Therapeutic viral vaccine

Abstract

To provide compositions for treating a viral infection in a subject and, in particular, a herpes virus infection.SOLUTION: Provided is a composition comprising an Fc receptor (FcR) derived from a virus or an immunogenic fragment thereof for use in a method for treating a subject infected with the virus, the ability of the FcR or immunogenic fragment thereof to bind to a human antibody Fc domain being reduced or abolished compared to the corresponding native viral FcR, and the method not comprising administration of an immunodominant viral antigen to the subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to viral Fc receptors or immunogenic fragments thereof for treating viral infections, particularly herpes virus infections, in a subject. [Background technology]

[0002] Herpes simplex viruses (HSV, including HSV1 and HSV2) are members of the Alphaherpesvirinae subfamily (α-herpesviruses) of the Herpesviridae family. Herpes simplex viruses are enveloped, double-stranded DNA viruses containing at least 74 genes encoding functional proteins. HSV1 and HSV2 infect mucosal epithelial cells and establish lifelong, persistent infections in sensory neurons innervating the mucosa where the primary infection occurred. Both HSV1 and HSV2 can periodically reactivate from established latency in neuronal cell bodies, resulting in either cold sores (herpes simplex) or genital herpes (GH).

[0003] The global prevalence of genital herpes is estimated at 417 million individuals between the ages of 15 and 49, with a disproportionately high disease burden in Africa. HSV1 is roughly as common as HSV2 as a cause of initial genital herpes in resource-rich countries. Recurrent infections are less common after HSV1 than HSV2 genital infections. Thus, HSV2 remains the predominant cause of recurrent genital herpes. While some infected individuals develop severe and frequent genital ulcers, while others have mild or asymptomatic infections, all individuals are at risk of transmitting genital herpes to their intimate partners.

[0004] Recurrent GH is the result of reactivation of HSV2 (and to some extent HSV1) from the sacral ganglia, followed by anterograde migration of viral capsids along neuronal axons, thus leading to assembly of viral particles, cell-cell fusion, viral spread, and infection of surrounding epithelial cells from the genital mucosa.

[0005] Antiviral agents, such as acyclovir, valacyclovir, and famciclovir, are used to treat GH in both primary and recurrent infections and regardless of HSV1 or HSV2 origin. These drugs do not eradicate the virus from the host because their biological mechanism of action blocks or interferes with the viral replication machinery. Randomized controlled trials have demonstrated that short-term treatment with any of these three drugs reduces the severity and duration of symptomatic recurrences by 1 to 2 days when initiated early after the onset of symptoms or clinical signs of recurrence. However, such intermittent regimens do not reduce the number of recurrences per year.

[0006] Current treatment options for HSV recurrence have limitations, including incomplete antiviral efficacy, short-term effectiveness, adherence to treatment regimens, emergence of antiviral resistance, cost of treatment, and side effects. Currently, no known strategies offer long-term prevention of symptomatic recurrence.

[0007] Human cytomegalovirus (HCMV) is a double-stranded DNA virus of the β-herpesvirinae subfamily of the Herpesviridae family. Congenital HCMV infection is a leading cause of hearing loss, vision loss, and neurological disorders in newborns. In addition, HCMV causes life-threatening illness in immunocompromised individuals, such as AIDS subjects and transplant recipients. Summary of the Invention

[0008] Thus, there is a need in the art for improved treatments for recurrent herpesvirus infections, particularly HSV2, HSV1 and HCMV infections.

[0009] In one aspect, the present invention provides an Fc receptor (FcR) or an immunogenic fragment thereof from said virus, preferably for use in therapy for treating a subject infected with said virus.

[0010] In one aspect, the present invention provides a recombinant viral FcR or immunogenic fragment thereof, wherein the ability of the viral FcR or immunogenic fragment thereof to bind to a human antibody Fc domain is reduced or abolished compared to the corresponding native viral Fc receptor.

[0011] In another aspect, the present invention provides a heterodimer comprising or consisting of an Fc receptor from an HSV virus or an immunogenic fragment thereof and a binding partner from said HSV virus or a fragment thereof for use in therapy.

[0012] In a further aspect, the present invention provides a nucleic acid encoding a viral Fc receptor or an immunogenic fragment or heterodimer thereof of the present invention.

[0013] In a further aspect, the present invention provides a vector comprising a nucleic acid according to the present invention.

[0014] In a further aspect, the present invention provides a cell comprising a viral Fc receptor or fragment thereof, heterodimer, nucleic acid or vector according to the invention.

[0015] In one aspect, the invention provides an immunogenic composition (or "therapeutic vaccine") comprising an Fc receptor or immunogenic fragment thereof from a virus as described herein or a nucleic acid and a pharmaceutically acceptable carrier. Suitably, the immunogenic composition can be prepared for administration to a subject by being suspended or dissolved in a pharmaceutically or physiologically acceptable carrier.

[0016] In one aspect, the invention provides a herpesvirus Fc receptor or an immunogenic fragment thereof, or a nucleic acid encoding said viral FcR or an immunogenic fragment thereof, for use in the treatment of a recurrent herpes infection, or in a method for preventing or reducing the frequency of recurrent herpesvirus infections, in a subject, preferably a human subject.

[0017] In one aspect, the invention provides HSV2 gE2 or an immunogenic fragment thereof, or a nucleic acid encoding said HSV2 gE2 or an immunogenic fragment thereof, for use in the treatment of a recurrent HSV2 infection, or for use in a method for preventing or reducing the frequency of recurrent HSV2 infection, in a subject, preferably a human subject.

[0018] In one aspect, the present invention provides an HSV2 gE2 / gI2 heterodimer or an immunogenic fragment thereof, or a nucleic acid encoding said HSV2 gE2 / gI2 heterodimer or an immunogenic fragment thereof, for use in the treatment of a recurrent HSV2 infection, or for use in a method for preventing or reducing the frequency of recurrent HSV2 infection, in a subject, preferably a human subject.

[0019] In one aspect, the invention provides HSV1 gE1 or an immunogenic fragment thereof, or a nucleic acid encoding said HSV1 gE1 or an immunogenic fragment thereof, for use in the treatment of recurrent HSV1 infection, or for use in a method for preventing or reducing the frequency of recurrent HSV1 infection, in a subject, preferably a human subject.

[0020] In one aspect, the present invention provides an HSV1 gE1 / gI1 heterodimer or an immunogenic fragment thereof, or a nucleic acid encoding said HSV1 gE1 / gI1 heterodimer or an immunogenic fragment thereof, for use in the treatment of recurrent HSV1 infection, or for use in a method for preventing or reducing the frequency of recurrent HSV1 infection, in a subject, preferably a human subject.

[0021] In one aspect, the invention provides a herpesvirus Fc receptor or an immunogenic fragment thereof, or a nucleic acid encoding said viral FcR or an immunogenic fragment thereof, as described herein, for use in the manufacture of an immunogenic composition.

[0022] In one aspect, the invention provides the use of a herpesvirus Fc receptor or an immunogenic fragment thereof, or a nucleic acid encoding said viral FcR or an immunogenic fragment thereof, as described herein, in the manufacture of a medicament for the treatment of a herpes infection or a herpes-related disease.

[0023] In one aspect, the present invention provides an HSV2 gE2 or HSV2 gE2 / gI2 heterodimer, an immunogenic fragment thereof, or a nucleic acid encoding said HSV2 gE2 or an immunogenic fragment thereof, as described herein, for use in the manufacture of an immunogenic composition.

[0024] In one aspect, the invention provides the use of HSV2 gE2 or HSV2 gE2 / gI2 heterodimer, an immunogenic fragment thereof, or a nucleic acid encoding said HSV2 gE2 or HSV2 gE2 / gI2 heterodimer or an immunogenic fragment thereof, as described herein, in the manufacture of a medicament for the treatment of HSV2 infection or an HSV2-associated disease.

[0025] In one aspect, the present invention provides an HSV1 gE1 or HSV1 gE1 / gI1 heterodimer, an immunogenic fragment thereof, or a nucleic acid encoding said HSV1 gE1 or HSV1 gE1 / gI1 heterodimer or an immunogenic fragment thereof, as described herein, for use in the manufacture of an immunogenic composition.

[0026] In one aspect, the invention provides the use of HSV1 gE1 or HSV1 gE1 / gI1 heterodimer, an immunogenic fragment thereof, or a nucleic acid encoding said HSV1 gE1 or HSV1 gE1 / gI1 heterodimer or an immunogenic fragment thereof, as described herein, in the manufacture of a medicament for the treatment of HSV1 infection or an HSV1-associated disease.

[0027] In one aspect, the invention provides a method of treating a herpesvirus infection or a herpesvirus-associated disease in a subject in need thereof, comprising administering to the subject an immunologically effective amount of a herpesvirus Fc receptor or an immunogenic fragment thereof, or a nucleic acid encoding said viral FcR or an immunogenic fragment thereof.

[0028] In one aspect, the present invention provides a method for treating an HSV2 infection or an HSV2-associated disease in a subject in need thereof, comprising administering to the subject an immunologically effective amount of HSV2 gE2 or an HSV2 gE2 / gI2 heterodimer, an immunogenic fragment thereof, or a nucleic acid encoding the HSV2 gE2 or HSV2 gE2 / gI2 heterodimer or an immunogenic fragment thereof.

[0029] In one aspect, the present invention provides a method for treating an HSV1 infection or an HSV1-associated disease in a subject in need thereof, comprising administering to the subject an immunologically effective amount of HSV1 gE1 or an HSV1 gE1 / gI1 heterodimer, an immunogenic fragment thereof, or a nucleic acid encoding the HSV1 gE1 or HSV1 gE1 / gI1 heterodimer or an immunogenic fragment thereof.

[0030] In one aspect, there is provided a kit comprising or consisting of a viral Fc receptor or immunogenic fragment thereof described herein and an adjuvant. [Brief explanation of the drawings]

