may in certain embodiments also express recombinant immunomodulating genes, such as IL-12, IL-15 or may express other HSV-1 or HSV-2 major antigens in levels comparable to, or higher than, the levels expressed by wild type HSV-1 or HSV-2 viruses.

The genome of the HSV-2 virus according to the invention lacks a sequence encoding a functional ICP0 protein. In certain embodiments, the second and third coding sequences (encoding HSV-2 gD, also referred to as gD2 herein) are inserted into the position where ICP0 genes are present in wild-type HSV-2, therewith disrupting the ICP0 genes (e.g. as inFIGS. 2 and 3herein, see also e.g. U.S. Pat. No. 8,809,047 for a description of such embodiments, incorporated by reference herein).

In some embodiments of various aspects, the genome of the HSV-2 virus according to the invention also lacks a sequence encoding a functional UL19 protein. In certain embodiments, the second and/or third coding sequences are inserted at the UL19 locus. In certain embodiments, the second and/or third coding sequences are inserted at the UL26/UL27 intergenic region. Insertion of a coding sequence into an intergenic region of the HSV genome, e.g., UL26 and UL27 intergenic region, does not disrupt or delete other HSV genes; this is an advantageous strategy.

In certain embodiments of various aspects, gD2 is in a bidirectional expression cassette, e.g. with UL9-C535C (e.g. as inFIG. 2herein), as for instance described in more detail in U.S. Pat. No. 8,809,047, incorporated by reference herein.

In certain embodiments of various aspects, gD2 is in a one-directional expression cassette, e.g. positioned at the UL19 locus, or at the UL26/UL27 intergenic region. In certain embodiments, one gD2 expression cassette is positioned at the UL19 locus and another gD2 expression cassette is positioned at the UL26/UL27 intergenic region, and preferably the nucleotide sequences of these two gD2 expression cassettes are different, while the gD2 protein sequences encoded by these two expression cassettes may be different but preferably are the same.

As used herein, the term “operably linked” refers to genetic elements that are joined together in a manner that enables them to carry out their normal functions. For example, a gene is operably linked to a promoter when its transcription is under the control of the promoter and this transcription results in the production of the product normally encoded by the gene. A tet operator sequence is operably linked to a promoter when the operator blocks transcription from the promoter in the presence of bound tet repressor but allows transcription in the absence of the repressor. The term “recombinant” refers to a virus that has nucleic acid sequences that are the result of recombining nucleic acid sequences and sequence elements and introducing these recombined sequences into the wildtype virus or into a recombinant ancestor virus.

In certain embodiments of various aspects, the first coding sequence of HSV-2 gB (also referred to as gB2 herein) is a codon optimized sequence. For the purposes of the present invention, a “codon optimized”, or “codon diversified” sequence is one in which one or more codons in the wild type sequence are replaced with an alternative codon that codes for the same amino acid and that results in increased protein production. In a preferred embodiment, the nucleic acid encoding HSV-2 gB2 is codon optimized for expression in mammalian cells, preferably human cells. Methods of codon-optimization are known and have been described previously (e.g. WO 96/09378). A sequence is considered codon optimized if at least one non-preferred codon as compared to a wild type sequence is replaced by a codon that is more preferred. Herein, a non-preferred codon is a codon that is used less frequently in an organism than another codon coding for the same amino acid, and a codon that is more preferred is a codon that is used more frequently in an organism than a non-preferred codon. The frequency of codon usage for a specific organism can be found in codon frequency tables, such as in http://www.kazusa.or.jp/codon. Preferably more than one non-preferred codon, e.g. more than 10%, 40%, 60%, 80% of non-preferred codons, preferably most (e.g. at least 90%) or all non-preferred codons, are replaced by codons that are more preferred. Preferably the most frequently used codons in an organism are used in a codon-optimized sequence. Replacement by preferred codons generally leads to higher expression. A non-limiting example of a codon-optimized sequence for HSV2 gB2 is provided herein in SEQ ID NO: 07. Optionally other coding sequences, e.g. one or more of the gD2 coding sequences, may also be codon-optimized.