[0031] [Figure 1] Annotated amino acid sequences of HSV2 gE (UniprotKB: A7U881) and HSV1 gE (UniprotKB: Q703E9). Sequence alignment using GAP in EBIO, gap weight: 8, length weight: 2, similarity: 78.68%, identity: 76.10%. Underlined: signal peptide (SP), bold underlined: transmembrane domain, italic underlined: Fc binding region, bold italic: region required for heterodimeric complex formation. [Figure 2]Annotated amino acid sequences of HSV2 gI (UniprotKB: A8U5L5) and HSV1 gI (UniprotKB: P06487). Sequence alignment using GAP in EBIO, gap weight: 8, length weight: 2, similarity: 73.37%, identity: 70.38%. Underlined: signal peptide (SP), bold underlined: transmembrane domain, bold italic: region required for heterodimeric complex formation. [Figure 3-1] Alignment of HSV2 gE ectodomain protein sequences. Black / dark grey / light grey shading: indicates 100% / 80% / 60% similarity, respectively, across all aligned sequences. [Figure 3-2] This is a continuation of Figure 3-1. [Figure 3-3] This is a continuation of Figure 3-2. [Figure 3-4] This is a continuation of Figure 3-3. [Figure 3-5] This is a continuation of Figure 3-4. [Figure 3-6] This is a continuation of Figure 3-5. [Figure 3-7] This is a continuation of Figure 3-6. [Figure 3-8] This is a continuation of Figure 3-7. [Figure 3-9] This is a continuation of Figure 3-8. [Figure 3-10] This is a continuation of Figure 3-9. [Figure 3-11] This is a continuation of Figure 3-10. [Figure 4-1] Alignment of HSV2 gI ectodomain protein sequences. Black / dark grey / light grey shading: indicates 100% / 80% / 60% similarity, respectively, across all aligned sequences. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 4-3] This is a continuation of Figure 4-2. [Figure 4-4] This is a continuation of Figure 4-3. [Figure 4-5] This is a continuation of Figure 4-4. [Figure 4-6] This is a continuation of Figure 4-5. [Figure 4-7]This is a continuation of Figure 4-6. [Figure 4-8] This is a continuation of Figure 4-7. [Figure 5] HSV-2 gE-specific CD4+ T cell responses elicited in CB6F1 mice after the first (day 14), second (day 28), or third (day 42) immunization with AS01-adjuvanted HSV-2 gE or HSV-2 gE / gI proteins. Circle, triangle, and diamond plots represent CD4+ T cell responses for individual mice at days 14 (14PI), 28 (14PII), and 42 (14PIII) after primary immunization, respectively. Dashed lines represent the 95th percentile (0.19%) of the NaCl data at different time points. [Figure 6] HSV-2 gE-specific CD4+ T cell responses elicited in CB6F1 mice after the second (day 28) or third (day 42) immunization with AS01-adjuvanted HSV-2 gE or HSV-2 gE / gI proteins from two independent experiments (Experiment B-Experiment A). Ten mice per group (six in Experiment B and four in Experiment A). Triangle and diamond plots represent CD4+ T cell responses for individual mice at days 28 (14PII) and 42 (14PIII) after primary immunization, respectively. The dashed line represents the 95th percentile (0.19%) of the NaCl data on both days. [Figure 7] HSV-2 gI-specific CD4+ T cell responses elicited in CB6F1 mice after the first (day 14), second (day 28), or third (day 42) immunization with AS01-adjuvanted HSV-2 gE / gI protein. Circle, triangle, and diamond plots represent CD4+ T cell responses for individual mice at days 14 (14PI), 28 (14PII), and 42 (14PIII) after primary immunization, respectively. Dashed lines represent the 95th percentile (0.32%) of the NaCl data at different time points. [Figure 8]HSV-2 gE-specific CD8+ T cell responses elicited in CB6F1 mice after the first (day 14), second (day 28), or third (day 42) immunization with AS01-adjuvanted HSV-2 gE or HSV-2 gE / gI proteins. Circle, triangle, and diamond plots represent CD8+ T cell responses for individual mice at days 14 (14PI), 28 (14PII), and 42 (14PIII) after primary immunization, respectively. Dashed lines represent the 95th percentile (0.12%) of the NaCl data at different time points. [Figure 9] HSV-2 gI-specific CD8+ T cell responses elicited in CB6F1 mice after the first (day 14), second (day 28), or third (day 42) immunization with AS01-adjuvanted HSV-2 gE / gI protein. Circle, triangle, and diamond plots represent CD8+ T cell responses for individual mice at days 14 (14PI), 28 (14PII), and 42 (14PIII) after primary immunization, respectively. Dashed lines represent the 95th percentile (0.43%) of the NaCl data at different time points. [Figure 10] Frequency of follicular B helper CD4+ T (Tfh) cells detected in draining lymph nodes 10 days after immunization with AS01-adjuvanted HSV-2 gE or HSV-2 gE / gI heterodimeric protein. Each plot represents an individual mouse, and the median response for each group is indicated by a horizontal line. [Figure 11] Frequency of activated B cells detected in draining lymph nodes 10 days after immunization with AS01-adjuvanted HSV-2 gE or HSV-2 gE / gI heterodimeric protein. Each plot represents an individual mouse, and the median response for each group is indicated by a horizontal line. [Figure 12]Total titers of HSV-2 gE-specific IgG antibodies measured by ELISA on sera collected after the first (day 14), second (day 28), or third (day 42) immunization with AS01-adjuvanted HSV-2 gE or HSV-2 gE / gI proteins. Circle, triangle, and diamond plots represent IgG antibody titers for individual mice at days 14 (14PI), 28 (14PII), and 42 (14PIII) after primary immunization, respectively. [Figure 13] Total titers of HSV-2 gE-specific IgG antibodies from two independent experiments (Experiment B-Experiment A) elicited after the first (day 14), second (day 28), or third (day 42) immunization with AS01-adjuvanted HSV-2 gE or HSV-2 gE / gI protein. Ten mice per group (six in Experiment B and four in Experiment A). Circle, triangle, and diamond plots represent gE-specific IgG antibody titers for individual mice from two independent experiments at days 14 (14PI), 28 (14PII), and 42 (14PIII) after primary immunization. [Figure 14] Total titers of HSV-2 gI-specific IgG antibodies measured by ELISA on sera collected after the first (day 14), second (day 28), or third (day 42) immunization with AS01-adjuvanted HSV-2 gE / gI heterodimer protein. Circle, triangle, and diamond plots represent IgG antibody titers for individual mice at days 14 (14PI), 28 (14PII), and 42 (14PIII) after primary immunization, respectively. [Figure 15] HSV-2 MS-specific neutralizing antibody titers in serum samples collected 14 days after the first, second, or third immunization with AS01-adjuvanted HSV-2 gE or gE / gI proteins. Each dot represents an individual mouse, and the median response is represented by the horizontal line. The dashed line indicates the threshold for positivity corresponding to the first sample dilution. Samples without neutralizing activity are indicated by a value of 5 (first sample dilution / 2). The sample used for the positive control (gD / AS01) was obtained from a different in vivo experiment. [Figure 16]Figure 1. The ability of gE / gI-specific antibodies to bind murine FcγRIV (mFCgRIV) was assessed 14 days after the first, second, or third immunization with AS01-adjuvanted HSV-2 gE or HSV-2 gE / gI proteins. Each dot represents the area under the curve (AUC) for an individual mouse, with the median response represented by the horizontal line. In the NaCl control group, negative AUC values ​​were arbitrarily set to 1. [Figure 17] Ratios of total proliferation rates of gE- and gI-specific CD4+ (A) and CD8+ (B) T cells in vaccinated and unvaccinated HSV2-infected guinea pigs. The black dotted line indicates the 95th percentile of proliferation rates obtained in the saline group when three antigens (gE, gI, β-actin) were combined. Each plot represents an individual data point. The geometric mean ratio (GMR) for each group is displayed on the x-axis and is represented by a black box on the graph. [Figure 18] HSV2 gE (A) and gI (B)-specific IgG antibody titers in serum from HSV2-infected guinea pigs after one, two, and three immunizations with AS01-adjuvanted HSV2 gE or HSV2 gE / gI proteins. Each dot represents an individual animal, and black error bars represent the geometric mean + 95% CI for each group. The geometric mean (GM) value for each group is displayed on the x-axis and is represented by a black square on the graph. [Figure 19] Comparison of groups and doses for the total titer (EU / mL) of HSV2 gE- or gI-specific IgG antibodies. A: Geometric mean ratios (and their 95% CI) of AS01-gE and AS01-gE / gI to the unvaccinated HSV2-infected group on days 33 (13PI), 46 (12PI), and 70 / 74 (22 / 26PI) after HSV2 infection. B: Geometric mean ratios of AS01-gE and AS01-gE / gI between each immunization dose. C: Geometric mean ratios of AS01-gE and AS01-gE / gI to the unvaccinated HSV2-infected group. D: Geometric mean ratios of AS01-gE and AS01-gE / gI between each immunization dose. [Figure 20]HSV2 MS-specific neutralizing antibody titers in serum from HSV2-infected guinea pigs after three immunizations with AS01-adjuvanted HSV-2 gE or HSV-2 gE / gI proteins. A: Each dot represents the titer of an individual animal, and the geometric mean (GM) of neutralizing titers is represented by the square dot. The threshold for positivity corresponds to the first sample dilution. Negative samples are represented by the first sample dilution / 2. B: The square dots represent the geometric mean ratio (GMR) + 95% CI for each group. The GMR for each group is also shown on the x-axis of the graph. [Figure 21] Individual cumulative lesion scores for the 34-70 day interval were calculated for each guinea pig, and the mean cumulative score for each group is also shown by the bold line. [Figure 22] Correlation of standardized cumulative scores between 0-14 days and 34-70 days. [Figure 23] Therapeutic evaluation of different AS01-formulated HSV2 recombinant protein candidates over [34-70] days in a guinea pig model of chronic genital herpes. A: Mean cumulative lesion scores (as described in statistical methodology) are shown for each group. B: Standardized cumulative lesion scores (as described in statistical methodology) are shown for individual animals (squares represent mean with 90% CI, circles represent individual data). C: Estimated reduction in mean standardized cumulative lesion score between vaccination and non-vaccination. [Figure 24] Direct comparison of standardized cumulative lesion scores between AS01-gE, AS01-gE / gI and AS01-gD2t-vaccinated groups over the [34-70] day interval. [Figure 25] Assessment of the total number of days with herpetic lesions over a period of [34-70] days following immunization with AS01-formulated HSV2 recombinant protein. A: The total number of days with lesions is shown for each animal in each group (round dots represent individual animals, and the mean response for each group is represented by square dots with a 95% confidence interval). B: The estimated mean difference in total number of days with lesions between vaccinated and unvaccinated groups is represented by square dots with a 90% confidence interval. [Figure 26] The distribution of clinical recurrences in each group over an interval of [34-47] days. [Figure 27] Therapeutic evaluation of different AS01-formulated HSV2 recombinant proteins over [34-47] and [48-70] days in a guinea pig model of chronic genital herpes. AB: Mean cumulative lesion score (as described in statistical methodology) is shown for each group and each period. CD: Standardized cumulative lesion score (as described in statistical methodology) is shown for individual animals (squares represent mean with 90% CI, circles represent individual data) and for each period. EF: Estimated reduction in mean standardized cumulative lesion score between vaccination and non-vaccination for each period. [Figure 28] Partial 3D model of the HSV2 gE-IgG Fc interface. Black: part of the gE Fc binding domain. Light grey: loops 1, 2 / 3: loops of IgG Fc that interact with gE. [Figure 29A] Evaluation of gE / gI expression in Expi293F™ cells at harvest. A) Unstained analysis by SDS-PAGE of cell culture supernatant. Untransfected cells (mock) are indicated by "(-) cells" and positive control samples are indicated by "+". The band of interest is observed between the 50 kDa and 75 kDa bands of the MW marker (Precision Plus Protein™ Unstained Protein Standard, Bio-Rad, Cat. No. 1610363). B) Western blot analysis of the samples described in A). Mouse Monoclonal Anti-poly Histidine-Peroxidase antibody (Sigma, Cat. No. A7058-1VL) was used at a 1 / 2000 dilution, followed by development with 1-Step™ Ultra TMB-Blotting Solution (ThermoFisher, Cat. No. 37574). [Figure 29B]Evaluation of gE / gI expression in Expi293F™ cells at harvest. A) Unstained analysis by SDS-PAGE of cell culture supernatant. Untransfected cells (mock) are indicated by "(-) cells" and positive control samples are indicated by "+". The band of interest is observed between the 50 kDa and 75 kDa bands of the MW marker (Precision Plus Protein™ Unstained Protein Standard, Bio-Rad, Cat. No. 1610363). B) Western blot analysis of the samples described in A). Mouse Monoclonal Anti-poly Histidine-Peroxidase antibody (Sigma, Cat. No. A7058-1VL) was used at a 1 / 2000 dilution, followed by development with 1-Step™ Ultra TMB-Blotting Solution (ThermoFisher, Cat. No. 37574). [Figure 30] Graphical representation of the binding rate constants of 25 mutants. x-axis: k on, y-axis: k off. Based on the k on / k off values ​​observed in the control, four regions are identified: fast binding, low binding, fast release, and slow release. [Figure 31] SDS-PAGE of different purified protein mutants. *: pooled samples from the void volume of size exclusion chromatography. [Figure 32] IgG binding curves for WT control and six mutant constructs of HSV2 gEgI. BLI measurements of human IgG binding to immobilized gEgI mutants compared to the WT protein control. From top to bottom: WT control - HSV44 - HSV61 - HSV57 - HSV45 - HSV49 - HSV41. The Y-axis represents BLI signal intensity in nm. [Figure 33] Protein content of gEgI mutants estimated from UPLC-SEC-UV measurements. All samples were analyzed in duplicate and reproducibility is shown. Values ​​for PhyTips-purified proteins are shown in dark gray, while values ​​for filter plate-purified proteins are shown in light gray. [Figure 34] Binding of hIgG by mutant candidates recorded by BLI (Octet). [Figure 35] Tm (°C) of mutant candidates recorded by nanoDSF at 330 nm. [Figure 36] Protein content of the candidate HSV1 mutants at the end of the purification scheme. [Figure 37] Overlay of human hIgG binding and DSF Tm data. Bars: human hIgG binding (nm) determined by Octet; Crosses: Tm (°C) determined by DSF. [Figure 38] Design of multiple ThHSV SAM vectors encoding the gEgI heterodimer. These vectors were cloned into the VEEV TC-83 SAM vector (an attenuated strain of Venezuelan Equine Encephalitis virus). A version of the HSV2 gE P317R mutant (Fc binding knockout) was also generated. A) Screening of various regulatory elements for driving gI expression. The regulatory elements selected were: i) the enterovirus 71 internal ribosome entry site (EV71 IRES), ii) two 2A peptide sequences (GSG-P2A: porcine teschovirus-1 2A with a GSG linker, F2A: 2A peptide (F2A) derived from foot-and-mouth disease virus), and iii) the 26S RNA promoter (26S promoter). The size (bp) of each regulatory element is indicated. B) The same construct as A), but with an HA tag at the C-terminus of the HSV2 gE and gI proteins. [Figure 39-1] DNA sequence of the plasmid expressing the RNA sequence of the SAM-gEgI construct. Top: SAM backbone; bottom: non-SAM sequence; underlined: 5'UTR of SAM; bold underlined: 3'UTR of SAM; grey shading: insert encoding the gEgI heterodimer. [Figure 39-2] This is a continuation of Figure 39-1. [Figure 39-3] This is a continuation of Figure 39-2. [Figure 40-1]Western blot analysis of gE and gI expression levels. BHK cells were electroporated with 100 ng of RNA. Cell culture supernatants were concentrated 10-fold and treated with PNGase to deglycosylate the proteins. Actin was used as a loading control. Left: Western blot images for the detection of gE (top) and gI (bottom). Right: Signal intensities for gE and gI band extraction. Primary rabbit anti-gE and anti-gI antibodies were used at a dilution of 1:1000, and mouse anti-actin was used at a dilution of 1:5000. Secondary Licor antibody was used at a dilution of 1:15000. Results for IRES P317R are not shown but are comparable to those for the wt IRES. [Figure 40-2] This is a continuation of Figure 40-1. [Figure 41A] Western blot analysis shows the expression level of gEgI and the stoichiometry of the construct. BHK cells were electroporated with 100 ng of RNA (HA-tagged construct). Cell culture supernatants were concentrated 10-fold and treated with PNGase to deglycosylate the proteins. Actin was used as a loading control. A) Western blot images for the detection of gE (left) and gI (right). B) Western blot images for the detection of gE-HA and gI-HA using anti-HA Ab. C) Signal intensities of the extracted gE-HA and gI-HA bands (from B), and normalization of the signals by gE intensity to determine the gE:gI ratio. Primary rabbit anti-gE, rabbit anti-gI, and mouse anti-HA antibodies were used at a dilution of 1:1000, and mouse / rabbit anti-actin was used at 1:5000. Secondary Licor antibody was used at 1:15000. [Figure 41B]Western blot analysis shows the expression level of gEgI and the stoichiometry of the construct. BHK cells were electroporated with 100 ng of RNA (HA-tagged construct). Cell culture supernatants were concentrated 10-fold and treated with PNGase to deglycosylate the proteins. Actin was used as a loading control. A) Western blot images for the detection of gE (left) and gI (right). B) Western blot images for the detection of gE-HA and gI-HA using anti-HA Ab. C) Signal intensities of the extracted gE-HA and gI-HA bands (from B), and normalization of the signals by gE intensity to determine the gE:gI ratio. Primary rabbit anti-gE, rabbit anti-gI, and mouse anti-HA antibodies were used at a dilution of 1:1000, and mouse / rabbit anti-actin was used at 1:5000. Secondary Licor antibody was used at 1:15000. [Figure 41C] Western blot analysis shows the expression level of gEgI and the stoichiometry of the construct. BHK cells were electroporated with 100 ng of RNA (HA-tagged construct). Cell culture supernatants were concentrated 10-fold and treated with PNGase to deglycosylate the proteins. Actin was used as a loading control. A) Western blot images for the detection of gE (left) and gI (right). B) Western blot images for the detection of gE-HA and gI-HA using anti-HA Ab. C) Signal intensities of the extracted gE-HA and gI-HA bands (from B), and normalization of the signals by gE intensity to determine the gE:gI ratio. Primary rabbit anti-gE, rabbit anti-gI, and mouse anti-HA antibodies were used at a dilution of 1:1000, and mouse / rabbit anti-actin was used at 1:5000. Secondary Licor antibody was used at 1:15000. [Figure 42] Agarose RNA gel. Expected MW: approximately 10.5 kb. M: Ambion® RNA Millennium™ marker. A) Candidate HSV2 SAM. B) Candidate HSV1 SAM. [Figure 43]Evaluation of gE and gI protein expression in HSV SAM constructs by WB analysis. A) HSV2 SAM candidates (963, 989). Analysis of BHK cell culture supernatants (SN) following electroporation of SAM. SN was analyzed directly (undiluted, ND) or after 2- and 4-fold dilutions (D2x and D4x, respectively). Untransfected SN was used as a negative control (mock). Purified HSV2 gEgI recombinant protein was used as a positive control. B) HSV2 SAM candidates (1188-1055). Analysis of BHK cell culture supernatants (SN) following electroporation of SAM. SN from BHK cells transfected with an unrelated SAM served as a negative control (Ctrl-), and purified HSV2 gEgI recombinant protein was used as a positive control. C) HSV1 SAM candidates (1203-1207). Analysis of BHK cell culture supernatants (SN) following electroporation of SAM. SN from BHK cells transfected with an unrelated SAM was used as a negative control (Ctrl-). Untransfected SN was used as an alternative negative control (mock). Purified HSV2 gEgI recombinant protein was used as a positive control. In both cases, anti-gE rabbit pAb (1:1000) and anti-gI rabbit pAb (1:1000) were used as primary antibodies. Anti-rabbit HRP Dako (P0448) (1:5000) was used as secondary antibody. GE Rainbow Ladder (RPN800E) was used as a MW marker. [Figure 44] HSV2 anti-gE or gI-specific IgG antibody titers detected by ELISA in the serum of CB6F1 mice immunized with 0.2 μg of AS01-adjuvanted non-mutated or mutated gEgI protein 14 days after the first and second immunizations. A. HSV2 anti-gE-specific IgG antibody titers. B. gI-specific IgG antibody titers. Each dot represents data from an individual animal, and horizontal error bars represent the geometric mean (GM) + 95% confidence interval (CI) for each group. The number of animals / group with valid results (N) and the GM for each group are indicated below the graph. [Figure 45]The titer levels of HSV2 MS-specific neutralizing antibodies detected in serum collected 14 days after two immunizations with 0.2 μg of AS01-adjuvanted HSV2 mutated and nonmutated gEgI. Sera from mice immunized with HSV2 gD-AS01 (2.5 μg) in a previous experiment were tested in duplicate and served as a positive control for the assay. Each dot represents data from an individual mouse, and horizontal error bars represent the geometric mean + 95% CI for each group. The number of animals / group with valid results (N) and the geometric mean (GM) for each group are indicated below the graph. The dashed line indicates the threshold for positivity corresponding to the first sample dilution. Samples without neutralizing activity are designated with a value of 5 (first sample dilution / 2). [Figure 46] Evaluation of the ability of AS01-adjuvanted HSV2 mutated and non-mutated gEgI to induce vaccine-specific antibodies capable of reducing human IgG Fc binding by gEgI protein. Mice were immunized with 0.2 μg of AS01-adjuvanted gEgI protein. A. HSV41 (insert gE_ARAA / gI). B. HSV45 (gE_P317R / gI). C. HSV57 (gE_P319D / gI). D. HSV61 (gE_R320D / gI). Each curve represents data obtained with one pool. [Figure 47] Figure 1 shows the levels of HSV2 gE- and gI-specific CD4+ / CD8+ T cell responses elicited after two immunizations of CB6F1 mice with 0.2 μg of AS01-adjuvanted HSV2 mutated or nonmutated gEgI protein. gEgI-specific CD4+ T (A) and CD8+ T (B) cell responses in the spleen at 28 days post-primary immunization (14P1I). Circles, triangles, and diamonds represent the individual percentages of CD4+ and CD8+ T cell responses detected against each antigen (HSV2 gE antigen, gI antigen, or β-actin). The black squares represent the geometric mean (GM) of the responses, and the dotted line indicates the 95th percentile obtained in the saline-treated group for the combination of three antigens (gE, gI, and β-actin). The number of animals / groups with valid results (N) and the GM for each group are indicated below the graph. [Figure 48]Geometric mean ratios of HSV2 gE- and gI-specific CD4+ T cell responses detected 14 days after two immunizations in mice immunized with 0.2 μg of mutated gEgI protein versus 0.2 μg of non-mutated gEgI protein. Horizontal error bars indicate the 90% confidence interval (CI) for each group. The geometric mean ratio (GMR), lower and upper CIs are shown below the graph. [Figure 49] Total titers of HSV2 gE (A) or gI (B)-specific IgG antibodies measured in serum samples collected after immunization with different mutant forms of AS01-adjuvanted HSV2 gEgI. Each symbol represents an individual animal in 14PI (dots), 14PII (triangles), or 14PIII (diamonds), and the horizontal bar represents the geometric mean (GM) for each group. The GM and number of animals (N) in each group are indicated on the x-axis. [Figure 50] HSV-2 MS-specific neutralizing antibody titers measured in serum samples collected 14 days after the third immunization with different mutant forms of AS01-adjuvanted HSV2 gE / gI. Each dot represents the titer of an individual animal. The threshold for positivity corresponds to the first sample dilution. Negative samples are indicated by the first sample dilution / 2. The number of mice in each group (N) and the geometric mean (GM) for each group are indicated below the x-axis of the graph. [Figure 51] Evaluation of the ability of vaccine-specific antibodies to reduce human IgG Fc binding by gEgI antigen in vitro 14 days after the third immunization with different mutant forms of AS01-adjuvanted HSV2 gE / gI. Each curve represents data from an individual mouse. A. AS01 / HSV2 gEgI V340W compared to NaCl; B. AS01 / HSV2 gEgI A248T compared to NaCl; C. AS01 / HSV2 gEgI A246W compared to NaCl; D. AS01 / HSV2 gEgI P318I compared to NaCl; E. AS01 / HSV2 gEgI A248T_V340W compared to NaCl. [Figure 52]Comparison of the ability of vaccine-specific antibodies to reduce human IgG Fc binding by gEgI antigen in vitro 14 days after the third immunization with different mutant forms of AS01-adjuvanted HSV2 gE / gI protein. Each dot represents the ED50 titer with 95% CI obtained from an individual mouse. The positive threshold corresponds to the first sample dilution. Negative samples are indicated by the first sample dilution / 2. The number of mice in each group (N) and the geometric mean (GM) for each group are indicated below the x-axis of the graph. [Figure 53] Evaluation of mouse FcγRIII binding activity to HSV2 gE / gI-positive cells 14 days after the third immunization with different mutant forms of AS01-adjuvanted HSV2 gE / gI protein. A-E: Each curve shows data obtained from a pool of two mouse sera immunized with a different AS01-HSV2 gEgI mutant compared to NaCl. F: Geometric mean compared to NaCl for each group vaccinated with AS01-HSV2 gEgI. [Figure 54] Percentage of vaccine-specific CD4+ / CD8+ T cell responses induced in CB6F1 mice 14 days after the third immunization with different mutant forms of AS01-adjuvanted HSV2 gE / gI protein. Circles, triangles, and diamonds represent the individual percentages of CD4+ (A) / CD8+ (B) T cell responses detected against HSV2 gE, HSV2 gI, or β-actin. The horizontal line represents the geometric mean (GM) of the responses, and the dotted line represents the 95th percentile (P95) obtained across all stimulations in the saline-treated group. The number of animals / groups with valid results (N) and the GM for each group are indicated below the graph. [Figure 55] Total titers of HSV2 gE- or gI-specific IgG antibodies measured in serum samples collected after one, two, or three immunizations with different mutant forms of SAM HSV2 gEgI vectors formulated in lipid nanoparticles (LNPs). Total titers of HSV2 gE (A) and HSV2 gI (B)-specific IgG antibodies by ELISA. Each symbol represents an individual animal in 21PI (dots), 21PII (triangles), and 21PIII (diamonds). The black bars represent the geometric mean (GM) for each group. The GM and number of animals (N) in each group are indicated on the x-axis. [Figure 56] HSV-2 MS-specific neutralizing antibody titers measured in serum samples collected 21 days after the third immunization with different LNP-formulated SAM-HSV2 gEgI mutants. Each symbol represents the titer of an individual animal, and each bar represents the geometric mean (GM) + 95% confidence interval (CI). The threshold for positivity corresponds to the first sample dilution. Negative samples are indicated by the first sample dilution / 2. The number of mice (N) in each group and the GM for each group are indicated below the x-axis of the graph. [Figure 57] Evaluation of the ability of vaccine-specific antibodies to reduce hIgG Fc binding by HSV2 gEgI antigen in vitro 21 days after the third immunization with different LNP-formulated SAM-HSV2 gEgI mutants. A. LNP / SAM-HSV2 gEgI V340W compared to NaCl group; B. LNP / SAM-HSV2 gEgI A248T compared to NaCl group; C. LNP / SAM-HSV2 gEgI A246W compared to NaCl group; D. LNP / SAM-HSV2 gEgI P318I compared to NaCl group; E. LNP / SAM-HSV2 gEgI A248T_V340W compared to NaCl group; F. LNP / SAM-HSV2 gEgI insert ARAA compared to NaCl group. [Figure 58] Comparison of the ability of vaccine-specific antibodies to reduce human IgG Fc binding by HSV2 gEgI antigen in vitro in CB6F1 mice 21 days after the third immunization with different LNP-formulated SAM-HSV2 gEgI mutants. Each dot represents the ED50 titer with 95% CI obtained from an individual mouse. The threshold for positivity corresponds to the first sample dilution. [Figure 59] Evaluation of mouse FcγRIII binding activity to HSV2 gE / gI-positive cells 21 days after the third immunization with different mutant forms of LNP-formulated SAM-HSV2 gEgI protein. AF: Each curve represents a pool of two mouse sera immunized with a different LNP-SAM HSV2 gEgI mutant compared to NaCl. G: Geometric mean compared to NaCl for each group vaccinated with LNP-SAM HSV2 gEgI. [Figure 60]Percentage of vaccine-specific CD4+ / CD8+ T cell responses induced in CB6F1 mice 21 days after the third immunization with different SAM HSV1 gEgI mutants formulated in lipid nanoparticles (LNPs). Circles, squares, and diamonds represent the individual percentages of CD4+ / CD8+ T cell responses detected against HSV2 gE, HSV2 gI, or β-actin. The horizontal line indicates the geometric mean (GM) of the responses, and the dotted line indicates the 95th percentile (P95) obtained in the saline-treated group for the combination of three antigens (gE, gI, and β-actin). The number of animals / groups with valid results (N) and the GM for each group are indicated below the graph. [Figure 61] Anti-HSV1 gEgI IgG antibody responses measured in serum samples after immunization with different forms of AS01-adjuvanted HSV1 gEgI protein. Each figure represents an individual animal at a different time point (circle = 13PI, triangle = 13PII, diamond = 14PIII), and the black bars represent the geometric mean for each group. The geometric mean (GM) and number of animals (N) for each group are shown on the x-axis. [Figure 62] Figure 1. HSV-1-specific neutralizing antibody titers measured in serum samples collected 14 days after the third immunization with different forms of AS01-adjuvanted HSV1 gEgI protein. Each dot represents the titer of an individual animal. The threshold for positivity corresponds to the first sample dilution. Negative samples are shown at first sample dilution / 2 (neutralizing titer = 5). The number of mice in each group (N) and the geometric mean (GM) for each group are indicated below the x-axis of the graph. [Figure 63]Evaluation of the ability of vaccine-specific antibodies to reduce hIgG Fc binding by HSV1 gEgI antigen in vitro 14 days after the third immunization with different forms of AS01-adjuvanted HSV1 gE / gI protein. A: Non-mutated AS01-HSV1 gEgI compared with NaCl; B: AS01-HSV1 gE_P319R / gI compared with NaCl; C: AS01-HSV1 gE_P321D / gI compared with NaCl; D: AS01-HSV1 gE_R322D / gI compared with NaCl; E: AS01-HSV1 gE_N243A_R322D / gI compared with NaCl; F: AS01-HSV1 gE_A340G_S341G_V342G / gI compared with NaCl. [Figure 64] Comparison of the ability of vaccine-specific antibodies to reduce hIgG Fc binding by HSV1 gEgI antigen in vitro 14 days after the third immunization with different forms of AS01-adjuvanted HSV1 gE / gI protein. Each dot represents the ED50 value for an individual mouse, and each bar represents GMT + 95% CI. The threshold for positivity corresponds to the first sample dilution. Negative samples are shown at the first sample dilution / 2 (ED50 value = 5). The number of mice in each group (N) and the geometric mean (GM) for each group are indicated below the x-axis of the graph. [Figure 65] Percentage of vaccine-specific CD4+ / CD8+ T cell responses elicited in CB6F1 mice 14 days after the third immunization with different forms of HSV1 gEgI protein adjuvanted with AS01. Circles, squares, and diamonds represent the individual percentages of CD4+ / CD8+ T cell responses detected against HSV1 gE, HSV2 gI, or β-actin. The horizontal line represents the geometric mean (GM) of the responses, and the dotted line represents the 95th percentile (P95) obtained across all stimulations in the saline-treated group. The number of animals / groups with valid results (N) and the geometric mean (GM) for each group are indicated below the graph. [Figure 66]HSV1 gEgI-specific IgG antibody responses measured 28 days after the first immunization or 21 days after the second immunization with different mutant forms of SAM HSV1 gEgI vector formulated in lipid nanoparticles (LNPs). Each figure represents an individual animal at a different time point (circle = 28 PI, triangle = 21 PI), and the black bars represent the geometric mean for each group. The geometric mean (GM) and number of animals (N) for each group are shown on the x-axis. [Figure 67] Figure 1. HSV-1-specific neutralizing antibody titers measured in serum samples collected 21 days after the second immunization with different mutant forms of LNP-formulated HSV1 gEgI vector. Each dot represents the titer of an individual animal, and the horizontal bar represents the geometric mean (GM) + 95% confidence interval (CI). The threshold for positivity corresponds to the first sample dilution. Negative samples are indicated at first sample dilution / 2 (neutralization titer = 5). The number of mice in each group (N) and the GM for each group are indicated below the x-axis of the graph. [Figure 68] Assessment of the ability of vaccine-specific antibodies to reduce hIgG Fc binding by HSV1 gEgI in vitro 21 days after the second immunization with different mutant forms of the LNP-formulated SAM-HSV1 gEgI vector. Each curve represents an individual mouse. LNP / SAM-HSV1 gE_P319R / gI compared to NaCl (A), LNP / SAM-HSV1 gE_P321D / gI compared to NaCl (B), LNP / SAM-HSV1 gE_R322D / gI compared to NaCl (C), LNP / SAM-HSV1 gE_N243A_R322D / gI compared to NaCl (D), and LNP / SAM-HSV1 gE_A340G_S341G_V342G / gI compared to NaCl (E). [Figure 69] Comparison of the ability of vaccine-specific antibodies to reduce human IgG Fc binding by HSV1 gEgI antigen in vitro 21 days after the second immunization with different mutant forms of LNP-formulated SAM HSV1 gEgI vector. Each dot represents the ED50 titer with 95% CI obtained from an individual mouse. The threshold for positivity corresponds to the first sample dilution. Negative samples are indicated by the first sample dilution / 2. The number of mice in each group (N) and the geometric mean (GM) for each group are indicated below the x-axis of the graph. [Figure 70] Percentage of vaccine-specific CD4+ / CD8+ T cell responses elicited in CB6F1 mice 21 days after the second immunization with different mutant forms of the SAM HSV1 gEgI vector formulated in LNP. Circles, squares, and diamonds represent the individual percentages of CD4+ (A) and CD8+ (B) T cell responses detected against each antigen (HSV1 gE antigen, HSV1 gI antigen, β-actin). Horizontal bars represent the geometric mean (GM) of the responses, and the dotted line represents the 95th percentile (P95) obtained across all stimulations in the saline-treated group. The number of animals / groups with valid results (N) and the GM for each group are indicated below the graph. [Figure 71] Total titers of anti-HSV-2 gE- or gI-specific IgG antibodies were measured in serum samples collected after immunization with different doses of LNP / SAM-gE_P317R / gI vaccine. Serum samples were collected on days 21 (21PI), 42 (21PII), and 63 (21PIII) and assessed for HSV-2 gE (A) or gI-specific (B) total IgG antibody titers by ELISA. Each symbol represents an individual animal on 21PI (dots), 21PII (squares), and 21PIII (triangles). Black bars represent the geometric mean (GM) with 95% confidence intervals (CI) for each group. The number of animals (N) in each group is indicated on the x-axis. [Figure 72] HSV-2 MS-specific neutralizing antibody titers measured in serum samples collected 21 days after the third immunization with different doses of LNP / SAM-gE_P317R / gI vaccine. Each symbol represents an individual animal, and the black bars represent the geometric mean (GM) with 95% confidence intervals (CI) for each group. The number of animals (N) in each group is indicated on the x-axis. [Figure 73]Evaluation of the ability of vaccine-specific antibodies to reduce human IgG Fc binding by gEgI antigen in vitro 21 days after the third immunization with different doses of LNP / SAM-gE_P317R / gI vaccine. Each curve represents data from an individual mouse. A: 5 μg LNP / SAM-gE_P317R / gI compared to NaCl; B: 1 μg LNP / SAM-gE_P317R / gI compared to NaCl; C: 0.1 μg LNP / SAM-gE_P317R / gI compared to NaCl; D: 0.01 μg LNP / SAM-gE_P317R / gI compared to NaCl. [Figure 74] Comparison of the ability of vaccine-specific antibodies to reduce human IgG Fc binding by HSV-2 gE / gI antigen in vitro 21 days after the third immunization with different doses of LNP / SAM-gE_P317R / gI vaccine. Each dot represents the ED50 titer with 95% CI obtained from an individual mouse. The threshold for positivity corresponds to the first sample dilution. Negative samples are indicated by the first sample dilution / 2. The number of mice (N) in each group is indicated below the x-axis of the graph. [Figure 75] Percentage of vaccine-specific CD4+ T cell responses induced in CB6F1 mice 21 days after the third immunization with different doses of LNP / SAM-gE_P317R / gI vaccine. The frequencies of CD4+ T cells secreting IL-2, IFN-γ, and TNF-α were measured by intracellular cytokine staining. The black line represents the geometric mean (GM) of responses with 95% confidence intervals (CI). [Figure 76] Percentage of vaccine-specific CD8+ T cell responses induced in CB6F1 mice 21 days after the third immunization with different doses of LNP / SAM-gE_P317R / gI vaccine. The frequencies of CD8+ T cells secreting IL-2, IFN-γ, and TNF-α were measured by intracellular cytokine staining. The black line represents the geometric mean (GM) of responses with 95% confidence intervals (CI). [Figure 77]Percentages of follicular B helper CD4+ T cells and activated B cells in the draining lymph nodes of LNP / SAM-gE_P317R / gI-vaccinated mice. Iliac draining lymph nodes were harvested on days 10 and 16 to assess the frequency of follicular B helper CD4+ T cells (Tfh-CD4+ / CXCR5+ / PD-1+ / Bcl6+) (A) and activated B cells (CD19+ / CXCR5+ / Bcl6+) (B). Each plot represents an individual mouse, and the black line represents the geometric mean (GM) of the response with 95% confidence intervals (CI). The number of mice (N) in each group is indicated below the x-axis of the graph. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention relates to the use of viral Fc receptors or immunogenic fragments thereof, in particular glycoprotein gE from HSV1 or HSV2 alone or with its binding partner gI, in therapeutic vaccines against viral infections, in particular against recurrent infections with HSV1 or HSV2 and the associated clinical and subclinical symptoms.