In one embodiment of various aspects, at least one of the first, second and third promoters are HSV-1 or HSV-2 immediate early promoters. ICP0 and ICP4 promoters are particularly preferred. In one embodiment, each of the first, second and third promoters are HSV-1 or HSV-2 immediate early promoters, e.g. ICP0 and ICP4. In certain embodiments, the second and third promoters operably linked to HSV-2 gD are tetO-containing ICP4 promoters. In certain embodiments of various aspects, at least one of the second and third promoters operably linked to HSV-2 gD2 is a tetO-containing HSV ICP27 promoter.

In one embodiment of various aspects, the first promoter, second promotor, or third promoter are a human cytomegalovirus (hCMV) immediate early promoter.

In embodiments of various aspects where the fourth coding sequence is present, this fourth coding sequence preferably encodes UL9-C535C. In a further embodiment, the fifth coding sequence is present and encodes UL9-C535C. As with the other coding sequences, the fourth and fifth coding sequences may be operably linked to an HSV-1 or HSV-2 immediate early promoter, but in these cases, hCMV immediate early promoters are most preferred.

In one aspect, the replication defective HSV-2 of the invention does not comprise a dominant negative UL9 mutant and is rendered replication incompetent by deletion of the essential expression of UL19, i.e. the replication defective HSV-2 does not express functional UL19 (VP5), does not express functional ICP0, and does not express functional gG2. In one embodiment of this aspect, recombinant gD2 is located in a one directional expression cassette and positioned at the UL19 locus and/or positioned in the UL26/27 intergenic region. In another embodiment, recombinant gD2 is located in a first one directional expression cassette positioned at the UL19 locus and in a second one directional expression cassette positioned in the UL26/27 intergenic region. In yet another embodiment, recombinant gD2 is located in a first one directional expression cassette positioned at the UL19 locus and is operably linked to a first promoter, wherein said first promoter is an HSV-1 or HSV-2 ICP0 or ICP4 promoter that is operably linked to a first tet-O sequence, and recombinant gD2 is located in a second one directional expression cassette positioned in the UL26/27 intergenic region and is operably linked to a second promoter, wherein said second promoter is an HSV-1 or HSV-2 ICP0 or ICP4 promoter that is operably linked to a second tet-O sequence.

In another embodiment of this aspect, at least one of the second and third promoters that are operably linked to gD2 in a tetO-containing HSV-1 or HSV-2 ICP27 promoter. In one embodiment both the second and the third promoter are a tetO-containing HSV-1 or HSV-2 ICP27 promoter.

In one embodiment of various aspects, the first coding sequence is operably linked to an ICP0 promoter which may optionally be modified so that the TATA element normally present (TATAAGTT (SEQ ID NO: 12)) corresponds to the hCMV TATA element (TATATAAG (SEQ ID NO: 13)), the second and third coding sequences are operably linked to ICP4 promoters, and the fourth and fifth (if present) coding sequences are operably linked to hCMV immediate early promoters, and each promoter is operably linked to a tetO sequence.

In order to obtain optimal results, the promoters used should have a TATA element and the tet operator sequences linked to the promoters should preferably have two op2 repressor binding sequences. The positioning of the operator sequence is important in achieving effective control over the promoter. Specifically, the first nucleotide in the operator sequence is located between six and twenty-four nucleotides 3′ to the last nucleotide in the TATA element (see e.g., U.S. Pat. No. 6,444,871 or 5,972,650). Structural sequences encoding, for example gB2, gD2 or UL9-C535C, are positioned 3′ to the operator.