[0033] Alphaherpesviruses, such as herpes simplex virus (HSV), have evolved specialized mechanisms that enable viral transmission in epithelial and neural tissues. Primary infection involves invasion of mucosal epithelial cells, followed by rapid viral transmission between these cells. During this phase of viral replication and transmission, the virus enters sensory neurons by fusion of the virion envelope with the neuronal membrane, resulting in delivery of capsids to the cytoplasm. The capsids undergo retrograde axonal transport on microtubules toward the neuronal cell body or nucleus in the ganglion, where latency is established. Later, upon neuronal stimulation, latent virus reactivates, producing viral particles that undergo rapid axonal transport on microtubules in the anterograde direction from the cell body to the axon tip. A critical phase in the life cycle of herpes simplex virus (HSV) and other alphaherpesviruses is their ability to reactivate from latency and subsequently transmit from infected neurons to epithelial tissues. This spread involves at least two steps: (i) anterograde transport to the axon tip, followed by (ii) exocytosis and extracellular spread from the axon to epithelial cells. HSV gE / gI is a heterodimer formed from two viral membrane glycoproteins, gE and gI. The HSV gE / gI heterodimer has been shown to facilitate virus spread. (Howard, Paul W., et al. "Herpes simplex virus gE / gI extracellular domains promote axonal transport and spread from neurons to epithelial cells." Journal of Virology 88.19 (2014): 11178-11186.)

[0034] When HSV1 or HSV2 reactivates within infected cells, the virus becomes more visible to the immune system and therefore more vulnerable. Normally, host IgG recognizes viral antigens on virions or on the cell surface of infected cells, and the host IgG Fc domain mediates important antibody effector activities by interacting with Fc gamma receptors on NK cells, granulocytes, and macrophages, thereby inducing antibody-dependent cellular cytotoxicity (ADCC), and by interacting with Fc gamma receptors on macrophages, monocytes, neutrophils, and dendritic cells, thereby inducing antibody-dependent cellular phagocytosis (ADCP).

[0035] HSV1 gE or HSV2 gE can form noncovalent heterodimeric complexes with HSV1 or HSV2 (respectively) glycoprotein I (gI). The gE-gI heterodimer functions as a viral Fc gamma receptor (FcγR) and has the ability to interact with the Fc portion of human IgG. Indeed, when presented on the cell surface of HSV-infected cells, HSV1 gE or HSV2 gE or HSV1 or HSV2 gE / gI heterodimers bind to host IgG through their Fc portions. The interaction between gE and gI is thought to increase the Fc binding affinity by approximately 100-fold compared to gE alone. This interaction has been implicated in immune evasion mechanisms. Indeed, human IgG, which can bind to HSV1 or HSV2 antigens (e.g., gD) on virions or infected cells through its IgG Fab domain, can also bind to the Fc-binding domain on viral gE through its Fc domain, resulting in endocytosis of the immune complex through a clathrin-mediated mechanism. This mechanism, called antibody bipolar bridging, is hypothesized to be a major immune evasion strategy that competes with the activation of innate immune cells. Through antibody Fc binding, viral FcγRs inhibit IgG Fc-mediated activities, including complement fixation and antibody-dependent cellular cytotoxicity (ADCC), allowing viruses to evade recognition by the immune system.(Ndjamen, Blaise, et al. "The herpes virus Fc receptor gE-gI mediates antibody bipolar bridging to clear viral antigens from the cell surface." PLoS pathogens 10.3 (2014): e1003961.; Dubin, G., et al. "Herpes simplex virus type 1 Fc receptor protects infected cells from antibody-dependent cellular cytotoxicity." Journal of virology 65.12 (1991): 7046-7050.; Sprague, Elizabeth R., et al. "Crystal structure of the HSV1 Fc receptor bound to Fc reveals a mechanism for antibody bipolar bridging." PLoS biology 4.6 (2006): e148.)。

[0036] An HSV2 prophylactic subunit gD2 vaccine did not effectively prevent HSV-2 disease or infection in human clinical trials (Johnston, Christine, Sami L. Gottlieb, and Anna Wald. "Status of vaccine research and development of vaccines for herpes simplex virus." Vaccine 34.26 (2016): 2948-2952). Another HSV2 vaccine candidate based on a truncated gD2 antigen and ICP4.2 antigen adjuvanted with Matrix-M2 reduced genital HSV2 shedding and lesion rates in a phase 2 trial (Van Wagoner, Nicholas, et al. "Effects of different doses of GEN-003, a therapeutic vaccine for genital herpes simplex virus-2, on viral shedding and lesions: results of a randomized placebo-controlled trial." The Journal of infectious diseases 218.12 (2018): 1890-1899). A trivalent adjuvanted vaccine containing the viral entry molecule (gD2) and two antigens that block HSV2 immune evasion, the complement-inhibiting gC2 and gE2, has shown efficacy in animal models.(Awasthi, Sita, et al. "Blocking herpes simplex virus 2 glycoprotein E immune evasion as an approach to enhance efficacy of a trivalent subunit antigen vaccine for genital herpes." Journal of virology 88.15 (2014): 8421-8432.; Awasthi, Sita, et al. "An HSV2 trivalent vaccine is immunogenic in rhesus macaques and highly efficacious in guinea pigs." PLoS pathogens 13.1 (2017): e1006141.; Awasthi, Sita, et al. "A trivalent subunit antigen glycoprotein vaccine as immunotherapy for genital herpes in the guinea pig genital infection model." Human vaccines & immunotherapeutics 13.12 (2017): 2785-2793.; Hook, Lauren M., et al. "A trivalent gC2 / gD2 / gE2 vaccine for herpes simplex virus generates antibody responses that block immune evasion domains on gC2 better than natural infection." Vaccine 37.4 (2019): 664-669.)。

[0037] The inventors hypothesized that by directing the immune response against the Fc-binding domain of gE, it is possible to prevent or interfere with the above-mentioned immune evasion mechanism (antibody bipolar bridging), thereby resulting in stronger natural immunity to viral proteins, particularly the immunodominant HSV1 gD or HSV2 gD antigens. The inventors also hypothesized that specific targeting of gE or gE / gI heterodimers by vaccination could enhance subdominant immune responses, particularly antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), and direct the immune system toward protective mechanisms. Without wishing to be bound by theory, the inventors therefore believe that specifically boosting the immune response by vaccination with gE alone or in combination with its heterodimeric binding partner, gI, may effectively treat subjects infected with HSV1 or HSV2.

[0038] For the treatment of subjects already infected with herpesvirus (seropositive subjects), whether the subject is symptomatic or asymptomatic, the inventors hypothesized that it would not be necessary to include an immunodominant antigen, such as HSV gD, in the therapeutic composition. Indeed, gD is a dominant antigen, and seropositive subjects, whether symptomatic or asymptomatic, already have high levels of naturally occurring neutralizing antibodies against gD (Cairns, Tina M., et al. "Patient-specific neutralizing antibody responses to herpes simplex virus are attributed to epitopes on gD, gB, or both and can be type specific." Journal of Virology 89.18 (2015): 9213-9231.). By inducing an immune response against a viral Fc receptor, e.g., HSV gE or HSV gE / gI, the inventors hypothesized that the gE / gI immune evasion mechanism would be bypassed, and that innate immunity, particularly innate antibody responses directed against immunodominant antigens, e.g., gD, would be able to more fully fulfill its role. The inventors also hypothesized that in addition to affecting immune evasion mechanisms, gE or gE / gI antigens might also induce humoral responses (anti-gE antibodies or anti-gE and anti-gI antibodies) that would lead to the destruction of infected cells by cytotoxic and / or phagocytic mechanisms (ADCC / ADCP). In seropositive subjects, the inventors hypothesized that ADCC and / or ADCP mechanisms might be more efficient at controlling early stages of viral replication than neutralizing antibody mechanisms (e.g., responses driven by the dominant HSV antigen gD). Finally, we also hypothesized that induction of CD4+ T cells by gE or gE / gI antigens may also be beneficial against recurrent HSV infection.

[0039] A similar immune evasion mechanism has been described for human cytomegalovirus (HCMV), which possesses gp34 and gp68 as viral Fc receptors (Corrales-Aguilar, Eugenia, et al. "Human cytomegalovirus Fcγ binding proteins gp34 and gp68 antagonize Fcγ receptors I, II, and III." PLoS pathogens 10.5 (2014): e1004131; Sprague, Elizabeth R., et al. "The human cytomegalovirus Fc receptor gp68 binds the Fc CH2-CH3 interface of immunoglobulin G." Journal of virology 82.7 (2008): 3490-3499.). The present inventors hypothesize that HCMV gp34 or gp68, or immunogenic fragments thereof, could serve as therapeutic vaccines for treating subjects infected with HCMV.

[0040] In a first aspect, the present invention provides a protein comprising or consisting of an Fc receptor from a virus, or an immunogenic fragment thereof, for use in treating a subject infected with said virus.

[0041] As used herein, an "Fc receptor" (or "FcR") is a protein found on the surface of certain cells and capable of binding to the Fc region of an antibody. Fc receptors are classified based on the type of antibody they recognize. Fc receptors that bind to IgG, the most common class of antibody, are called "Fc-gamma receptors" (or "FcγR"), Fc receptors that bind IgA are called "Fc-alpha receptors" (or "FcαR"), and Fc receptors that bind IgE are called "Fc-epsilon receptors" (or "FcεR"). As used herein, Fc receptors that are displayed on the surface of cells from a given multicellular organism as a result of expression of endogenous genes are referred to as "host Fc receptors." Host Fc receptors are found, inter alia, on the surface of host immune effector cells, such as B lymphocytes, follicular dendritic cells, natural killer (NK) cells, macrophages, neutrophils, eosinophils, basophils, human platelets, and mast cells. Binding of host Fc receptors to the Fc region of antibodies bound to infected cells or invading pathogens through their Fab regions triggers phagocytosis or destruction of the infected cells or invading pathogens by antibody-mediated cellular phagocytosis (ADCP) or antibody-dependent cellular cytotoxicity (ADCC).

[0042] Suitably, the FcR or immunogenic fragment thereof is in subunit form, meaning that it is not part of the whole virus. Suitably, the FcR or immunogenic fragment thereof is isolated.

[0043] Some viruses, particularly herpes viruses, express viral Fc receptors that bind to the Fc portion of host IgG, thereby preventing IgG from binding to host Fc receptors on immune effector cells and allowing the virus to evade the host's ADCC or ADCP immune response. As used herein, a "viral Fc receptor" (or "virus-derived Fc receptor") is an Fc receptor of viral origin. As used herein, a "viral FcγR" is an FcγR of viral origin.

[0044] In one embodiment, the viral Fc receptor is from a herpes virus.

[0045] As used herein, a "herpesvirus" is a member of the Herpesviridae family, which includes herpes simplex virus (HSV) types 1 and 2 (HSV1 and HSV2, respectively), human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and varicella-zoster virus (VZV).

[0046] In a preferred embodiment, the viral Fc receptor is from a herpesvirus selected from HSV2, HSV1 and HCMV.

[0047] In one embodiment, the viral Fc receptor is a viral FcγR. Preferably, the viral FcγR is selected from HSV2 gE2, HSV1 gEl, HCMV gp34, and HCMV gp68.

[0048] As used herein, "HSV2 gE2" (or "HSV2 gE") refers to the HSV2 gE glycoprotein encoded by the HSV2 gene US8, displayed on the surface of infected cells, and functioning as a viral FcγR. Suitably, the HSV2 gE2 is selected from the HSV2 gE glycoproteins shown in Table 1, or variants thereof that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.

[0049] [Table 1] JPEG2025163028000002.jpg103162

[0050] In a preferred embodiment, the HSV2 gE2 is gE from HSV2 strain SD90e having the amino acid sequence set forth in SEQ ID NO: 1 (Genbank accession number AHG54732.1, UniProtKB accession number: A7U881), or a variant therefrom that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical thereto.

[0051] As used herein, a "variant" is a peptide sequence that differs in sequence from a reference antigen sequence but retains at least one essential property of the reference antigen. Sequence changes in a peptide variant can be limited or conservative, such that the sequences of the reference peptide and variant are closely similar overall and, in many regions, identical. A variant and the reference antigen can differ in amino acid sequence by one or more substitutions, additions, or deletions, in any combination. A variant of an antigen can be a naturally occurring, e.g., allelic, variant, or a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and polypeptides can be generated by mutagenesis techniques or by direct synthesis. In a preferred embodiment, the essential property retained by the variant is the ability to induce an immune response, suitably a humoral or T-cell response, similar to that induced by the reference antigen. Suitably the variant induces a humoral or T cell response in mice that is no more than 10-fold weaker, more suitably no more than 5-fold weaker, no more than 2-fold weaker or no less than the immune response induced by the reference antigen.

[0052] As used herein, "HSV1 gEl" (or "HSV1 gE") refers to the HSV1 gE glycoprotein encoded by the HSV1 gene US8, displayed on the surface of infected cells, and functioning as a viral FcγR. Suitably, HSV1 gEl is gE from HSV1 strain KOS321 (UniProtKB Accession Number: Q703E9) having the amino acid sequence set forth in SEQ ID NO: 3, or a variant thereof that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.

[0053] As used herein, "HCMV gp34" refers to the HCMV gp34 glycoprotein that is displayed on the surface of infected cells and functions as a viral FcγR. Suitably, the HCMV gp34 is gp34 from HCMV strain AD169 (UniProtKB Accession Number: P16809, SEQ ID NO: 5) having the amino acid sequence set forth in SEQ ID NO: 5, or a variant therefrom that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.

[0054] As used herein, "HCMV gp68" refers to the HCMV gp68 glycoprotein that is displayed on the surface of infected cells and functions as a viral FcγR. Suitably, the HCMV gp68 is gp68 from HCMV strain AD169 (UniProtKB Accession Number: P16739, SEQ ID NO: 6) having the amino acid sequence set forth in SEQ ID NO: 6, or a variant therefrom that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.

[0055] In one embodiment, an immunogenic fragment of a viral Fc receptor is used.

[0056] As used herein, an "immunogenic fragment" refers to a fragment of a reference antigen that contains one or more epitopes (e.g., linear, conformational, or both) that can stimulate a host's immune system to mount a humoral and / or cellular antigen-specific immune response (i.e., an immune response that specifically recognizes a naturally occurring polypeptide, e.g., a viral or bacterial protein). An "epitope" is a portion of an antigen that determines its immunological specificity. T-cell and B-cell epitopes can be identified empirically (e.g., using PEPSCAN or similar methods). In a preferred embodiment, an immunogenic fragment induces an immune response, suitably a humoral or T-cell response, that is similar to the immune response induced by the reference antigen. Suitably, the immunogenic fragment induces a humoral or T-cell response in mice that is no more than 10-fold weaker, more suitably no more than 5-fold weaker, no more than 2-fold weaker, or no weaker than the immune response induced by the reference antigen.

[0057] As used herein, an "immunogenic fragment of a viral Fc receptor" refers to a fragment of a naturally occurring viral Fc receptor having at least 10, 15, 20, 30, 40, 50, 60, 100, 200, 300, or more amino acids, or a peptide having an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity to a naturally occurring viral Fc receptor (or a fragment of a naturally occurring viral Fc receptor having at least about 10, 15, 20, 30, 40, 50, 60, or more amino acids). Thus, an immunogenic fragment of an antigenic viral Fc receptor can be a fragment of at least 10 amino acids of a naturally occurring viral Fc receptor and can include one or more amino acid substitutions, deletions, or additions.

[0058] Any of the encoded viral Fc receptor immunogenic fragments can further include an initial methionine residue, if desired.

[0059] Suitably, the viral Fc receptor or immunogenic fragment thereof does not comprise a functional transmembrane domain. Suitably, the viral Fc receptor or immunogenic fragment thereof does not comprise a cytoplasmic domain. Preferably, the viral Fc receptor or immunogenic fragment thereof does not comprise a functional transmembrane domain or a cytoplasmic domain. In other words, in a preferred embodiment, the viral Fc receptor or immunogenic fragment consists of a viral FcR ectodomain (or extracellular domain). More preferably, the viral Fc receptor or immunogenic fragment comprises or consists of an HSV2 gE2 ectodomain, an HSV1 gE1 ectodomain, an HCMV gp34 ectodomain, or an HCMV gp68 ectodomain.

[0060] In a preferred embodiment, the viral FcR ectodomain is an HSV2 gE2 ectodomain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:7 (corresponding to amino acid residues 1-419 of SEQ ID NO:1), or a sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto. Suitably, the viral FcR ectodomain has a sequence selected from the sequences set forth in Table 1 or Figure 3. In a preferred embodiment, the viral FcR ectodomain is an HSV2 gE2 ectodomain that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:7.

[0061] Suitably, the viral Fc receptor HSV2 ectodomain may comprise one or more amino acid residue substitutions, deletions or insertions relative to the amino acid sequence set out in SEQ ID NO:7, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residue substitutions, deletions or insertions.

[0062] In another embodiment, the viral FcR ectodomain is an HSV2 gE2 ectodomain comprising or consisting of an amino acid sequence corresponding to amino acid residues 1 to 417 of SEQ ID NO: 1, or a sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical thereto. Suitably, the viral Fc receptor HSV2 ectodomain comprises one or more amino acid residue substitutions, deletions or insertions, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residue substitutions, deletions or insertions, relative to the amino acid sequence corresponding to amino acid residues 1 to 417 of SEQ ID NO: 1.

[0063] In another embodiment, the viral FcR ectodomain is an HSV1 gEl ectodomain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:9 (corresponding to amino acid residues 1 to 421 of SEQ ID NO:3), or a sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto. In a preferred embodiment, the viral FcR ectodomain is an HSV1 gEl ectodomain that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:9.

[0064] Suitably, the viral Fc receptor HSV1 ectodomain may comprise one or more amino acid residue substitutions, deletions or insertions relative to the amino acid sequence set out in SEQ ID NO:9, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residue substitutions, deletions or insertions.

[0065] In another embodiment, the viral FcR HSV1 ectodomain comprises or consists of an amino acid sequence corresponding to amino acid residues 1 to 419 of SEQ ID NO: 3, or a sequence which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical thereto. Suitably, the viral Fc receptor HSV1 ectodomain comprises one or more amino acid residue substitutions, deletions or insertions, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residue substitutions, deletions or insertions, relative to the amino acid sequence corresponding to amino acid residues 1 to 419 of SEQ ID NO: 3.

[0066] In another embodiment, the viral FcR ectodomain is an HCMV gp34 ectodomain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:11 (corresponding to amino acid residues 1-180 of SEQ ID NO:5), or a sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto. In a preferred embodiment, the viral FcR ectodomain is an HCMV gp34 ectodomain that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:11.

[0067] Suitably, the viral Fc receptor HCMV gp34 ectodomain may comprise one or more amino acid residue substitutions, deletions or insertions, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residue substitutions, deletions or insertions relative to the amino acid sequence shown in SEQ ID NO:11.

[0068] In another embodiment, the viral FcR ectodomain is an HCMV gp68 ectodomain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 12, or a sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto. In a preferred embodiment, the viral FcR ectodomain is an HCMV gp68 ectodomain that is at least at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 12.

[0069] Suitably, the viral Fc receptor HCMV gp68 ectodomain may comprise one or more amino acid residue substitutions, deletions or insertions, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residue substitutions, deletions or insertions, relative to the amino acid sequence set forth in SEQ ID NO: 12 (corresponding to amino acid residues 1 to 271 of SEQ ID NO: 6).

[0070] In another embodiment, the immunogenic fragment of a viral FcR comprises or consists of an Fc-binding domain from the viral FcR or a variant thereof.

[0071] In one embodiment, the immunogenic fragment of HSV2 FcR comprises or consists of an Fc binding domain from HSV2 gE, e.g., an amino acid sequence corresponding to amino acid residues 233 to 378 of SEQ ID NO: 1, or a sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical thereto. Suitably, the viral Fc receptor HSV2 Fc binding domain comprises one or more amino acid residue substitutions, deletions or insertions, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residue substitutions, deletions or insertions, relative to the amino acid sequence corresponding to amino acid residues 233 to 378 of SEQ ID NO: 1.

[0072] In one embodiment, the immunogenic fragment of HSV1 FcR comprises or consists of an Fc binding domain from HSV1 gE, e.g., an amino acid sequence corresponding to amino acid residues 235 to 380 of SEQ ID NO: 3, or a sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto. Suitably, the viral Fc receptor HSV1 Fc binding domain comprises one or more amino acid residue substitutions, deletions, or insertions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions, deletions, or insertions, relative to the amino acid sequence corresponding to amino acid residues 235 to 380 of SEQ ID NO: 3.

[0073] In a preferred embodiment, the ability of the viral Fc receptor or immunogenic fragment thereof to bind to a human antibody Fc domain is reduced or eliminated compared to the corresponding native viral Fc receptor. Suitably, the viral Fc receptor or immunogenic fragment thereof comprises one or more amino acid substitutions, deletions or insertions compared to the native sequence of the viral Fc receptor or immunogenic fragment thereof, which substitutions, deletions or insertions reduce or eliminate the binding affinity between the viral FcR or immunogenic fragment thereof and the antibody Fc domain compared to the native viral Fc receptor.

[0074] The binding affinity between a viral FcR or an immunogenic fragment thereof and an antibody Fc domain can be determined by methods well known to those skilled in the art. For example, the association rate (k on ), dissociation rate (k off ), equilibrium dissociation constant (K D =k off / k on ) and the equilibrium association constant (K A =1 / K D =k on / k off ) is determined by biolayer interferometry as described in Example 3.

[0075] In a preferred embodiment, the kappa binding affinity between a viral FcR or an immunogenic fragment thereof and human IgG is onis the k between the corresponding native viral FcR and human IgG on Lower than (slow binder).

[0076] In a preferred embodiment, the kappa binding affinity between a viral FcR or an immunogenic fragment thereof and human IgG is off is the k between the corresponding native viral FcR and human IgG off Higher than (fast releaser).

[0077] In a more preferred embodiment, the kappa ratio between a viral FcR or an immunogenic fragment thereof and human IgG is on is the k between the corresponding native viral FcR and human IgG on and the k between the viral FcR or its immunogenic fragment and human IgG is lower than off is the k between the corresponding native viral FcR and human IgG off Higher than (slow binder / fast releaser).

[0078] In a preferred embodiment, the equilibrium dissociation constant (K D ) is the K between the corresponding native viral FcR and human IgG D Higher than.

[0079] The relative affinity between viral FcR or its immunogenic fragment and human IgG is calculated using the K determined for the native viral FcR. D The K determined for the viral FcR or its immunogenic fragment D It can be determined by dividing by

[0080] In a preferred embodiment, the relative affinity between the viral FcR or immunogenic fragment thereof and human IgG is less than 100%, e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, or 10% of the affinity between the corresponding native viral FcR and human IgG. In a more preferred embodiment, the relative affinity between the viral FcR or immunogenic fragment thereof and human IgG is less than 15%, more preferably even less than 10%, of the affinity between the corresponding native viral FcR and human IgG.

[0081] In a preferred embodiment, the equilibrium dissociation constant (K D ) is 2 × 10 -7 Higher than M, preferably 5 x 10 -7 M, preferably 1×10 -6 Higher than M.

[0082] Alternatively, the ability of a viral Fc receptor or an immunogenic fragment thereof to bind to a human antibody Fc domain can be assessed by measuring the response (expressed in nm) in a biolayer interferometry assay, as described in Examples 3 and 4.

[0083] In a preferred embodiment, the response in a bio-layer interferometry assay corresponding to binding between a viral Fc receptor or an immunogenic fragment thereof and human IgG is less than 80%, suitably less than 70%, 60%, 50%, 40% of the response obtained with the corresponding native viral Fc receptor. In a preferred embodiment, the response in a bio-layer interferometry assay corresponding to binding between a viral Fc receptor or an immunogenic fragment thereof and human IgG is less than 0.4 nm, suitably less than 0.3 nm, 0.2 nm or 0.1 nm.

[0084] Suitably, the HSV2 gE2 or immunogenic fragment thereof comprises one or more mutations (insertion, substitution or deletion) at a position selected from N241, H245, A246, A248, R314, P317, P318, P319, F322, R320, A337, S338 or V340 of the HSV2 gE2 sequence shown in SEQ ID NO:1.

[0085] Exemplary mutations that can be used herein to reduce or eliminate binding affinity between HSV2 gE2 or an immunogenic fragment thereof and an antibody Fc domain include single point substitution mutations of the sequence shown in SEQ ID NO: 1 selected from the following: H245A, H245K, P317R, P319A, P319R, P319G, P319K, P319T, A337G, P319D, P319S, S 338D, N241A, R320D, H245E, H245V, H245R, H245D, H245Q, H245G, H245I, H245K, H24 5S, H245T, A246W, A248K, A248T, A248G, R314A, R314N, R314D, R314Q, R314E, R314 G, R314I, R314L, R314K, R314M, R314F, R314P, R314S, R314T, R314Y, R314V, P317N, P317G, P317I, P317L, P317K, P317F, P317S, P318R, P318D, P318Q, P318I, P318S, P 318T, P318Y, P319L, R320A, R320S, R320N, R320Q, R320E, R320G, R320H, R320I, R32 0L, R320M, R320P, R320T, R320V, F322A, F322N, F322I, F322K, F322P, F322T, S338G, S338E, S338L, S338T, V340A, V340R, V340D, V340Q, V340M, V340F, V340P and V340W.

[0086] Exemplary mutations that can be used herein to reduce or eliminate binding affinity between HSV2 gE2 or an immunogenic fragment thereof and an antibody Fc domain also include double point substitution mutations of the sequence shown in SEQ ID NO: 1 selected from the following:H245A and P319A; H245A and P319R; H245A and P319G; H245A and P319K; H245A and P319T; N241A and R320D; N241A and P319D; A246W and P317K; A246W and P317F; A246W and P317S; A246W and R320D; A246W and R320G; A246W and R320T; A248K and V340R; A248K and V340M; A248K and V340W; A248T and V340R; A248T and V340M; A248T and V340W; A24 8G and V340R;A248G and V340M;A248G and V340W;A248K and F322A;A248K and F322I;A248K and F322P;A248T and F322A;A248T and F322I;A248T and F322P;A248G and F322A;A248G and F322I;A248G and F322P;H245A and R320D;H245A and R320G;H245A and R320T;H245G and R320D;H245G and R320G;H245G and R320T;H245S and R320D;H245S and and R320G; H245S and R320T; H245A and P319G; H245A and P319L; H245G and P319G; H245G and P319L; H245S and P319G; H245S and P319L; R314G and P318R; R314G and P318D; R314G and P318I; R314L and P318R; R314L and P318D; R314L and P318I; R314P and P318R; R314P and P318D; R314P and P318I; R314G and F322A; R314G and F322I; R314G and F3 22P;R314L and F322A;R314L and F322I;R314L and F322P;R314P and F322A;R314P and F322I;R314P and F322P;R314G and V340R;R314G and V340M;R314G and V340W;R314L and V340R;R314L and V340M;R314L and V340W;R314P and V340R;R314P and V340M;R314P and V340W;P317K and V340R;P317K and V340M;P317K and V340W;P317F and V340R;P317F and V340M;P317F and V340W;P317S and V340R;P317S and V340M;P317S and V340W;P317K and S338G;P317K and S338H;P317K and S338L;P317F and S338G;P317F and S338H;P317F and S338L;P317S and S338G;P317S and S338H;P317S and S338L;P318R and S338G;P318R and S338H; P318R and S338L; P318D and S338G; P318D and S338H; P318D and S338L; P318I and S338G; P318I and S338H; P318I and S338L; P319G and V340R; P319G and V340M; P319G and V340W; P319L and V340R; P319L and V340M; P319L and V340W; P317R and P319D; P317R and R320D; P319D and R320D. ;

[0087] Exemplary mutations that can be used herein to reduce or eliminate binding affinity between HSV2 gE2 or an immunogenic fragment thereof and an antibody Fc domain also include deletion mutations at positions P319 and / or R320 of the sequence shown in SEQ ID NO: 1, either alone or in combination with substitution mutations, particularly mutations selected from the following: P319 deletion; R320 deletion; P319 deletion / R320 deletion; P319 deletion / R320 deletion / P317G / P318G; P319 deletion / R320 deletion / P318E; P319 deletion / R320 deletion / P318G; P319 deletion / R320 deletion / P318K; P319 deletion / R320 deletion / P317R / P318E; P319 deletion / R320 deletion / P317R / P318G; P319 deletion / R320 deletion / P317R / P318K; P319 deletion / R320 deletion / P317G / P318K.

[0088] Exemplary mutations that can be used herein to reduce or eliminate binding affinity between HSV2 gE2 or an immunogenic fragment thereof and an antibody Fc domain also include insertion mutations selected from the following: insertion of the peptide sequence LDIGE between amino acid residues Y275 and E276 of SEQ ID NO: 1 (275_insert_LDIGE), insertion of the peptide sequence ADIGL between amino acid residues S289 and P290 of SEQ ID NO: 1 (289_insertADIGL), insertion of the peptide sequence ARAA between amino acid residues A337 and S338 of SEQ ID NO: 1 (337_insert_ARAA), insertion of the peptide sequence ARAA between amino acid residues S338 and T339 of SEQ ID NO: 1 (338_insert_ARAA), and Insertion of the peptide sequence ADIT between amino acid residues H346 and A347 of SEQ ID NO: 1 (346_insert_ADIT).

[0089] In a preferred embodiment, the HSV2 gE2 or immunogenic fragment thereof comprises a mutation or combination of mutations with respect to the sequence shown in SEQ ID NO: 1 selected from the following: 289_インサートADIGL;338_インサートARAA;H245K;P317R;P319R;P319G;P319K;H24 5A_P319R;H245A_P319G;H245A_P319K;H245A_P319T;P319D;S338D;R320D; N241A_R320D;A248K_V340M;P318Y;A248K_V340R;A248T_V340W;A248K_V3 40W;A246W_R320G;A246W_P317K;A246W_R320D;A246W_R320T;V340W;A248G _V340W;H245G_R320D;P318D;A246W_P317F;P319G_V340W;A248T_V340M;P 317K_V340W;V340F;V340D;H245A_R320D;P317F_V340W;A246W_P317S;H245 S_R320D;R314G_P318D;A248T;P318S;P317K;P317S_V340W;H245D;R314P_ V340W;R314L_318D;P319L_V340W;P317F;P318D_S338G;R314G_V340W;P317 K_S338H;R314L_V340W;P318R;P318Q;P317F_S338G;R314G_P318I;H245G_ P319G;P317L;P318I;A248T_F322A;H245E;P318T;P318R_S338G;P318D_S33 8H;P317F_S338H;A248T_V340R;A248T_F322I;H245A_R320G;P318R_S338H;H245S_R320G;P317K_S338G;A248T_F322P;V340R;R314L_P318R;H245S_R3 20T;R314G_P318R;R320E;H245G_R320G;H245A_R320T;A246W;P318I_S338 G;P317K_V340M;P317I;R320H;R314P_P318I;P318I_S338H;P317F_V340M;H 245A_P319G;H245A_P319L;R320P;H245G_R320T;R314L_V340R;P319G_V34 0R;R314G_F322I;R314L_P318I;R320A;R314N;P317F_V340R;P318D_S338L;A248G_V340R;R314E;R314P_P318D;H245S_P319G;V340Q;A248K_F322I;R320G;H245S_P319L;R314F;P319L;P317K_S338L;P319L_V3 40M;P317G;R320S;R320Q;R314P_V340R;V340A;H245G_P319L;R320T;R314P_P318R;A248G_F322I;R320N;P317N;R314D;R314Y;R314 P_F322I; P319G_V340M; P317S_V340R; R314V; P317R_P319D; P317R_R320D; P319D_R320D; Δ319_Δ320; P317G_P318G_Δ319_Δ320; P318E_Δ319_Δ320; P318G_Δ319_Δ320; P318K_Δ319_Δ320; P317R_P318E_Δ319_320; P317R_P318G_Δ319_Δ320 and P317G_P318K_Δ319_Δ320. (Δ indicates deleted residues).

[0090] In a more preferred embodiment, the HSV2 gE2 or immunogenic fragment thereof comprises a mutation or combination of mutations with respect to the sequence shown in SEQ ID NO: 1 selected from 338_insert ARAA; P317R; P319D; R320D; A248T_V340W; V340W; A248T; P318I and A246W.

[0091] For the exemplary substitution, selection, and insertion mutations listed above, corresponding mutations in other HSV2 gE2 sequences, such as those listed in Table 1 and shown on the alignment provided in Figure 3, are also within the scope of the invention.

[0092] All possible combinations of the exemplary single and double substitution mutations and insertion mutations listed above are also within the scope of the present invention.

[0093] Suitably, the HSV1 gEl or immunogenic fragment thereof comprises one or more mutations (insertion, substitution or deletion) at positions selected from H247, P319 and P321 of the HSV1 gEl sequence shown in SEQ ID NO:3.

[0094] Exemplary mutations that can be used herein to reduce or eliminate binding affinity between HSV1 gEl or an immunogenic fragment thereof and an antibody Fc domain include single point substitution mutations of the sequence set forth in SEQ ID NO:3 selected from: H247A, H247K, P319R, P321A, P321R, P321G, P321K, P321T, A339G, P321D, P321S, A340D, N243A, and R322D, and double point substitution mutations of the sequence set forth in SEQ ID NO:3 selected from: These include H247A / P321A, H247A / P321R, H247A / P321G, H247A / P321K, H247A / P321T, N243A / R322D, N243A / P321D, H247G / P319G, P319G / P321G, and A340G / S341G / V342G.

[0095] Exemplary mutations that can be used herein to reduce or eliminate binding affinity between HSV1 gE1 or an immunogenic fragment thereof and an antibody Fc domain also include insertion mutations selected from the following: insertion of the peptide sequence LDIGE between amino acid residues Y277 and E278 of SEQ ID NO: 3 (277_insert_LDIGE); insertion of the peptide sequence ADIGL between amino acid residues S291 and P292 of SEQ ID NO: 3 (291_insert_ADIGL); insertion of the peptide sequence ARAA between amino acid residues A339 and A340 of SEQ ID NO: 3 (339_insert_ARAA); insertion of the peptide sequence ARAA between amino acid residues A340 and S341 of SEQ ID NO: 3 (340_insert_ARAA); and Insertion of the peptide sequence ADIT between amino acid residues D348 and A349 of SEQ ID NO: 3 (348_insert_ADIT).

[0096] In a preferred embodiment, the HSV1 gEl or immunogenic fragment thereof comprises a mutation or combination of mutations relative to the sequence shown in SEQ ID NO: 3 selected from the following: P321K; P321D; R322D; N243A_R322D; N243A_P321D; A340G_S341G_V342G; H247G_P319G; P321R; H247A_P321K; 291_insert ADIGL; 339_insert ARAA; P319R; P319G_P321G and H247A_P321R.

[0097] In a more preferred embodiment, the HSV1 gEl or immunogenic fragment thereof comprises a mutation or combination of mutations with respect to the sequence shown in SEQ ID NO: 3 selected from P321D; R322D; A340G_S341G_V342G and P319R.

[0098] For the exemplary single and double substitution mutations and insertion mutations listed above, corresponding mutations in other HSV1 gE1 sequences are also within the scope of the present invention.

[0099] All possible combinations of the exemplary single and double substitution mutations and insertion mutations listed above are also within the scope of the present invention.

[0100] In a preferred embodiment, when the viral Fc receptor or immunogenic fragment thereof is a viral FcR ectodomain, the ability of the ectodomain to bind to an antibody Fc domain is reduced or eliminated compared to a native viral Fc receptor. Suitably, the viral FcR ectodomain comprises one or more amino acid substitutions, deletions or insertions compared to the native sequence of the viral FcR ectodomain, which substitutions, deletions or insertions reduce or eliminate the binding affinity between the viral FcR ectodomain and the antibody Fc domain compared to the native viral FcR.

[0101] The binding affinity between a viral FcR ectodomain and an antibody Fc domain can be determined by the methods described above.

[0102] In a preferred embodiment, the kappa binding affinity between the viral FcR ectodomain and human IgG is on is the k between the corresponding native viral FcR ectodomain and human IgG on Lower than (slow binder).

[0103] In a preferred embodiment, the kappa binding affinity between the viral FcR ectodomain and human IgG is off is the k between the corresponding native viral FcR ectodomain and human IgG off Higher than (fast releaser).

[0104] In a more preferred embodiment, the kappa ratio between the viral FcR ectodomain and human IgG is on is the k between the corresponding native viral FcR ectodomain and human IgG on The k between the viral FcR ectodomain and human IgG was lower than off is the k between the corresponding native viral FcR ectodomain and human IgG off Higher than (slow binder / fast releaser).

[0105] In a preferred embodiment, the equilibrium dissociation constant (K D ) is the K between the corresponding native viral FcR ectodomain and human IgG. D Higher than.

[0106] In a preferred embodiment, the relative affinity between a viral FcR ectodomain and human IgG is less than 100%, e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, or 10% of the affinity between a corresponding native viral FcR ectodomain and human IgG. In a more preferred embodiment, the relative affinity between a viral FcR ectodomain and human IgG is less than 15%, more preferably even less than 10%, of the affinity between a corresponding native viral FcR ectodomain and human IgG.

[0107] In a preferred embodiment, the equilibrium dissociation constant (K D ) is 2 × 10 -7 Higher than M, preferably 5 x 10 -7 M, preferably 1×10 -6 Higher than M.

[0108] Alternatively, the ability of a viral FcR ectodomain to bind to a human antibody Fc domain can be assessed by measuring the response (expressed in nm) in a bio-layer interferometry assay, as described in Examples 3 and 4. In preferred embodiments, the response in the bio-layer interferometry assay corresponding to binding between a viral FcR ectodomain and human IgG is less than 80%, suitably less than 70%, 60%, 50%, or 40% of the response obtained with the corresponding native viral FcR ectodomain. In preferred embodiments, the response in the bio-layer interferometry assay corresponding to binding between a viral FcR ectodomain and human IgG is less than 0.6 nm, suitably less than 0.5 nm, 0.4 nm, 0.3 nm, or 0.2 nm.

[0109] Suitably, the HSV2 gE2 ectodomain comprises one or more mutations (insertion, substitution or deletion) at a position selected from N241, H245, A246, A248, R314, P317, P318, P319, F322, R320, A337, S338 or V340 of the HSV2 gE2 ectodomain sequence shown in SEQ ID NO:7.

[0110] Exemplary mutations that can be used herein to reduce or eliminate binding affinity between the HSV2 gE2 ectodomain and an antibody Fc domain include single point substitution mutations of the sequence shown in SEQ ID NO:7 selected from the following: H245A, H245K, P317R, P319A, P319R, P319G, P319K, P319T, A337G, P319D, P319S, S 338D, N241A, R320D, H245E, H245V, H245R, H245D, H245Q, H245G, H245I, H245K, H24 5S, H245T, A246W, A248K, A248T, A248G, R314A, R314N, R314D, R314Q, R314E, R314 G, R314I, R314L, R314K, R314M, R314F, R314P, R314S, R314T, R314Y, R314V, P317N, P317G, P317I, P317L, P317K, P317F, P317S, P318R, P318D, P318Q, P318I, P318S, P 318T, P318Y, P319L, R320A, R320S, R320N, R320Q, R320E, R320G, R320H, R320I, R32 0L, R320M, R320P, R320T, R320V, F322A, F322N, F322I, F322K, F322P, F322T, S338G, S338E, S338L, S338T, V340A, V340R, V340D, V340Q, V340M, V340F, V340P and V340W.