Accordingly, in certain embodiments of various aspects, a) the first, second, third, and optionally the fourth and fifth promoters of the replication-defective dominant negative HSV-2 virus each have a TATA element; b) each of the first, second, third, and optional fourth and fifth tet-O sequences comprise two op2 repressor binding sites, wherein the first nucleotide in said tet operator is between 6 and 24 nucleotides 3′ to the last nucleotide in said TATA element; c) the first sequence, encoding HSV-2 gB, lies 3′ to said first tet-O sequence, and said first tet-O sequence is operably linked to said first promoter; d) the second sequence, encoding HSV-2 gD, lies 3′ to the second tet-O sequence and said second tet-O sequence is operably linked to the second promoter; e) the third sequence, encoding HSV-2 gD, lies 3′ to said third tet-O sequence and said third tet-O sequence is operably linked to the third promoter; and in some embodiments, f) a fourth sequence, encoding a dominant negative mutant HSV-1 or HSV-2 UL9 protein, lies 3′ to said fourth tet-O sequence and the fourth tet-O sequence is operably linked to said fourth promoter; and, in some embodiments, a fifth sequence, encoding a dominant negative mutant HSV-1 or HSV-2 UL9 protein, lies 3′ to said fifth tet-O sequence, and said fifth tet-O sequence is operably linked to said fifth promoter.

In one specific aspect of the invention, a replication-defective HSV-2 recombinant virus, is provided that comprises within its genome: (a) a first coding sequence, comprising a codon optimized HSV-2 gB2 sequence operably linked to a first promoter, wherein said first promoter is an HSV-1 or HSV-2 ICP0 or ICP4 promoter that is operably linked to a first tet-O sequence and wherein said first coding sequence is preferably displaces the gG2 gene; (b) a second coding sequence, comprising a codon-optimized HSV gD2 sequence operably linked to a second promoter, wherein said second promoter is an HSV-1 ICP4 promoter that is operably linked to a second tet-O sequence and wherein said second coding sequence is located at the UL26/UL27 intergenic region; (c) a third coding sequence, comprising a codon-diversified HSV gD2 sequence operably linked to a third promoter, wherein said third promoter is an HSV-1 ICP27 promoter or an HSV-1 ICP4 promoter that is operably linked to a third tet-O sequence, wherein the third coding sequence displaces the UL19 gene; and wherein said genome does not comprise a sequence encoding a functional ICP0 protein, and does not comprise a sequence encoding HSV-2 gG2 protein.

In one specific aspect, a replication-defective, dominant-negative HSV-2 recombinant virus, is provided that comprises within its genome: (a) a first coding sequence, comprising a codon optimized HSV-2 gB2 sequence operably linked to a first promoter, wherein said first promoter is an HSV-1 or HSV-2 ICP0 or ICP4 promoter that is operably linked to a first tet-O sequence; (b) a second coding sequence, comprising an HSV-2 gD2 sequence operably linked to a second promoter, wherein said second promoter is an HSV-1 or HSV-2 ICP0 or ICP4 promoter that is operably linked to a second tet-O sequence; (c) a third coding sequence, comprising an HSV-2 gD2 sequence operably linked to a third promoter, wherein said third promoter is an HSV-1 or HSV-2 ICP0 or ICP4 promoter that is operably linked to a third tet-O sequence; (d) a fourth coding sequence, encoding a UL9-C535C protein, and operably linked to a fourth promoter, wherein said fourth promoter is an hCMV immediate early promoter that is operably linked to a fourth tet-O sequence; (d) a fifth sequence, encoding a UL9-C535C protein, and operably linked to a fifth promoter, wherein said fifth promoter is an hCMV immediate early promoter that is operably linked to a fifth tet-O sequence; and wherein said genome does not comprise a sequence encoding a functional ICP0 protein. In certain embodiments, i) said first, second, third, fourth and fifth promoters each have a TATA element; ii) each of said first, second, third, fourth and fifth tet-O sequences comprise two op2 repressor binding sites, wherein the first nucleotide in said tet operator is between 6 and 24 nucleotides 3′ to the last nucleotide in said TATA element; and iii) said first, second, third, fourth and fifth coding sequences each lie 3′ to their respective tet-O sequences, and said tet-O sequences are operably linked to their respective promoters.