[0111] Exemplary mutations that can be used herein to reduce or eliminate binding affinity between the HSV2 gE2 ectodomain and an antibody Fc domain also include double point substitution mutations of the sequence shown in SEQ ID NO:7 selected from the following: H245A and P319A; H245A and P319R; H245A and P319G; H245A and P319K; H245A and P319T; N241A and R320D; N241A and P319D; A246W and P317K; A246W and P317F; A246W and P317S; A246W and R320D; A246W and R320G; A246W and R320T; A248K and V340R; A248K and V340M; A248K and V340W; A248T and V340R; A248T and V340M; A248T and V340W; A248G and V3 40R;A248G and V340M;A248G and V340W;A248K and F322A;A248K and F322I;A248K and F322P;A248T and F322A;A248T and F322I;A248T and F322P;A248G and F322A;A248G and F322I;A248G and F322P;H245A and R320D;H245A and R320G;H245A and R320T;H245G and R320D;H245G and R320G;H245G and R320T;H245S and R320D;H245S and and R320G; H245S and R320T; H245A and P319G; H245A and P319L; H245G and P319G; H245G and P319L; H245S and P319G; H245S and P319L; R314G and P318R; R314G and P318D; R314G and P318I; R314L and P318R; R314L and P318D; R314L and P318I; R314P and P318R; R314P and P318D; R314P and P318I; R314G and F322A; R314G and F322I; R31 4G and F322P;R314L and F322A;R314L and F322I;R314L and F322P;R314P and F322A;R314P and F322I;R314P and F322P;R314G and V340R;R314G and V340M;R314G and V340W;R314L and V340R;R314L and V340M;R314L and V340W;R314P and V340R;R314P and V340M;R314P and V340W;P317K and V340R;P317K and V340M;P317K and V340W;P317F and V340R; P317F and V340M; P317F and V340W; P317S and V340R; P317S and V340M; P317S and V340W; P317K and S338G; P317K and S338H; P317K and S338L; P317F and S338G; P317F and S338H; P317F and S338L; P317S and S338G; P317S and S338H; P317S and S338L; P318R and S338G; P318R and S338H; P318R and S338L; P318D and S338G; P318D and S338H; P318D and S338L; P318I and S338G; P318I and S338H; P318I and S338L; P319G and V340R; P319G and V340M; P319G and V340W; P319L and V340R; P319L and V340M; and P319L; V340W; P317R and P319D; P317R and R320D; P319D and R320D. ;

[0112] Exemplary mutations that can be used herein to reduce or eliminate binding affinity between the HSV2 gE2 ectodomain and an antibody Fc domain also include deletion mutations at positions P319 and / or R320 of the sequence shown in SEQ ID NO: 7, either alone or in combination with substitution mutations, particularly mutations selected from the following: P319 deletion; R320 deletion; P319 deletion / R320 deletion; P319 deletion / R320 deletion / P317G / P318G; P319 deletion / R320 deletion / P318E; P319 deletion / R320 deletion / P318G; P319 deletion / R320 deletion / P318K; P319 deletion / R320 deletion / P317R / P318E; P319 deletion / R320 deletion / P317R / P318G; P319 deletion / R320 deletion / P317R / P318K; P319 deletion / R320 deletion / P317G / P318K.

[0113] Exemplary mutations that can be used herein to reduce or eliminate binding affinity between the HSV2 gE2 ectodomain and an antibody Fc domain also include insertion mutations selected from the following: insertion of the peptide sequence LDIGE between amino acid residues Y275 and E276 of SEQ ID NO: 7 (275_insert_LDIGE), insertion of the peptide sequence ADIGL between amino acid residues S289 and P290 of SEQ ID NO: 7 (289_insertADIGL), insertion of the peptide sequence ARAA between amino acid residues A337 and S338 of SEQ ID NO: 7 (337_insert_ARAA), an insert peptide sequence ARAA between amino acid residues S338 and T339 of SEQ ID NO: 7 (338_insert_ARAA), and The inserted peptide sequence ADIT (346_insert_ADIT) between amino acid residues H346 and A347 of SEQ ID NO: 7.

[0114] In a preferred embodiment, the HSV2 gE2 ectodomain comprises a mutation or combination of mutations with respect to the sequence shown in SEQ ID NO: 7 selected from the following: 289_インサートADIGL;338_インサートARAA;H245K;P317R;P319R;P319G;P319K;H24 5A_P319R;H245A_P319G;H245A_P319K;H245A_P319T;P319D;S338D;R320D; N241A_R320D;A248K_V340M;P318Y;A248K_V340R;A248T_V340W;A248K_V3 40W;A246W_R320G;A246W_P317K;A246W_R320D;A246W_R320T;V340W;A248G _V340W;H245G_R320D;P318D;A246W_P317F;P319G_V340W;A248T_V340M;P 317K_V340W;V340F;V340D;H245A_R320D;P317F_V340W;A246W_P317S;H245 S_R320D;R314G_P318D;A248T;P318S;P317K;P317S_V340W;H245D;R314P_ V340W;R314L_318D;P319L_V340W;P317F;P318D_S338G;R314G_V340W;P317 K_S338H;R314L_V340W;P318R;P318Q;P317F_S338G;R314G_P318I;H245G_ P319G;P317L;P318I;A248T_F322A;H245E;P318T;P318R_S338G;P318D_S33 8H;P317F_S338H;A248T_V340R;A248T_F322I;H245A_R320G;P318R_S338H;H245S_R320G;P317K_S338G;A248T_F322P;V340R;R314L_P318R;H245S_R3 20T;R314G_P318R;R320E;H245G_R320G;H245A_R320T;A246W;P318I_S338 G;P317K_V340M;P317I;R320H;R314P_P318I;P318I_S338H;P317F_V340M;H 245A_P319G;H245A_P319L;R320P;H245G_R320T;R314L_V340R;P319G_V34 0R;R314G_F322I;R314L_P318I;R320A;R314N;P317F_V340R;P318D_S338L;A248G_V340R;R314E;R314P_P318D;H245S_P319G;V340Q;A248K_F322I;R320G;H245S_P319L;R314F;P319L;P317K_S338L;P319L_V3 40M;P317G;R320S;R320Q;R314P_V340R;V340A;H245G_P319L;R320T;R314P_P318R;A248G_F322I;R320N;P317N;R314D;R314Y;R314 P_F322I;P319G_V340M;P317S_V340R;R314V;P317R_P319D;P317R_R320D;P319D_R320D;Δ319_Δ320;P317G_P318G_Δ319_Δ320;P318 E_Δ319_Δ320;P318G_Δ319_Δ320;P318K_Δ319_Δ320;P317R_P318E_Δ319_320;P317R_P318G_Δ319_Δ320 and P317G_P318K_Δ319_Δ320. ;

[0115] In a more preferred embodiment, the HSV2 gE2 ectodomain comprises a mutation or combination of mutations with respect to the sequence set forth in SEQ ID NO: 7 selected from 338_insert ARAA; P317R; P319D; R320D; A248T_V340W; V340W; A248T; P318I and A246W.

[0116] For the exemplary substitution, deletion, and insertion mutations listed above, corresponding mutations in other HSV2 gE2 ectodomain sequences, such as those listed in Table 1 and shown on the alignment provided in Figure 3, are also within the scope of the invention.

[0117] All possible combinations of the exemplary single and double substitution mutations and insertion mutations listed above are also within the scope of the present invention.

[0118] Suitably, the HSV1 gE1 ectodomain comprises one or more mutations (insertion, substitution or deletion) at positions selected from H247, P319 and P321 of the HSV1 gE1 ectodomain sequence shown in SEQ ID NO:9.

[0119] Exemplary mutations that can be used herein to reduce or eliminate binding affinity between the HSV1 gEl ectodomain and an antibody Fc domain include single point substitution mutations of the sequence set forth in SEQ ID NO:9 selected from: H247A, H247K, P319R, P321A, P321R, P321G, P321K, P321T, A339G, P321D, P321S, A340D, N243A, and R322D, and double point substitution mutations of the sequence set forth in SEQ ID NO:9 selected from: These include H247A / P321A, H247A / P321R, H247A / P321G, H247A / P321K, H247A / P321T, N243A / R322D, N243A / P321D, H247G / P319G, P319G / P321G, and A340G / S341G / V342G.

[0120] Exemplary mutations that can be used herein to reduce or eliminate binding affinity between the HSV1 gE1 ectodomain and an antibody Fc domain also include insertion mutations selected from the following: insertion of the peptide sequence LDIGE between amino acid residues Y277 and E278 of SEQ ID NO: 9 (277_insert_LDIGE); insertion of the peptide sequence ADIGL between amino acid residues S291 and P292 of SEQ ID NO: 9 (291_insertADIGL); insertion of the peptide sequence ARAA between amino acid residues A339 and A340 of SEQ ID NO: 9 (339_insert_ARAA); insertion of the peptide sequence ARAA between amino acid residues A340 and S341 of SEQ ID NO: 9 (340_insert_ARAA); and Insertion of the peptide sequence ADIT between amino acid residues D348 and A349 of SEQ ID NO: 9 (348_insert_ADIT).

[0121] In a preferred embodiment, the HSV1 gEl ectodomain comprises a mutation or combination of mutations with respect to the sequence shown in SEQ ID NO:9 selected from the following: P321K; P321D; R322D; N243A_R322D; N243A_P321D; A340G_S341G_V342G; H247G_P319G; P321R; H247A_P321K; 291_insert ADIGL; 339_insert ARAA; P319R; P319G_P321G and H247A_P321R.

[0122] In a more preferred embodiment, the HSV1 gEl ectodomain comprises a mutation or combination of mutations with respect to the sequence set forth in SEQ ID NO: 9 selected from P321D; R322D; A340G_S341G_V342G and P319R.

[0123] For the exemplary single and double substitution mutations and insertion mutations listed above, corresponding mutations in other HSV1 gE1 ectodomain sequences are also within the scope of the present invention.

[0124] All possible combinations of the exemplary single and double substitution mutations and insertion mutations listed above are also within the scope of the present invention.

[0125] In a preferred embodiment, the viral Fc receptor or immunogenic fragment thereof is part of a heterodimer with a binding partner or fragment thereof from said virus.

[0126] As used herein, a "binding partner" is a viral protein (or glycoprotein) or fragment thereof that forms a non-covalent heterodimeric complex with an Fc receptor or immunogenic fragment thereof.

[0127] In a preferred embodiment, the viral Fc receptor is HSV2 gE2 or an immunogenic fragment thereof and the binding partner is HSV2 gI2 or a fragment thereof.

[0128] As used herein, "HSV2 gI2" (or "HSV2 gI") refers to the HSV2 gI glycoprotein encoded by the HSV2 gene US7 and displayed on the surface of infected cells, where it associates with HSV2 gE2 to form a heterodimer. Suitably, the HSV2 gI2 is selected from the HSV2 gI glycoproteins shown in Table 2, or a variant thereof that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical thereto.

[0129] [Table 2] JPEG2025163028000004.jpg178132

[0130] In a preferred embodiment, the HSV2 gI2 is gI from HSV2 strain SD90e having the amino acid sequence set forth in SEQ ID NO: 2 (Genbank accession number AHG54731.1, UniProtKB accession number: A8U5L5, SEQ ID NO: 2), or a variant therefrom that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical thereto.

[0131] In another preferred embodiment, the viral Fc receptor is HSV1 gE1 or an immunogenic fragment thereof and the binding partner is HSV1 gI1 or a fragment thereof.

[0132] As used herein, "HSV1 gll" (or "HSV1 gI") refers to the HSV1 gI glycoprotein encoded by the HSV1 gene US7 and displayed on the surface of infected cells, where it associates with HSV1 gEl to form a heterodimer. Suitably, HSV1 gll is gI from HSV1 strain 17 having the amino acid sequence set forth in SEQ ID NO: 4 (UniProtKB Accession No: P06487, SEQ ID NO: 4), or a variant thereof that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.

[0133] In one embodiment, a fragment of a viral FcR binding partner is used.

[0134] As used herein, the term "fragment" as applied to a protein or peptide refers to a subsequence of a larger protein or peptide. A "fragment" of a protein or peptide is at least about 10 amino acids in length (in the case of a plurality of amino acids that naturally occur as contiguous amino acids, e.g., a single linear epitope), e.g., at least about 15, 20, 30, 40, 50, 60, 100, 200, 300 or more amino acids in length (and any integer value in between).

[0135] In a preferred embodiment, the viral FcR binding partner fragment is an immunogenic fragment.

[0136] As used herein, a "fragment of a viral FcR binding partner" refers to a fragment of a naturally occurring viral FcR binding partner having at least 10, 15, 20, 30, 40, 50, 60, 100, 200, 300, or more amino acids, or a peptide having an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity to a naturally occurring viral FcR binding partner (or a fragment of a naturally occurring viral FcR binding partner having at least about 10, 15, 20, 30, 40, 50, 60, or more amino acids). Thus, a fragment of a viral FcR binding partner can be a fragment of at least 10 amino acids of a naturally occurring viral FcR binding partner and can include one or more amino acid substitutions, deletions, or additions.

[0137] Any of the encoded viral FcR binding partner fragments can further include an initial methionine residue, if desired.

[0138] A transmembrane protein is a type of integral membrane protein that has the ability to span across a cell membrane under normal culture conditions. As used herein, a transmembrane domain is a section of a transmembrane protein that is within the cell membrane under normal culture conditions. As used herein, a cytoplasmic domain is a section of a transmembrane protein that is on the cytoplasmic side of the cell membrane under normal culture conditions. As used herein, an ectodomain is a section of a transmembrane protein that is on the outside of the cell membrane under normal culture conditions.

[0139] Suitably, the viral FcR binding partner or fragment thereof does not comprise a transmembrane domain. Suitably, the viral FcR binding partner or immunogenic fragment thereof does not comprise a cytoplasmic domain. Preferably, the viral FcR binding partner or immunogenic fragment thereof does not comprise a transmembrane domain or a cytoplasmic domain. In other words, in a preferred embodiment, the viral FcR binding partner or immunogenic fragment consists of the viral FcR binding partner ectodomain (or extracellular domain). More preferably, the viral FcR binding partner or immunogenic fragment is selected from the HSV2 gI2 ectodomain and the HSV1 gI1 ectodomain.

[0140] In a preferred embodiment, the viral FcR binding partner ectodomain is an HSV2 gI2 ectodomain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:8 (corresponding to amino acid residues 1-256 of SEQ ID NO:2), or a sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto. Suitably, the viral FcR ectodomain has a sequence selected from the sequences set forth in Table 2 or Figure 4. In a preferred embodiment, the viral FcR ectodomain is an HSV2 gE2 ectodomain that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO:8.

[0141] Suitably, the viral FcR binding partner HSV2 gI2 ectodomain may comprise one or more amino acid residue substitutions, deletions or insertions, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residue substitutions, deletions or insertions relative to the amino acid sequence set out in SEQ ID NO:8.

[0142] In another embodiment, the viral FcR binding partner is an HSV2 gI2 ectodomain comprising or consisting of an amino acid sequence corresponding to amino acid residues 1 to 262 of SEQ ID NO: 2, or a sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical thereto. Suitably, the viral FcR binding partner HSV2 gI2 ectodomain comprises one or more amino acid residue substitutions, deletions or insertions, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residue substitutions, deletions or insertions, relative to the amino acid sequence corresponding to amino acid residues 1 to 262 of SEQ ID NO: 2.

[0143] In another embodiment, the viral FcR binding partner ectodomain is an HSV1 gI1 ectodomain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:10 (corresponding to amino acid residues 1-270 of SEQ ID NO:4), or a sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto. In a preferred embodiment, the viral FcR ectodomain is an HSV1 gE1 ectodomain that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:10.

[0144] Suitably, the viral FcR binding partner HSV1 gll ectodomain may comprise one or more amino acid residue substitutions, deletions or insertions, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residue substitutions, deletions or insertions relative to the amino acid sequence shown in SEQ ID NO: 10.

[0145] In another embodiment, the viral FcR binding partner is an HSV1 gI1 ectodomain comprising or consisting of an amino acid sequence corresponding to amino acid residues 1 to 276 of SEQ ID NO: 4, or a sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical thereto. Suitably, the viral FcR binding partner HSV1 gI1 ectodomain comprises one or more amino acid residue substitutions, deletions or insertions, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residue substitutions, deletions or insertions, relative to the amino acid sequence corresponding to amino acid residues 1 to 276 of SEQ ID NO: 4.

[0146] Antibodies to gD, gB, and gC, and to a lesser extent gH / gL, have been detected in HSV-infected subjects. The predominant neutralizing response was to gD (Cairns, Tina M., et al. "Dissection of the antibody response against herpes simplex virus glycoproteins in naturally infected humans." Journal of Virology 88.21 (2014): 12612-12622.).

[0147] In one embodiment, the viral Fc receptor or immunogenic fragment thereof is not administered to the subject in combination with an immunodominant viral antigen.

[0148] Immunodominance is an immunological phenomenon in which an immune response is initiated only against a subset of antigenic peptides produced by a pathogen. Immunodominance has been demonstrated for antibody-mediated and cell-mediated immunity. As used herein, an "immunodominant antigen" is an antigen that contains an immunodominant epitope. In contrast, a "subdominant antigen" is an antigen that does not contain an immunodominant epitope, or in other words, contains only a subdominant epitope. As used herein, an "immunodominant epitope" is an epitope that is predominantly targeted or targeted to a greater extent by neutralizing antibodies during an immune response to the same pathogen, compared to other epitopes from the pathogen. As used herein, a "subdominant epitope" is an epitope that is not targeted or targeted to a lesser extent by neutralizing antibodies during an immune response to the same pathogen, compared to other epitopes from the pathogen. For example, gD2 is the immunodominant antigen of HSV2, and gD1 is the immunodominant antigen of HSV1. In contrast, gB2, gC2, gE2 / gI2, and gH2 / gL2 are subdominant antigens of HSV2, and gB1, gCl, gE1 / gI1, and the gH1 / gL1 heterodimer are subdominant antigens of HSV1.

[0149] Suitably, when the viral Fc receptor is HSV2 gE2 or HSV1 gE1, the Fc receptor or immunogenic fragment thereof is not administered to the subject together with HSV2 gD2 or HSV1 gD1, or a fragment thereof comprising an immunodominant epitope. In a specific embodiment when the viral Fc receptor is HSV2 gE2, the viral Fc receptor or immunogenic fragment thereof is not administered to the subject together with HSV2 gD2 or a fragment thereof comprising an immunodominant epitope. In another specific embodiment when the viral Fc receptor is HSV1 gE1, the viral Fc receptor or immunogenic fragment thereof is not administered to the subject together with HSV1 gD1 or a fragment thereof comprising an immunodominant epitope.

[0150] In one embodiment, the viral Fc receptor is not varicella zoster virus (VZV) gE.

[0151] The glycoprotein gC from HSV1 and HSV2 is also involved in immune evasion mechanisms by inhibiting complement (Awasthi, Sita, et al. "Blocking herpes simplex virus 2 glycoprotein E immune evasion as an approach to enhance efficacy of a trivalent subunit antigen vaccine for genital herpes." Journal of Virology 88.15 (2014): 8421-8432.).

[0152] In one embodiment, the viral Fc receptor is HSV2 gE2 and is administered to the subject along with HSV2 gC2 or an immunogenic fragment thereof.

[0153] In one embodiment, the viral Fc receptor is HSV1 gE1 and is administered to the subject along with HSV1 gC1 or an immunogenic fragment thereof.

[0154] In one aspect, the present invention provides a recombinant viral FcR or immunogenic fragment thereof, wherein the ability of the viral FcR or immunogenic fragment thereof to bind to a human antibody Fc domain is reduced or abolished compared to the corresponding native viral Fc receptor.

[0155] Suitably, the recombinant viral Fc receptor or immunogenic fragment thereof comprises one or more amino acid substitutions, deletions or insertions compared to the native sequence of the viral Fc receptor or immunogenic fragment thereof, which substitutions, deletions or insertions reduce or eliminate the binding affinity between the viral FcR or immunogenic fragment thereof and the antibody Fc domain compared to the native viral Fc receptor.

[0156] In a preferred embodiment, the kappa binding affinity between the recombinant viral FcR or immunogenic fragment thereof and human IgG is on is the k between the corresponding native viral FcR and human IgG onIn a preferred embodiment, the k between the recombinant viral FcR or immunogenic fragment thereof and human IgG is lower than off is the k between the corresponding native viral FcR and human IgG off In a more preferred embodiment, the k between the recombinant viral FcR or immunogenic fragment thereof and human IgG is higher than on is the k between the corresponding native viral FcR and human IgG on The k between the recombinant viral FcR or its immunogenic fragment and human IgG is lower than off is the k between the corresponding native viral FcR and human IgG off Higher than (slow binder / fast releaser).

[0157] In a preferred embodiment, the equilibrium dissociation constant (K D ) is the K between the corresponding native viral FcR and human IgG D Higher than.

[0158] In a preferred embodiment, the relative affinity between the recombinant viral FcR or immunogenic fragment thereof and human IgG is less than 100%, e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, or 10% of the affinity between the corresponding native viral FcR and human IgG. In a more preferred embodiment, the relative affinity between the recombinant viral FcR or immunogenic fragment thereof and human IgG is less than 15%, more preferably even less than 10%, of the affinity between the corresponding native viral FcR and human IgG.

[0159] In a preferred embodiment, the equilibrium dissociation constant (K D ) is 2 × 10 -7 Higher than M, preferably 5 x 10 -7 M, preferably 1×10 -6 Higher than M.

[0160] Alternatively, the ability of a viral FcR or an immunogenic fragment thereof to bind to a human antibody Fc domain can be assessed by measuring the response (expressed in nm) in a biolayer interferometry assay, as described in Examples 3 and 4.

[0161] In a preferred embodiment, the response in a bio-layer interferometry assay corresponding to binding between the viral FcR or an immunogenic fragment thereof and human IgG is less than 80%, suitably less than 70%, 60%, 50%, 40% of the response obtained with the corresponding native viral FcR. In a preferred embodiment, the response in a bio-layer interferometry assay corresponding to binding between the viral FcR or an immunogenic fragment thereof and human IgG is less than 0.4 nm, suitably less than 0.3 nm, 0.2 nm or 0.1 nm.

[0162] In a preferred embodiment, the recombinant viral FcR or immunogenic fragment thereof is HSV2 gE2 or an immunogenic fragment thereof. Suitably, the recombinant HSV2 gE2 or immunogenic fragment thereof comprises one or more mutations (insertion, substitution or deletion) at positions selected from N241, H245, A246, A248, R314, P317, P318, P319, F322, R320, A337, S338 or V340 of the HSV2 gE2 sequence shown in SEQ ID NO:1.