In another aspect of the invention, a replication defective HSV recombinant virus (e.g. HSV-1 or HSV-2) is provided that comprises a modified HSV-1 or HSV-2 ICP0 promoter comprising a hCMV TATA element, wherein said modified promoter is operably linked to a transgene. In one embodiment, the modified promoter comprises SEQ ID NO: 08. In one embodiment, the transgene encodes HSV-2 glycoprotein B (gB2). Replication-defective genomic HSV vectors are known in the art and are described in, e.g., Burton et al,Current Opinion in Molecular Therapeutics7(4):326-336 (2005); Mundle et al.PLoS ONE8(2): e57224, (2013); Akhrameyeva et al.J. Virol.85(10): 5036-5047 (2011); and Johnston et al.,Vaccine32 (14):1553-1560 (2014). Such recombinant viruses may be used in both vaccine applications and in gene therapy applications to express a transgene of interest.

In still another aspect, the invention is directed to a vaccine that can be used prophylactically or therapeutically against HSV-2 infection and which comprises one or more of the recombinant viruses described above in unit dose form. The term “unit dose form” refers to a single drug administration entity, e.g., as a syringe, tablet, or capsule. Preferably the “unit dose form” will be a solution in which the drug (e.g., a vaccine virus described herein) is suspended at a concentration that provides a therapeutic or prophylactic effect when a selected volume (unit dose) is administered to a patient by injection and will be found within an injection vial. It is believed that the minimum effective dose in a human should be between about 1×106and 1×108plaque-forming units (PFU). Thus, a unit dose should have at least this amount of virus, with 1×106-1×109PFU or 1×107-1×109PFU being typical. Vaccines may be stored in a lyophilized form and reconstituted in a pharmaceutically acceptable carrier prior to administration. Alternatively, preparations may be stored in the vehicle itself. The volume of a single dose of the vaccine will vary but, in general, should be between about 0.1 mL and 10 mL and, more typically, between about 0.2 mL and 5 mL, e.g., about 0.5 mL, 1 mL or 2 mL.

The invention also includes methods of immunizing subjects against HSV-1 or HSV-2 infection and the conditions resulting from such infection (e.g., genital herpes ulcers) by administering to the subjects the vaccines described above. The vaccines may also be given to patients that have been infected to prevent or reduce outbreaks of the virus. Any method for administering a vaccine to a patient which does not result in the destruction of vaccine virus is compatible with the present invention. Generally, administration will be by parenteral means such as by intramuscular or intradermal injection. The dosage and scheduling of administration of vaccines can be determined using methods that are routine in the art. The preparations may be administered in either single or multiple injections.

In another aspect, the invention relates to a method for producing any of the viruses described herein comprising: (a) infecting complementing cells with the virus, wherein the complementing cells express a functional gene product or products that are needed for replication of the virus and for which sequences encoding such are lacking from the virus genome; (b) culturing the complementing cells such that the virus replicates; and (c) harvesting said replicated virus from the complementing cells.

In one embodiment, the complementing cells further express a tetR to repress expression from the tetO-regulated promoters. In certain embodiments, the complementing cells express at least one functional gene product selected from ICP0, UL9, or UL19 (VP5). In certain embodiments, the complementing cells express tetR and ICP0. In certain embodiments, the complementing cells express tetR, ICP0 and UL19 (VP5).

As used herein, a “functional gene product or products that are needed for replication of the virus” refers to any gene product or products required for, e.g., the genetic replication of the virus in a host cell. These gene product(s) include, but are not limited to, gene products required for transcription of immediate-early, early, or late gene products (e.g., α-TIF); immediate-early, early, or late gene products; gene products required for virion host shutoff (e.g., VHS, and UL41); and gene products required for production of, e.g., the capsid, viral envelope, or viral surface receptors. One skilled in the art will be able to determine those gene product(s) required for replication of the viruses described herein.

As used herein, a “subject” means a human or animal. In one embodiment, the animal is a vertebrate such as a primate, rodent, domestic animal, or game animal. In one embodiment, the subject is human. The terms, “patient”, “individual” and “subject” are used interchangeably herein.