[0163] Exemplary mutations that can be used herein to reduce or eliminate binding affinity between recombinant HSV2 gE2 or an immunogenic fragment thereof and an antibody Fc domain include single point substitution mutations of the sequence shown in SEQ ID NO: 1 selected from the following: H245A, H245K, P317R, P319A, P319R, P319G, P319K, P319T, A337G, P319D, P319S, S 338D, N241A, R320D, H245E, H245V, H245R, H245D, H245Q, H245G, H245I, H245K, H24 5S, H245T, A246W, A248K, A248T, A248G, R314A, R314N, R314D, R314Q, R314E, R314 G, R314I, R314L, R314K, R314M, R314F, R314P, R314S, R314T, R314Y, R314V, P317N, P317G, P317I, P317L, P317K, P317F, P317S, P318R, P318D, P318Q, P318I, P318S, P 318T, P318Y, P319L, R320A, R320S, R320N, R320Q, R320E, R320G, R320H, R320I, R32 0L, R320M, R320P, R320T, R320V, F322A, F322N, F322I, F322K, F322P, F322T, S338G, S338E, S338L, S338T, V340A, V340R, V340D, V340Q, V340M, V340F, V340P and V340W.

[0164] Exemplary mutations that can be used herein to reduce or eliminate binding affinity between recombinant HSV2 gE2 or an immunogenic fragment thereof and an antibody Fc domain also include double point substitution mutations of the sequence shown in SEQ ID NO: 1 selected from the following: H245A and P319A; H245A and P319R; H245A and P319G; H245A and P319K; H245A and P319T; N241A and R320D; N241A and P319D; A246W and P317K; A246W and P317F; A246W and P317S; A246W and R320D; A246W and R320G; A246W and R320T; A248K and V340R; A248K and V340M; A248K and V340W; A248T and V340R; A248T and V340M; A248T and V340W; A24 8G and V340R;A248G and V340M;A248G and V340W;A248K and F322A;A248K and F322I;A248K and F322P;A248T and F322A;A248T and F322I;A248T and F322P;A248G and F322A;A248G and F322I;A248G and F322P;H245A and R320D;H245A and R320G;H245A and R320T;H245G and R320D;H245G and R320G;H245G and R320T;H245S and R320D;H245S and and R320G; H245S and R320T; H245A and P319G; H245A and P319L; H245G and P319G; H245G and P319L; H245S and P319G; H245S and P319L; R314G and P318R; R314G and P318D; R314G and P318I; R314L and P318R; R314L and P318D; R314L and P318I; R314P and P318R; R314P and P318D; R314P and P318I; R314G and F322A; R314G and F322I; R314G and F3 22P;R314L and F322A;R314L and F322I;R314L and F322P;R314P and F322A;R314P and F322I;R314P and F322P;R314G and V340R;R314G and V340M;R314G and V340W;R314L and V340R;R314L and V340M;R314L and V340W;R314P and V340R;R314P and V340M;R314P and V340W;P317K and V340R;P317K and V340M;P317K and V340W;P317F and V340R;P317F and V340M;P317F and V340W;P317S and V340R;P317S and V340M;P317S and V340W;P317K and S338G;P317K and S338H;P317K and S338L;P317F and S338G;P317F and S338H;P317F and S338L;P317S and S338G;P317S and S338H;P317S and S338L;P318R and S338G;P318R and S338H; P318R and S338L; P318D and S338G; P318D and S338H; P318D and S338L; P318I and S338G; P318I and S338H; P318I and S338L; P319G and V340R; P319G and V340M; P319G and V340W; P319L and V340R; P319L and V340M; P319L and V340W; P317R and P319D; P317R and R320D; P319D and R320D. ;

[0165] Exemplary mutations that can be used herein to reduce or eliminate binding affinity between recombinant HSV2 gE2 or an immunogenic fragment thereof and an antibody Fc domain also include deletion mutations at positions P319 and / or R320 of the sequence shown in SEQ ID NO: 1, either alone or in combination with substitution mutations, particularly mutations selected from the following: P319 deletion; R320 deletion; P319 deletion / R320 deletion; P319 deletion / R320 deletion / P317G / P318G; P319 deletion / R320 deletion / P318E; P319 deletion / R320 deletion / P318G; P319 deletion / R320 deletion / P318K; P319 deletion / R320 deletion / P317R / P318E; P319 deletion / R320 deletion / P317R / P318G; P319 deletion / R320 deletion / P317R / P318K; P319 deletion / R320 deletion / P317G / P318K.

[0166] Exemplary mutations that can be used herein to reduce or eliminate binding affinity between recombinant HSV2 gE2 or an immunogenic fragment thereof and an antibody Fc domain also include insertion mutations selected from the following: insertion of the peptide sequence LDIGE between amino acid residues Y275 and E276 of SEQ ID NO: 1 (275_insert_LDIGE), insertion of the peptide sequence ADIGL between amino acid residues S289 and P290 of SEQ ID NO: 1 (289_insertADIGL), insertion of the peptide sequence ARAA between amino acid residues A337 and S338 of SEQ ID NO: 1 (337_insert_ARAA), insertion of the peptide sequence ARAA between amino acid residues S338 and T339 of SEQ ID NO: 1 (338_insert_ARAA), and Insertion of the peptide sequence ADIT between amino acid residues H346 and A347 of SEQ ID NO: 1 (346_insert_ADIT).

[0167] In a preferred embodiment, the recombinant HSV2 gE2 or immunogenic fragment thereof comprises a mutation or combination of mutations with respect to the sequence shown in SEQ ID NO: 1 selected from the following: 289_インサートADIGL;338_インサートARAA;H245K;P317R;P319R;P319G;P319K;H24 5A_P319R;H245A_P319G;H245A_P319K;H245A_P319T;P319D;S338D;R320D; N241A_R320D;A248K_V340M;P318Y;A248K_V340R;A248T_V340W;A248K_V3 40W;A246W_R320G;A246W_P317K;A246W_R320D;A246W_R320T;V340W;A248G _V340W;H245G_R320D;P318D;A246W_P317F;P319G_V340W;A248T_V340M;P 317K_V340W;V340F;V340D;H245A_R320D;P317F_V340W;A246W_P317S;H245 S_R320D;R314G_P318D;A248T;P318S;P317K;P317S_V340W;H245D;R314P_ V340W;R314L_318D;P319L_V340W;P317F;P318D_S338G;R314G_V340W;P317 K_S338H;R314L_V340W;P318R;P318Q;P317F_S338G;R314G_P318I;H245G_ P319G;P317L;P318I;A248T_F322A;H245E;P318T;P318R_S338G;P318D_S33 8H;P317F_S338H;A248T_V340R;A248T_F322I;H245A_R320G;P318R_S338H;H245S_R320G;P317K_S338G;A248T_F322P;V340R;R314L_P318R;H245S_R3 20T;R314G_P318R;R320E;H245G_R320G;H245A_R320T;A246W;P318I_S338 G;P317K_V340M;P317I;R320H;R314P_P318I;P318I_S338H;P317F_V340M;H 245A_P319G;H245A_P319L;R320P;H245G_R320T;R314L_V340R;P319G_V34 0R;R314G_F322I;R314L_P318I;R320A;R314N;P317F_V340R;P318D_S338L;A248G_V340R;R314E;R314P_P318D;H245S_P319G;V340Q;A248K_F322I;R320G;H245S_P319L;R314F;P319L;P317K_S338L;P319L_V3 40M;P317G;R320S;R320Q;R314P_V340R;V340A;H245G_P319L;R320T;R314P_P318R;A248G_F322I;R320N;P317N;R314D;R314Y;R314 P_F322I;P319G_V340M;P317S_V340R;R314V;P317R_P319D;P317R_R320D;P319D_R320D;Δ319_Δ320;P317G_P318G_Δ319_Δ320;P318 E_Δ319_Δ320;P318G_Δ319_Δ320;P318K_Δ319_Δ320;P317R_P318E_Δ319_320;P317R_P318G_Δ319_Δ320 and P317G_P318K_Δ319_Δ320. ;

[0168] In a more preferred embodiment, the HSV2 gE2 or immunogenic fragment thereof comprises a mutation or combination of mutations with respect to the sequence shown in SEQ ID NO: 1 selected from 338_insert ARAA; P317R; P319D; R320D; A248T_V340W; V340W; A248T; P318I and A246W.

[0169] For the exemplary single and double substitutions, selections and mutations and insertion mutations listed above, corresponding mutations in other HSV2 gE2 sequences, such as those listed in Table 1 and shown on the alignment provided in Figure 3, are also within the scope of the invention.

[0170] All possible combinations of the exemplary single and double substitution mutations and insertion mutations listed above are also within the scope of the present invention.

[0171] In a preferred embodiment, the recombinant HSV2 gE2 or immunogenic fragment thereof is a recombinant HSV2 gE2 ectodomain as described herein.

[0172] In another embodiment, the recombinant viral FcR or immunogenic fragment thereof is a recombinant HSV1 gEl or immunogenic fragment thereof. Suitably, the recombinant HSV1 gEl or immunogenic domain thereof comprises one or more mutations (insertion, substitution or deletion) at positions selected from H247, P319 and P321 of the HSV1 gEl sequence shown in SEQ ID NO:3.

[0173] Exemplary mutations that can be used herein to reduce or eliminate binding affinity between a recombinant HSV1 gEl or an immunogenic fragment thereof and an antibody Fc domain include single point substitution mutations of the sequence set forth in SEQ ID NO:3 selected from the following: H247A, H247K, P319R, P321A, P321R, P321G, P321K, P321T, A339G, P321D, P321S, A340D, N243A, and R322D, and double point substitution mutations of the sequence set forth in SEQ ID NO:3 selected from the following: These include H247A / P321A, H247A / P321R, H247A / P321G, H247A / P321K, H247A / P321T, N243A / R322D, N243A / P321D, H247G / P319G, P319G / P321G, and A340G / S341G / V342G.

[0174] Exemplary mutations that can be used herein to reduce or eliminate binding affinity between recombinant HSV1 gEl or an immunogenic fragment thereof and an antibody Fc domain also include insertion mutations selected from the following: insertion of the peptide sequence LDIGE between amino acid residues Y277 and E278 of SEQ ID NO: 3 (277_insert_LDIGE); insertion of the peptide sequence ADIGL between amino acid residues S291 and P292 of SEQ ID NO: 3 (291_insert_ADIGL); insertion of the peptide sequence ARAA between amino acid residues A339 and A340 of SEQ ID NO: 3 (339_insert_ARAA); insertion of the peptide sequence ARAA between amino acid residues A340 and S341 of SEQ ID NO: 3 (340_insert_ARAA); and Insertion of the peptide sequence ADIT between amino acid residues D348 and A349 of SEQ ID NO: 3 (348_insert_ADIT).

[0175] In a preferred embodiment, the HSV1 gEl or immunogenic fragment thereof comprises a mutation or combination of mutations with respect to the sequence shown in SEQ ID NO:3 selected from the following: P321K; P321D; R322D; N243A_R322D; N243A_P321D; A340G_S341G_V342G; H247G_P319G; P321R; H247A_P321K; 291_insert ADIGL; 339_insert ARAA; P319R; P319G_P321G and H247A_P321R.

[0176] In a more preferred embodiment, the HSV1 gEl or immunogenic fragment thereof comprises a mutation or combination of mutations with respect to the sequence shown in SEQ ID NO: 3 selected from P321D; R322D; A340G_S341G_V342G and P319R.

[0177] For the exemplary single and double substitution mutations and insertion mutations listed above, corresponding mutations in other HSV1 gE1 sequences are also within the scope of the present invention.

[0178] All possible combinations of the exemplary single and double substitution mutations and insertion mutations listed above are also within the scope of the present invention.

[0179] In a preferred embodiment, the recombinant HSV1 gE1 or immunogenic fragment thereof is a recombinant HSV1 gE1 ectodomain as described herein.

[0180] In a preferred embodiment, the recombinant viral FcR or immunogenic fragment thereof is part of a heterodimer with a binding partner or fragment thereof derived from said virus.

[0181] In a preferred embodiment, the recombinant viral Fc receptor is recombinant HSV2 gE2 or an immunogenic fragment thereof, and the binding partner is HSV2 gI2 or a fragment thereof as described herein.

[0182] In another preferred embodiment, the recombinant viral Fc receptor is recombinant HSV1 gE1 or an immunogenic fragment thereof and the binding partner is HSV1 gI1 or a fragment thereof or a fragment thereof described herein.

[0183] In another aspect, the present invention provides a heterodimer comprising or consisting of an Fc receptor from an HSV virus or an immunogenic fragment thereof and a binding partner from said HSV virus or a fragment thereof for use in therapy.

[0184] In one embodiment of the heterodimer, the viral Fc receptor is HSV2 gE2 and the binding partner is HSV2 gI2, hi another embodiment, the viral Fc receptor is HSV1 gE1 and the binding partner is HSV1 gI1.

[0185] In another aspect, the present invention provides a pharmaceutical composition comprising an Fc receptor derived from an HSV virus or an immunogenic fragment thereof, a binding partner derived from said HSV virus or a fragment thereof, and a pharmaceutically acceptable carrier.

[0186] In one embodiment of the pharmaceutical composition, the viral Fc receptor is HSV2 gE2 and the binding partner is HSV2 gI2. In another embodiment of the pharmaceutical composition, the viral Fc receptor is HSV1 gE1 and the binding partner is HSV1 gI1.

[0187] In another aspect, the present invention provides an immunogenic composition comprising an Fc receptor or immunogenic fragment thereof derived from a virus as described herein and a pharmaceutically acceptable carrier. Suitably, the immunogenic composition can be prepared for administration by being suspended or dissolved in a pharmaceutically or physiologically acceptable carrier. Preferably, the immunogenic composition of the present invention is suitable for use as a therapeutic vaccine.

[0188] A "pharmaceutically acceptable carrier" includes any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (e.g., oil droplets or liposomes). Such carriers are well known to those skilled in the art. The composition may also contain a pharmaceutically acceptable diluent, such as water, saline, glycerol, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. Sterile, pyrogen-free, phosphate-buffered saline is a typical carrier. The appropriate carrier may, in large part, depend upon the route of administration.

[0189] Suitably, the viral Fc receptor or fragment thereof will be administered to the subject by any route known in the art, including intramuscular, intravaginal, intravenous, intraperitoneal, subcutaneous, transdermal, intradermal, nasal, intratumoral or oral administration.

[0190] In one embodiment, the subject is a vertebrate, e.g., a mammal, e.g., a human, a non-human primate, or a veterinary mammal (domestic or pet animal). In a preferred embodiment, the subject is a human.

[0191] In a preferred embodiment, the subject is infected (i.e., seropositive) with a virus, e.g., a herpesvirus, e.g., HSV2, HSV1, or HCMV, before being treated with the viral FcR or immunogenic fragment thereof. A subject infected with a virus before being treated with the viral FcR or immunogenic fragment thereof may exhibit clinical signs of infection (symptomatic subject) or may not exhibit clinical signs of viral infection (asymptomatic subject). In one embodiment, a symptomatic subject has exhibited several episodes (relapses) with clinical symptoms of infection over time separated by periods without clinical symptoms.

[0192] In one aspect, the invention provides a herpesvirus Fc receptor or an immunogenic fragment thereof, or a nucleic acid encoding said viral FcR or an immunogenic fragment thereof, for use in the treatment of a recurrent herpes infection, or in a method for preventing or reducing the frequency of recurrent herpesvirus infections, in a subject, preferably a human subject.

[0193] In one aspect, the invention provides HSV2 gE2 or an immunogenic fragment thereof, or a nucleic acid encoding said HSV2 gE2 or an immunogenic fragment thereof, for use in the treatment of a recurrent HSV2 infection, or for use in a method for preventing or reducing the frequency of recurrent HSV2 infection, in a subject, preferably a human subject.

[0194] In one aspect, the present invention provides an HSV2 gE2 / gI2 heterodimer or an immunogenic fragment thereof, or a nucleic acid encoding said HSV2 gE2 / gI2 heterodimer or an immunogenic fragment thereof, for use in the treatment of a recurrent HSV2 infection, or for use in a method for preventing or reducing the frequency of recurrent HSV2 infection, in a subject, preferably a human subject.

[0195] In one aspect, the invention provides HSV1 gE1 or an immunogenic fragment thereof, or a nucleic acid encoding said HSV1 gE1 or an immunogenic fragment thereof, for use in the treatment of recurrent HSV1 infection, or for use in a method for preventing or reducing the frequency of recurrent HSV1 infection, in a subject, preferably a human subject.

[0196] In one aspect, the present invention provides an HSV1 gE1 / gI1 heterodimer or an immunogenic fragment thereof, or a nucleic acid encoding said HSV1 gE1 / gI1 heterodimer or an immunogenic fragment thereof, for use in the treatment of recurrent HSV1 infection, or for use in a method for preventing or reducing the frequency of recurrent HSV1 infection, in a subject, preferably a human subject.

[0197] In one aspect, the invention provides a herpesvirus Fc receptor or an immunogenic fragment thereof, or a nucleic acid encoding said viral FcR or an immunogenic fragment thereof, as described herein, for use in the manufacture of an immunogenic composition.

[0198] In one aspect, the invention provides the use of a herpesvirus Fc receptor or an immunogenic fragment thereof, or a nucleic acid encoding said viral FcR or an immunogenic fragment thereof, as described herein, in the manufacture of a medicament for the treatment of a herpes infection or a herpes-related disease.

[0199] In one aspect, the present invention provides an HSV2 gE2 or HSV2 gE2 / gI2 heterodimer, an immunogenic fragment thereof, or a nucleic acid encoding said HSV2 gE2 or an immunogenic fragment thereof, as described herein, for use in the manufacture of an immunogenic composition.

[0200] In one aspect, the invention provides the use of HSV2 gE2 or HSV2 gE2 / gI2 heterodimer, an immunogenic fragment thereof, or a nucleic acid encoding said HSV2 gE2 or HSV2 gE2 / gI2 heterodimer or an immunogenic fragment thereof, as described herein, in the manufacture of a medicament for the treatment of HSV2 infection or an HSV2-associated disease.

[0201] In one aspect, the present invention provides an HSV1 gE1 or HSV1 gE1 / gI1 heterodimer, an immunogenic fragment thereof, or a nucleic acid encoding said HSV1 gE1 or HSV1 gE1 / gI1 heterodimer or an immunogenic fragment thereof, as described herein, for use in the manufacture of an immunogenic composition.

[0202] In one aspect, the invention provides the use of HSV1 gE1 or HSV1 gE1 / gI1 heterodimer, an immunogenic fragment thereof, or a nucleic acid encoding said HSV1 gE1 or HSV1 gE1 / gI1 heterodimer or an immunogenic fragment thereof, as described herein, in the manufacture of a medicament for the treatment of HSV1 infection or an HSV1-associated disease.

[0203] In one aspect, the invention provides a method of treating a herpesvirus infection or a herpesvirus-associated disease in a subject in need thereof, comprising administering to the subject an immunologically effective amount of a herpesvirus Fc receptor or an immunogenic fragment thereof, or a nucleic acid encoding said viral FcR or an immunogenic fragment thereof.

[0204] In one aspect, the present invention provides a method for treating an HSV2 infection or an HSV2-associated disease in a subject in need thereof, comprising administering to the subject an immunologically effective amount of HSV2 gE2 or an HSV2 gE2 / gI2 heterodimer, an immunogenic fragment thereof, or a nucleic acid encoding the HSV2 gE2 or HSV2 gE2 / gI2 heterodimer or an immunogenic fragment thereof.

[0205] In one aspect, the present invention provides a method for treating an HSV1 infection or an HSV1-associated disease in a subject in need thereof, comprising administering to the subject an immunologically effective amount of HSV1 gE1 or an HSV1 gE1 / gI1 heterodimer, an immunogenic fragment thereof, or a nucleic acid encoding the HSV1 gE1 or HSV1 gE1 / gI1 heterodimer or an immunogenic fragment thereof.

[0206] As used herein, the terms "treat" and "treatment," and words derived therefrom, do not imply that a "cure" of a condition will be treated in all individuals or that treatment will be 100% effective in any given population. Rather, there are various degrees of treatment that those skilled in the art will recognize as having a beneficial therapeutic effect. In this regard, the methods and uses of the present invention can provide any level of treatment for herpesvirus infection, particularly HSV2- or HSV1-associated disease, in a subject in need of such treatment, and can include a reduction in the severity, duration, or number of recurrences over time of one or more conditions or symptoms of herpesvirus infection, particularly HSV2- or HSV1-associated disease.

[0207] As used herein, "therapeutic immunization" or "therapeutic vaccination" refers to administering an immunogenic composition of the invention to a subject, preferably a human subject, known to be infected with a virus, e.g., a herpes virus, particularly HSV2 or HSV1, at the time of administration, to treat the viral infection or virus-related disease. As used herein, "prophylactic immunization" or "prophylactic vaccination" refers to administering an immunogenic composition of the invention to a subject, preferably a human subject, not infected with a virus, e.g., a herpes virus, particularly HSV2 or HSV1, at the time of administration, to prevent the viral infection or virus-related disease.

[0208] In the present invention, treatment of HSV infection aims to prevent reactivation events from latent HSV infection or control viral replication at an early stage to reduce viral shedding and clinical manifestations following primary HSV infection, i.e., recurrent HSV infection. Thus, treatment prevents either or both symptomatic and asymptomatic reactivation of HSV (also known as recurrent HSV infection), including asymptomatic viral shedding. Therefore, treatment can reduce the severity, duration, and / or number of episodes of recurrent HSV infection following reactivation in symptomatic individuals. Prevention of asymptomatic reactivation and shedding from mucosal sites can also reduce or prevent transmission of infection to individuals naive to the HSV virus (i.e., HSV2, HSV1, or both). This includes preventing sexual transmission of HSV, particularly HSV2, but also potentially HSV1. Thus, the immunogenic constructs of the invention may achieve any of the following useful goals: preventing or reducing asymptomatic viral shedding, reducing or preventing symptomatic disease recurrence, reducing the duration or severity of symptomatic disease, reducing the frequency of recurrences, extending the time to recurrence, improving the proportion of subjects who are recurrence-free at a given time point, reducing the use of antiviral agents, preventing transmission between sexual partners. In the case of HCMV, an HCMV Fc receptor-based vaccine can control congenital HCMV infection, particularly in HCMV-seropositive subjects.

[0209] In particular, the viral-derived Fc receptors or immunogenic fragments thereof and immunogenic compositions described herein are useful as therapeutic vaccines to treat recurrent viral infections in subjects in need of such treatment, preferably human subjects.

[0210] Suitably, the viral-derived Fc receptors or immunogenic fragments thereof and immunogenic compositions described herein are not part of a prophylactic vaccine.

[0211] The methods of use provided herein can be directed to both HSV2 and HSV1 infections (and thus to both HSV2 and HSV1 associated diseases, i.e., genital herpes and herpes labialis, respectively), or to HSV2 infections (and thus primarily aimed at treating genital herpes) or to HSV1 infections (and thus primarily aimed at treating herpes labialis).

[0212] By "immunologically effective amount," it is intended that administration of this amount of antigen (or immunogenic composition containing the antigen) to a subject, either as a single dose or as part of a series, is effective to induce a measurable immune response in the subject to the administered antigen. This amount will vary depending on the health and physical condition of the individual being treated, their age, the taxonomic group of the individual being treated (e.g., human, non-human primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the composition or vaccine, the treating physician's assessment of the medical condition, the severity of the disease, the potency of the administered compound, the mode of administration, and other relevant factors. The vaccines disclosed herein are typically therapeutic. In some embodiments, the immunogenic compositions disclosed herein are capable of inducing an effective immune response against herpesvirus infection, i.e., a response sufficient to treat or prevent herpesvirus infection, e.g., recurrent HSV infection. Further uses of immunogenic compositions or vaccines comprising the nucleic acid constructs described herein are provided hereinafter. It will be readily apparent that the viral Fc receptors or immunogenic fragments thereof and immunogenic compositions described herein are suitable for use in regimens involving repeated delivery of viral Fc receptors or immunogenic fragments thereof over time for therapeutic purposes. Suitably, a prime-boost regimen can be used. Prime-boost refers to the induction of two separate immune responses in the same individual: (i) a first priming of the immune system, followed by (ii) a second or boosting of the immune system several weeks or months after the primary immune response has been established. Preferably, the boosting composition is administered about 2 to about 12 weeks after the priming composition is administered to the subject, e.g., about 2, 3, 4, 5, or 6 weeks after the priming composition. In one embodiment, the boosting composition is administered one or two months after the priming composition. In one embodiment, the first boosting composition is administered one or two months after the priming composition, and the second boosting composition is administered one or two months after the first boosting composition.

[0213] The dosage will depend primarily on factors such as the route of administration, the condition being treated, and the age, weight, and health of the subject, and thus may vary from subject to subject. For example, a therapeutically effective adult dosage of a viral-derived Fc receptor or immunogenic fragment thereof can contain 1 to 250 μg, e.g., 2 to 100 μg, of viral FcR or an immunogenic fragment thereof, e.g., about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg of viral FcR or an immunogenic fragment thereof.

[0214] When a viral FcR binding partner or fragment thereof is administered to a subject together with a viral FcR or immunogenic fragment thereof, a therapeutically effective adult dosage of the viral FcR binding partner or fragment thereof can contain 5 to 250 μg, e.g., 10 to 100 μg, of the viral FcR binding partner or fragment thereof, for example, about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg of the viral FcR binding partner or fragment thereof.

[0215] In a preferred embodiment, when a viral FcR binding partner or fragment thereof is administered to a subject together with a viral FcR or immunogenic fragment thereof, the doses of the viral FcR immunogenic fragment and the viral FcR binding partner or fragment thereof are in a stoichiometric ratio of about 1:1.

[0216] Generally, a human dose will be between 0.1 ml and 2 ml in volume. Thus, the compositions described herein can be formulated, for example, in human dose volumes of about 0.1, 0.15, 0.2, 0.5, 1.0, 1.5, or 2.0 ml per individual component or combined immunogenic component.

[0217] Those skilled in the art will be able to adjust these dosages depending on the route of administration and the subject being treated.

[0218] The therapeutic immune response to the virus-derived Fc receptor or immunogenic fragment thereof can be monitored to determine the need for a booster, if any. Following assessment of the immune response (e.g., CD4+ T cell response, CD8+ T cell response, serum antibody titers), optional booster immunizations can be administered.

[0219] Suitable in vitro or in vivo test methods for assessing immune responses to viral Fc receptors or fragments thereof according to the present invention are known to those skilled in the art. For example, viral Fc receptors or fragments thereof can be tested for their effect on the proliferation or induction of effector functions of specific lymphocyte types of interest, such as B cells, T cells, T cell lines, and T cell clones. For example, spleen cells from immunized mice can be isolated and assessed for the ability of cytotoxic T lymphocytes to lyse autologous target cells containing viral Fc receptors or fragments thereof according to the present invention. In addition, T helper cell differentiation can be analyzed by measuring proliferation or production of TH1 (IL-2, TNF-α, and IFN-γ) cytokines in CD4+ T cells by cytoplasmic cytokine staining and flow cytometry analysis. Viral Fc receptors or fragments thereof according to the present invention can also be tested for their ability to induce humoral immune responses, as evidenced, for example, by examining B cell activation in draining lymph nodes and measuring B cell production of antibodies specific to the HSV antigen of interest in serum. Such assays can be performed, for example, using peripheral B lymphocytes from immunized individuals.

[0220] In a further aspect, the present invention provides a nucleic acid encoding a viral Fc receptor or immunogenic fragment or heterodimer thereof of the present invention. In a preferred embodiment, the nucleic acid of the present invention is for use in therapy, suitably for use in treating a subject infected with a virus.

[0221] The term "nucleic acid" generally refers to a polymeric form of nucleotides of any length, containing deoxyribonucleotides, ribonucleotides, and / or their analogs. This includes DNA, RNA, and DNA / RNA hybrids. It also includes analogs of DNA or RNA, such as those containing modified backbones (e.g., peptide nucleic acid (PNA) or phosphorothioates) or modified bases. Thus, nucleic acids of the present disclosure include mRNA, DNA, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, and the like. When the nucleic acid is in the form of RNA, it may or may not have a 5' cap. Nucleic acid molecules disclosed herein can be in a variety of forms (e.g., single-stranded, double-stranded). Nucleic acid molecules can be circular or branched, but are typically linear.

[0222] As used herein, nucleic acids are preferably provided in purified or substantially purified form, i.e., substantially free from other nucleic acids (e.g., free from naturally occurring nucleic acids), and are generally at least about 50% pure (by weight), usually at least about 90% pure.

[0223] The nucleic acid molecules of the present invention can be produced by any suitable means, including recombinant production, chemical synthesis, or other synthetic means. Suitable production techniques are well known to those skilled in the art. Typically, the nucleic acids of the present invention will be in recombinant form, i.e., not naturally occurring. For example, the nucleic acid can contain one or more heterologous nucleic acid sequences (e.g., a sequence encoding another antigen and / or a regulatory sequence, such as a promoter or internal ribosome entry site) in addition to the nucleic acid sequence encoding the viral Fc receptor or a fragment or heterodimer thereof. The sequence or chemical structure of the nucleic acid can be modified compared to the naturally occurring sequence encoding the viral Fc receptor or a fragment or heterodimer thereof.

[0224] The sequence of a nucleic acid molecule can be modified, for example, to improve the efficiency of expression or replication of the nucleic acid, or to provide greater stability or resistance to degradation.

[0225] Nucleic acid molecules encoding viral Fc receptors or fragments or heterodimers thereof can be codon-optimized. "Codon optimization" refers to modifications to codon usage that can improve the translation efficiency and / or half-life of a nucleic acid. A polyA tail (e.g., of about 30 or more adenosine residues) can be attached to the 3' end of an RNA to improve its half-life. The 5' end of an RNA can be capped with a modified ribonucleotide or derivative thereof comprising the structure m7G(5')ppp(5')N (Cap 0 structure), which can be incorporated during RNA synthesis or enzymatically engineered after RNA transcription (e.g., by using vaccinia virus capping enzyme (VCE), which is composed of mRNA triphosphatase, guanylyltransferase, and guanine-7-methyltransferase, and which catalyzes the construction of the N7-monomethylated Cap 0 structure). The Cap 0 structure plays an important role in maintaining the stability and translation efficiency of an RNA molecule. The 5' cap of an RNA molecule can be further modified by 2'-O-methyltransferase, which results in the generation of the Cap 1 structure (m7Gppp [m2'-O]N), which can further improve translation efficiency.

[0226] Nucleic acids can contain one or more nucleotide analogs or modified nucleotides. As used herein, "nucleotide analog" or "modified nucleotide" refers to a nucleotide that contains one or more chemical modifications (e.g., substitutions) in or on the nitrogenous base of a nucleoside (e.g., cytosine (C), thymine (T), or uracil (U)), adenine (A), or guanine (G)). A nucleotide analog can contain additional chemical modifications in or on the sugar moiety (e.g., ribose, deoxyribose, modified ribose, modified deoxyribose, six-membered sugar analog, or open-chain sugar analog) or phosphate of the nucleoside. The preparation of nucleotides and modified nucleotides and nucleosides is well known in the art, see the following references: U.S. Patent Nos. 4,373,071, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642. Many modified nucleosides and nucleotides are commercially available.

[0227] Modified nucleobases that can be incorporated into modified nucleosides and nucleotides and present in mRNA molecules include: m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine); Am (2-1-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio-N6-isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonylcarbamoyladenosine); ms2t6A (2-methylthio-N6-threonylcarbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); hn6A (N6-hydroxynorvalylcarbamoyladenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalylcarbamoyladenosine); Ar(p) (2'-O-ribosyladenosine (phosphate)); I (inosine); mi1 (1-methylinosine); m'1m (1,2'-O-dimethylinosine); m3C (3-methylcytidine); Cm (2T-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); L5C (5-formylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm (N4-acetyl-2O-methylcytidine); k2C (lysidine); m1G (1-methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2,N2,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wybutosine);mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactactosylqueuosine); manQ (mannosylqueuosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine); m5Um (5,2'-O-dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2-thiouridine); s2Um (2-thio-2'-O-methyluridine); acp3U (3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U (5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5-(carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonylmethyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2'-O-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethyl-1-aminomethyl-2-LO-methyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6-dimethyladenosine); Tm (2'-O-methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6,T-O-dimethyladenosine);rn62Am (N6,N6,0-2-trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2,N2,7-trimethylguanosine); m3Um (3,2T-0-dimethyluridine); m5D (5-methyldihydrouridine); L5Cm (5-formyl-2'-0-methylcytidine); m1Gm (1,2'-0-dimethylguanosine); m'Am (1,2-0-dimethyladenosine) irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine); iniG-14 (4-demethylguanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(Ci-Ce)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-Ce)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(Ci-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (abasic residue), m5C, m5U, m6A, s2U, W, or 2'-O-methyl-U. Many of these modified nucleobases and their corresponding ribonucleosides are available from commercial suppliers;

[0228] An exemplary effective amount of nucleic acid component can be between 1 ng and 100 μg, e.g., between 1 ng and 1 μg (e.g., 100 ng and 1 μg), or between 1 μg and 100 μg, e.g., 10 ng, 50 ng, 100 ng, 150 ng, 200 ng, 250 ng, 500 ng, 750 ng, or 1 μg. An effective amount of nucleic acid can also include between 1 μg and 500 μg, e.g., between 1 μg and 200 μg, e.g., between 10 and 100 μg, e.g., 1 μg, 2 μg, 5 μg, 10 μg, 20 μg, 50 μg, 75 μg, 100 μg, 150 μg, or 200 μg. Alternatively, an exemplary effective amount of nucleic acid can be between 100 μg and 1 mg, e.g., 100 μg to 500 μg, e.g., 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, or 1 mg.

[0229] In a preferred embodiment, the nucleic acid encodes a heterodimer according to the invention, wherein expression of the viral FcR or immunogenic fragment thereof is under the control of a subgenomic promoter, suitably the 26S subgenomic promoter set forth in SEQ ID NO: 126 or a variant therefrom that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical thereto.

[0230] In a preferred embodiment, the viral FcR or immunogenic fragment thereof and its binding partner or fragment thereof are separated by an internal ribosome entry site (IRES) sequence. In a preferred embodiment, the IRES sequence is an IRES EV71 sequence. In a preferred embodiment, the IRES sequence comprises or consists of the sequence set forth in SEQ ID NO: 127, or a variant thereof that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.

[0231] In another embodiment, the sequences encoding the viral FcR or immunogenic fragment thereof and its binding partner or fragment thereof are separated by two 2A "self-cleaving" peptide sequences. In one embodiment, the 2A "self-cleaving" peptide sequence is a GSG-P2A sequence that suitably comprises or consists of the sequence set forth in SEQ ID NO: 124, or a variant thereof that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto. In one embodiment, the 2A "self-cleaving" peptide sequence is an F2A sequence that suitably comprises or consists of the sequence set forth in SEQ ID NO: 125, or a variant thereof that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.

[0232] In yet another embodiment, the sequences encoding the viral FcR or immunogenic fragment thereof and its binding partner or fragment thereof are separated by two subgenomic promoters, hi one embodiment, the subgenomic promoter is a 26S subgenomic promoter, suitably comprising or consisting of the sequence set forth in SEQ ID NO: 126, or a variant therefrom that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical thereto.

[0233] The nucleic acid molecule of the present invention may be, for example, RNA or DNA, such as plasmid DNA. In a preferred embodiment, the nucleic acid molecule is an RNA molecule. In a more preferred embodiment, the RNA molecule is a self-amplifying RNA molecule ("SAM").

[0234] Self-amplifying (or self-replicating) RNA molecules are well known in the art and can be generated by using replication elements, such as those derived from alphaviruses, to replace structural viral proteins with nucleotide sequences encoding proteins of interest. Self-amplifying RNA molecules are typically positive-strand molecules that can be directly translated after delivery to cells. This translation provides an RNA-dependent RNA polymerase that generates both antisense and sense transcripts from the delivered RNA. In this manner, the delivered RNA results in the generation of multiple daughter RNAs. These daughter RNAs, as well as colinear subgenomic transcripts, can themselves be translated to result in the in situ expression of the encoded polypeptide, or can be transcribed to produce further transcripts with the same sense as the delivered RNA, which are translated to result in the in situ expression of the antigen. The overall result of transcription of this sequence is a massive amplification of the number of introduced replicon RNAs, such that the encoded antigen becomes the predominant polypeptide product of the cell. One suitable system for achieving self-replication in this manner is the use of alphavirus-based replicons. These replicons are positive-strand RNAs that direct the translation of replicases (or replicase transcriptases) after delivery to cells. The replicases are translated as polyproteins that self-cleave to form a replication complex, which creates genomic copies of the positive-strand delivered RNA. These negative-strand transcripts can themselves be transcribed to provide additional copies of the positive-strand parent RNA and also to provide subgenomic transcripts encoding antigens. Translation of the subgenomic transcripts then leads to in situ expression of the antigen by infected cells. Suitable alphavirus replicons can use replicases from Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, etc. Mutant or wild-type viral sequences can be used; for example, the attenuated TC83 mutant of VEEV has been used as a replicon. See WO 2005 / 113782.

[0235] In one embodiment, the self-amplifying RNA molecules described herein encode an RNA-dependent RNA polymerase capable of transcribing RNA from the self-amplifying RNA molecule and a viral Fc receptor or fragment or heterodimer thereof. The polymerase can be, for example, an alphavirus replicase comprising one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP4.

[0236] In a preferred embodiment, the self-amplifying RNA molecule is an RNA replicon derived from an alphavirus.

[0237] While native alphavirus genomes encode structural virion proteins in addition to nonstructural replicase polyproteins, in certain embodiments, self-amplifying RNA molecules do not encode alphavirus structural proteins. Thus, preferred self-amplifying RNAs are capable of producing genomic RNA copies of themselves within cells, but are unable to produce RNA-containing virions. Their inability to produce these virions means that, unlike wild-type alphaviruses, the self-amplifying RNA molecules are unable to perpetuate themselves in an infectious form. The alphavirus structural proteins required for persistence in wild-type viruses are absent from the self-amplifying RNAs of the present disclosure, and their place is occupied by a gene encoding the immunogen of interest, resulting in a subgenomic transcript encoding the immunogen rather than the structural alphavirus virion proteins. Thus, self-amplifying RNA molecules useful in the present invention can have two open reading frames: a first (5') open reading frame encoding the replicase, and a second (3') open reading frame encoding the antigen. In some embodiments, the RNA can have additional (eg, downstream) open reading frames, for example, to encode additional antigens or to encode accessory polypeptides.

[0238] Suitably, the self-amplifying RNA molecules disclosed herein have a 5' cap (e.g., 7-methylguanosine), which can enhance in vivo translation of the RNA. The self-amplifying RNA molecule can have a 3' polyA tail. The self-amplifying RNA molecule can also include a polyA polymerase recognition sequence (e.g., AAUAAA) near its 3' end. The self-amplifying RNA molecule can have a variety of lengths, but is typically between 5,000 and 25,000 nucleotides in length. The self-amplifying RNA molecule is typically single-stranded.

[0239] Suitably, the self-replicating RNA comprises or consists of a VEEV TC-83 replicon encoding viral nonstructural proteins 1-4 (nsP1-4) from 5' to 3', followed by a subgenomic promoter, and a construct (or insert) encoding the gEgI heterodimer. In a preferred embodiment, the insert comprises or consists of a gE ectodomain sequence under the control of the above-mentioned subgenomic promoter, followed by an IRES regulatory sequence, followed by a gI ectodomain sequence. In a preferred embodiment, the IRES sequence is an IRES EV71 sequence. In a preferred embodiment, the IRES sequence comprises or consists of the sequence set forth in SEQ ID NO: 127, or a variant thereof that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.

[0240] DNA encoding an empty SAM is shown in Figure 39 and SEQ ID NO: 130. The corresponding empty SAM is shown in SEQ ID NO: 133. The construct will be inserted immediately after nucleotide 7561. Thus, the SAM can comprise three regions from 5' to 3': the first region comprises the sequence up to the insertion point (e.g., nucleotides 1-7561 of SEQ ID NO: 133, herein SEQ ID NO: 134), the second region comprises the insert encoding the gEgI heterodimer, and the third region comprises the sequence after the insertion point (e.g., nucleotides 7562-7747 of SEQ ID NO: 133, herein SEQ ID NO: 135). Thus, the DNA encoding the SAM can comprise three regions from 5' to 3': the first region comprises the sequence up to the insertion point (e.g., nucleotides 1 to 7561 of SEQ ID NO: 130, herein SEQ ID NO: 131), the second region comprises the insert encoding the gEgI heterodimer, and the third region comprises the sequence after the insertion point (e.g., nucleotides 7562 to 9993 of SEQ ID NO: 130, herein SEQ ID NO: 132).

[0241] In one embodiment, the VEEV TC-83 replicon has the DNA sequence shown in Figure 39 and SEQ ID NO: 130, and the construct encoding the gEgI heterodimeric antigen is inserted immediately after residue 7561. In one embodiment, the VEE TC-83 replicon has the RNA sequence shown in SEQ ID NO: 133, and the construct encoding the antigen is inserted immediately after residue 7561.

[0242] Self-amplifying RNA can be conveniently prepared by in vitro transcription (IVT). IVT can use a (cDNA) template that is created and propagated in bacteria in plasmid form or that is synthetically created (e.g., by gene synthesis and / or polymerase chain reaction (PCR) engineering methods). For example, a DNA-dependent RNA polymerase (e.g., bacteriophage T7, T3, or SP6 RNA polymerase) can be used to transcribe the self-amplifying RNA from a DNA template. Appropriate capping and poly(A) addition reactions can be used, if necessary (although the replicon's poly(A) is typically encoded within the DNA template). These RNA polymerases can have stringent requirements for the transcribed 5' nucleotide(s); in some embodiments, these requirements must match those of the encoded replicase to ensure that the IVT-transcribed RNA can efficiently serve as a substrate for that self-encoded replicase.

[0243] Self-amplifying RNAs can contain one or more nucleotides with modified nucleobases (in addition to any 5' cap structure). RNAs used in the present invention ideally contain only phosphodiester internucleoside linkages, but in some embodiments can contain phosphoramidate, phosphorothioate, and / or methylphosphonate linkages.

[0244] The nucleic acid molecules of the present invention can be associated with viral or non-viral delivery systems. The delivery system (also referred to herein as a delivery vehicle) can have an adjuvant effect that enhances the immunogenicity of the encoded viral Fc receptor or fragment or heterodimer thereof. For example, the nucleic acid molecule can be encapsulated in liposomes, non-toxic biodegradable polymer microparticles, or viral replicon particles (VRPs), or can be complexed with particles of a cationic oil-in-water emulsion. In some embodiments, the nucleic acid molecule is associated with a non-viral delivery material, for example, to form a cationic nanoemulsion (CNE) delivery system or a lipid nanoparticle (LNP) delivery system. In some embodiments, the nucleic acid molecule is associated with a non-viral delivery system, i.e., the nucleic acid molecule is substantially free of viral capsids. Alternatively, the nucleic acid molecule can be associated with a viral replicon particle. In other embodiments, the nucleic acid molecule can comprise naked nucleic acid, such as naked RNA (e.g., mRNA).

[0245] In a preferred embodiment, the RNA molecule or self-amplifying RNA molecule is associated with a non-viral delivery material, for example to form a cationic nanoemulsion (CNE) or lipid nanoparticle (LNP).

[0246] CNE delivery systems and methods for their preparation are described in WO2012 / 006380. In the CNE delivery system, antigen-encoding nucleic acid molecules (e.g., RNA) are complexed with cationic oil-in-water emulsion particles. Cationic oil-in-water emulsions can be used to deliver negatively charged molecules, such as RNA molecules, to cells. The emulsion particles contain an oil core and cationic lipids. The cationic lipids can interact with the negatively charged molecules, thereby immobilizing them on the emulsion particles. Further details of useful CNEs can be found in WO2012 / 006380, WO2013 / 006834, and WO2013 / 006837 (the contents of each of which are incorporated herein in their entirety).

[0247] Thus, in one embodiment, an RNA molecule, e.g., a self-amplifying RNA molecule, encoding a viral Fc receptor or a fragment or heterodimer thereof can be complexed with particles of a cationic oil-in-water emulsion. The particles typically comprise an oil core (e.g., vegetable oil or squalene) in a liquid phase at 25°C, a cationic lipid (e.g., a phospholipid), and optionally a surfactant (e.g., sorbitan trioleate, polysorbate 80). Polyethylene glycol may also be included. In some embodiments, the CNE comprises squalene and a cationic lipid, e.g., 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP). In some preferred embodiments, the delivery system is a non-viral delivery system, e.g., a CNE, and the nucleic acid molecule comprises a self-amplifying RNA (mRNA). This can be particularly effective in eliciting humoral and cellular immune responses.

[0248] LNP delivery systems and non-toxic biodegradable polymeric microparticles, as well as methods for their preparation, are described in WO2012 / 006376 (LNP and microparticle delivery systems); Geall et al. (2012) PNAS USA. Sep 4; 109(36): 14604-9 (LNP delivery systems); and WO2012 / 006359 (microparticle delivery systems). LNPs are non-virion liposomal particles that can encapsulate nucleic acid molecules (e.g., RNA). While the particles may contain some external RNA (e.g., on the surface of the particle), at least half (ideally all) of the RNA is encapsulated. Liposomal particles can be formed, for example, from a mixture of zwitterionic, cationic, and anionic lipids, which may be saturated or unsaturated, such as DSPC (zwitterionic, saturated), DlinDMA (cationic, unsaturated), and / or DMG (anionic, saturated). Preferred LNPs for use in the present invention include amphipathic lipids capable of forming liposomes, optionally in combination with at least one cationic lipid (e.g., DOTAP, DSDMA, DODMA, DLinDMA, DLenDMA, etc.) A mixture of DSPC, DlinDMA, PEG-DMG, and cholesterol is particularly effective. Other useful LNPs are described in: WO2012 / 006376; ​​WO2012 / 030901; WO2012 / 031046; WO2012 / 031043; WO2012 / 006378; WO2011 / 076807; WO2013 / 033563; WO2013 / 006825; WO2014 / 136086; WO2015 / 095340; WO2015 / 095346; WO2016 / 037053. In some embodiments, the LNP is an RV01 liposome. See the following references: WO2012 / 006376 and Geall et al. (2012) PNAS USA. Sep 4; 109(36): 14604-9.

[0249] Dosage will depend primarily on factors such as the route of administration, the condition being treated, and the age, weight, and health of the subject, and may therefore vary from subject to subject. For example, a therapeutically effective adult dosage of a nucleic acid of the invention may contain 0.5 to 50 μg, e.g., 1 to 30 μg, e.g., about 1, 3, 5, 10, 15, 20, 25, or 30 μg, of nucleic acid.

[0250] In a further aspect, the present invention provides a vector comprising a nucleic acid according to the present invention.

[0251] Vectors for use in the present invention can be any suitable nucleic acid molecule, including naked DNA or RNA, plasmids, viruses, cosmids, phage vectors, e.g., lambda vectors, artificial chromosomes, e.g., BACs (bacterial artificial chromosomes), or episomes. Alternatively, vectors can be transcription and / or expression units for cell-free in vitro transcription or expression, e.g., T7-compatible systems. Vectors can be used alone or in combination with other vectors, e.g., adenoviral sequences or fragments, or in combination with elements derived from non-adenoviral sequences. Suitably, the vectors are substantially altered compared to wild-type sequences (e.g., genes or functional regions are deleted and / or inactivated) so that, when introduced into a host cell, they replicate and express the inserted polynucleotide sequence.

[0252] In a further aspect, the present invention provides a cell comprising a viral Fc receptor or fragment thereof, heterodimer, nucleic acid or vector according to the invention.

[0253] The viral Fc receptor or immunogenic fragment thereof, viral FcR binding partner or fragment thereof, or heterodimer according to the present invention is suitably produced by recombinant techniques. By "recombinant" is meant that the polynucleotide is the product of at least one cloning, restriction, or ligation step, or other procedure that results in a polynucleotide that is distinct from that found in nature. A recombinant vector is a vector that contains a recombinant polynucleotide.

[0254] In one embodiment, the heterodimer according to the present invention is expressed from a multicistronic vector. Suitably, the heterodimer is expressed from a single vector in which the nucleic acid sequences encoding the viral FcR or immunogenic fragment thereof and its binding partner or fragment thereof are separated by an internal ribosome entry site (IRES) sequence (Mokrejs, Martin, et al. "IRESite: the database of experimentally verified IRES structures (www.iresite.org)." Nucleic acids research 34.suppl_1 (2006): D125-D130.). In a preferred embodiment, the IRES is the IRES EV71 sequence. In a preferred embodiment, the IRES comprises or consists of the sequence set forth in SEQ ID NO: 127, or a variant thereof that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.

[0255] Alternatively, the two nucleic acid sequences can be separated by a viral 2A or "2A-like" sequence, resulting in the production of two separate polypeptides. 2A sequences are known from a variety of viruses, including foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus, and swine virus-1. See, e.g., Szymczak et al., Nature Biotechnology 22:589-594 (2004); Donnelly et al., J Gen Virol.; 82(Pt 5): 1013-25 (2001).

[0256] Optionally, to facilitate expression and recovery, the Fc receptor or immunogenic fragment thereof and / or viral FcR binding partner or fragment thereof can include a signal peptide at its N-terminus. The signal peptide can be selected from among numerous signal peptides known in the art and is typically selected to facilitate production and processing in the system chosen for recombinant expression. In one embodiment, the signal peptide is one that naturally occurs in the native viral Fc protein or binding partner. The signal peptide of HSV2 gE from strain SD90e is located at residues 1-20 of SEQ ID NO:1. Signal peptides for gE proteins from other HSV strains can be identified by sequence alignment. The signal peptide of HSV2 gI from strain SD90e is located at residues 1-20 of SEQ ID NO:2. Signal peptides for gI proteins from other HSV strains can be identified by sequence alignment.

[0257] Optionally, the Fc receptor or immunogenic fragment thereof and / or viral FcR binding partner or fragment thereof can include the addition of an amino acid sequence constituting a tag that can facilitate detection (e.g., an epitope tag for detection with a monoclonal antibody) and / or purification (e.g., a polyhistidine tag that allows purification on a nickel chelate resin) of the protein. In certain embodiments, a cleavable linker can be used, which allows the tag to be separated from the purified complex, for example, by the addition of an agent that can cleave the linker. Several different cleavable linkers are known to those of skill in the art.

[0258] When the host cells herein are cultured under appropriate conditions, the nucleic acids can express both the Fc receptor or immunogenic fragment thereof, the viral FcR binding partner or fragment thereof, and / or the heterodimer peptide. The Fc receptor or immunogenic fragment thereof, the viral FcR binding partner or fragment thereof, and / or the heterodimer can then be secreted from the host cell. Suitable host cells include, for example: Insect cells (e.g., Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni), mammalian cells (e.g., human, non-human primate, horse, cow, sheep, dog, cat, and rodent (e.g., hamster)), avian cells (e.g., chicken, duck, and goose), bacteria (e.g., E. coli, Bacillus subtilis, and Streptococcus spp.), yeast cells (e.g., Saccharomyces cerevisiae), cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica), Tetrahymena cells (e.g., Tetrahymena thermophila), or combinations thereof. Suitably, the host cell should be one that has an enzyme that mediates glycosylation. Bacterial hosts are generally not suitable for such modified proteins unless the host cells have been modified to introduce glycosylation enzymes; instead, eukaryotic hosts such as insect, avian or mammalian cells must be used.

[0259] Suitable insect cell expression systems, such as baculovirus systems, are known to those skilled in the art and are described, for example, in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (clonal isolates derived from the parental Stinging Looper BTI-TN-5B1-4 cell line (Invitrogen)).

[0260] Avian cell expression systems are also known to those skilled in the art and are described, for example, in U.S. Patent Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168; and 6,500,668; European Patent No. EP 0787180B; European Patent Application No. EP03291813.8; WO 03 / 043415; and WO 03 / 076601. Suitable avian cells include, for example, chicken embryonic stem cells (e.g., EBx® cells), chicken embryonic fibroblasts, chicken embryonic germ cells, duck cells (e.g., AGE1.CR and AGE1.CR.pIX cell lines (ProBioGen), which are described, for example, in Vaccine 27:4975-4982 (2009) and WO2005 / 042728), EB66 cells, and the like.

[0261] Preferably, the host cells are mammalian cells (eg, human, non-human primate, equine, bovine, ovine, canine, feline, and rodent (eg, hamster)). Suitable mammalian cells include, for example, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK-293 cells, usually transformed with sheared adenovirus type 5 DNA), NIH-3T3 cells, 293-T cells, Vero cells, HeLa cells, PERC.6 cells (ECACC Deposit No. 96022940), Hep G2 cells, MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), fetal rhesus lung cells (ATCC CL-160), Madin-Darby bovine kidney ("MDBK") cells, Madin-Darby canine kidney ("MDCK") cells (e.g., MDCK(NBL2), ATCC CCL34; or MDCK33016, DSM ACC 2219), baby hamster kidney (BHK) cells such as BHK21-F, HKCC cells, and the like.

[0262] In certain embodiments, the recombinant nucleic acids encoding the viral Fc receptor or immunogenic fragment thereof, viral FcR binding partner or fragment thereof, and / or heterodimer are codon-optimized for expression in a selected prokaryotic or eukaryotic host cell.

[0263] Viral Fc receptors or immunogenic fragments thereof, viral FcR binding partners or fragments thereof, and / or heterodimers can be recovered and purified from recombinant cell culture by any of several methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography (e.g., using any of the tagging systems described herein), hydroxyapatite chromatography, and lectin chromatography. Protein refolding steps can be used, if desired, in completing configuration of the mature protein. Finally, high-performance liquid chromatography (HPLC) can be used for final purification steps.In addition to the above references, various purification methods are well known in the art, including, for example, those described in: Sandana (1997) Bioseparation of Proteins, Academic Press, Inc.; and Bollag et al. (1996) Protein Methods, 2nd Edition, Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook, Humana Press, NJ; Harris and Angal (1990) Protein Purification Applications: A Practical Approach, IRL Press at Oxford, Oxford, UK; Scopes (1993) Protein Purification: Principles and Practice, 3rd Edition, Springer Verlag, NY; Janson and Ryden (1998) Protein Purification: Principles, High Resolution Methods and Applications, Second Edition, Wiley-VCH, NY; and Walker (1998) Protein Protocols on CD-ROM, Humana Press, NJ.

[0264] The terms "purification" or "purifying" refer to a process that removes components whose presence is undesired from a composition or host cell or culture. Purification is a relative term and does not require that all traces of the undesired component be removed from the composition. In the context of vaccine production, purification includes processes such as centrifugation, dialysis, ion exchange chromatography, and size exclusion chromatography, affinity purification, or precipitation. Thus, the term "purified" does not require absolute purity; rather, it is intended as a relative term. A substantially pure nucleic acid or protein preparation can be purified such that the desired nucleic acid or protein represents at least 50% of the total nucleic acid content of the preparation. In certain embodiments, a substantially pure nucleic acid or protein will represent at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% or more of the total nucleic acid or protein content of the preparation. Immunogenic molecules, antigens, or antibodies that have not been subjected to any purification steps (i.e., molecules as found in nature) are not suitable for pharmaceutical (e.g., vaccine) use.

[0265] Suitably, the recovery yield of viral Fc receptor or immunogenic fragment thereof, viral FcR binding partner or fragment thereof, and / or heterodimer is greater than 2 mg per liter, preferably greater than 5, 10, 15 or 20 mg per liter, more preferably even greater than 25 mg per liter.

[0266] Suitably, the aggregation level of the viral Fc receptor or immunogenic fragment thereof, viral FcR binding partner or fragment thereof, and / or heterodimer is below 20%, preferably below 15 or 10%, more preferably even below 5%.

[0267] In a preferred embodiment, the viral Fc receptor or fragment thereof is administered to the subject together with an adjuvant. As used herein, "adjuvant" refers to a composition that enhances the immune response to an antigen in an intended subject, e.g., a human subject.

[0268] Examples of suitable adjuvants include, but are not limited to, inorganic adjuvants (e.g., inorganic metal salts, such as aluminum phosphate or aluminum hydroxide), organic adjuvants (e.g., saponins, such as QS21, or squalene), oil-based adjuvants (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), oil-in-water emulsions, cytokines (e.g., IL-1β, IL-2, IL-7, IL-12, IL-18, GM-CFS, and IFN-γ), particle adjuvants (e.g., immune stimulating complexes (ISCOMS), liposomes, or biodegradable microspheres), virosomes, bacterial adjuvants (e.g., monophosphoryl lipid A, e.g., 3-de-O-acylated monophosphoryl lipid A), and the like. (3D-MPL, or muramyl peptides), synthetic adjuvants (e.g., non-ionic block copolymers, muramyl peptide analogs, or synthetic lipid A), synthetic polynucleotide adjuvants (e.g., polyarginine or polylysine), Toll-like receptor (TLR) agonists (including agonists for TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, ​​TLR-7, TLR-8, and TLR-9), and immunostimulatory oligonucleotides containing unmethylated CpG dinucleotides ("CpG").

[0269] In a preferred embodiment, the adjuvant comprises a TLR agonist and / or an immunologically active saponin. Preferably still, the adjuvant comprises or consists of a TLR agonist and a saponin in a liposomal formulation. The ratio of TLR agonist to saponin can be 5:1, 4:1, 3:1, 2:1, or 1:1.

[0270] The use of TLR agonists in adjuvants is well known in the art and is reviewed, for example, by Lahiri et al. (2008) Vaccine 26:6777. TLRs that can be stimulated to achieve an adjuvant effect include TLR2, TLR4, TLR5, TLR7, TLR8, and TLR9. Agonists of TLR2, TLR4, TLR7, and TLR8, particularly TLR4 agonists, are preferred.

[0271] Suitable TLR4 agonists include lipopolysaccharides, such as monophosphoryl lipid A (MPL) and 3-O-deacylated monophosphoryl lipid A (3D-MPL). U.S. Patent No. 4,436,727 discloses MPL and its preparation. U.S. Patent No. 4,912,094 and Reexamination Certificate No. B1 4,912,094 disclose 3D-MPL and its preparation method. Another TLR4 agonist is glucopyranosyl lipid adjuvant (GLA), a synthetic lipid A-like molecule (see, e.g., Fox et al. (2012) Clin. Vaccine Immunol 19:1633). In further embodiments, the TLR4 agonist may be a synthetic TLR4 agonist, such as a synthetic disaccharide molecule similar in structure to MPL and 3D-MPL, or the TLR4 agonist may be a synthetic monosaccharide molecule, such as an aminoalkyl glucosaminide phosphate (AGP) compound, as disclosed in, for example, WO9850399, WO0134617, WO0212258, WO3065806, WO04062599, WO06016997, WO0612425, WO03066065, and WO0190129. Such molecules are also described in the scientific and patent literature as lipid A mimics. Lipid A mimics suitably share some functional and / or structural activity with lipid A and, in one aspect, are recognized by the TLR4 receptor. The AGPs described herein are sometimes referred to in the art as lipid A mimics. In a preferred embodiment, the TLR4 agonist is 3D-MPL. TLR4 agonists, such as 3-O-deacylated monophosphoryl lipid A (3D-MPL) and their use as adjuvants in vaccines, are described, for example, in WO 96 / 33739 and WO2007 / 068907 and reviewed in Alving et al. (2012) Curr Opin in Immunol 24:310.

[0272] Suitably the adjuvant comprises an immunologically active saponin, for example an immunologically active saponin fraction such as QS21.

[0273] Adjuvants containing saponins have been described in the art. Saponins are described in Lacaille-Dubois and Wagner (1996) "A review of the biological and pharmacological activities of saponins," Phytomedicine, Vol. 2:363. Saponins are known as adjuvants in vaccines. For example, Quil A (derived from the bark of the South American tree Quillaja Saponaria Molina) was reported to have adjuvant activity by Dalsgaard et al. ("Saponin adjuvants," Archiv. fur die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, 243) in 1974. A purified fraction of Quil A that is nontoxic and retains adjuvant activity was isolated by HPLC (Kensil et al. (1991) J. Immunol. 146:431). Quil A fraction is also described in US Pat. No. 5,057,540 and in "Saponins as vaccine adjuvants", Kensil, CR, Crit Rev Ther Drug Carrier Syst, 1996, 12 (1-2):1-55.

[0274] Two such Quil A fractions suitable for use in the present invention are QS7 and QS21 (also known as QA-7 and QA-21). QS21 is a preferred immunologically active saponin fraction for use in the present invention. QS21 is reviewed in Kensil (2000) In O'Hagan: Vaccine Adjuvants: Preparation Methods and Research Protocols, Homana Press, Totowa, New Jersey, Chapter 15. Particulate adjuvant systems comprising fractions of Quil A, such as QS21 and QS7, are described, for example, in WO 96 / 33739, WO 96 / 11711, and WO2007 / 068907.

[0275] In addition to other ingredients, the adjuvant preferably contains a sterol. The presence of a sterol can further reduce the reactogenicity of a composition containing a saponin. See, for example, EP 0 822 831. Suitable sterols include beta-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol. Cholesterol is particularly suitable. Suitably, the immunologically active saponin fraction is QS21, and the ratio of QS21:sterol is 1:100 to 1:1 w / w, for example 1:10 to 1:1 w / w, for example 1:5 to 1:1 w / w.

[0276] In a preferred embodiment, the adjuvant comprises a TLR4 agonist and an immunologically active saponin, hi a more preferred embodiment, the TLR4 agonist is 3D-MPL and the immunologically active saponin is QS21.

[0277] In some embodiments, the adjuvant is provided in the form of an oil-in-water emulsion, for example, comprising squalene, alpha-tocopherol, and a surfactant (see, e.g., WO 95 / 17210), or in the form of liposomes, preferably liposomes.

[0278] The term "liposome" as used herein refers to a mono- or multi-layered (particularly 2, 3, 4, 5, 6, 7, 8, 9, or 10-layered, depending on the number of lipid membranes formed) lipid structure surrounding an aqueous interior. Liposomes and liposome formulations are well known in the art. Liposome preparations are described, for example, in WO 96 / 33739 and WO 2007 / 068907. Lipids capable of forming liposomes include any substance with fat or fat-like properties. Lipids that can constitute the lipids in liposomes can be selected from the group including glycerides, glycerophospholipids, glycerophosphinolipids, glycerophosphonolipids, sulfolipids, sphingolipids, phospholipids, isoprenolides, steroids, stearins, sterols, archelipids, synthetic cationic lipids, and carbohydrate-containing lipids. In certain embodiments of the present invention, the liposomes comprise phospholipids.Suitable phospholipids include (but are not limited to): phosphocholine (PC), which is an intermediate in the synthesis of phosphatidylcholine; natural phospholipid derivatives: egg phosphocholine, egg phosphocholine, soybean phosphocholine, hydrogenated soybean phosphocholine, sphingomyelin as a natural phospholipid; and synthetic phospholipid derivatives: Phosphocholines (didecanoyl-La-phosphatidylcholine [DDPC], dilauroylphosphatidylcholine [DLPC], dimyristoylphosphatidylcholine [DMPC], dipalmitoylphosphatidylcholine [DPPC], distearoylphosphatidylcholine [DSPC], dioleoylphosphatidylcholine [DOPC], 1-palmitoyl, 2-oleoylphosphatidylcholine [POPC], dielaidoylphosphatidylcholine [DEPC]), phosphoglycerols (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol [DMPG], 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol [DPPG], 1,2-distearoyl-sn-glycero-3-phosphoglycerol [DSPG], 1-palmitoyl-sn-glycero-3-phosphoglycerol [DSPG], These include 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (1,2-dioleoyl-sn-glycero-3-phosphoglycerol [POPG]), phosphatidic acids (1,2-dimyristoyl-sn-glycero-3-phosphatidic acid [DMPA], dipalmitoylphosphatidic acid [DPPA], distearoyl-phosphatidic acid [DSPA]), phosphoethanolamines (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine [DMPE], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine [DPPE], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine [DSPE], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine [DOPE]), phosphoserine, and polyethylene glycol [PEG] phospholipids.

[0279] Liposome size may vary from 30 nm to several microns, depending on the phospholipid composition and the method used for their preparation. In certain embodiments of the invention, liposome sizes range from 50 nm to 500 nm, and in further embodiments, from 50 nm to 200 nm. Dynamic laser light scattering is a method used to measure liposome size that is well known to those skilled in the art.

[0280] In particularly suitable embodiments, the liposomes used in the present invention comprise DOPC and a sterol, particularly cholesterol. Thus, in certain embodiments, the compositions of the present invention comprise QS21 in the form of liposomes in any amount described herein, wherein the liposomes comprise DOPC and a sterol, particularly cholesterol.

[0281] In a more preferred embodiment, the adjuvant comprises 3D-MPL and QS21 in a liposomal formulation.

[0282] In one embodiment the adjuvant comprises 25 to 75, for example 35 to 65 micrograms (for example about or exactly 50 micrograms) of 3D-MPL and 25 to 75, for example 35 to 65 (for example about or exactly 50 micrograms) of QS21 in a liposomal formulation.

[0283] In another embodiment the adjuvant comprises 12.5 to 37.5, for example 20 to 30 micrograms (for example about or exactly 25 micrograms) of 3D-MPL and 12.5 to 37.5, for example 20 to 30 micrograms (for example about or exactly 25 micrograms) of QS21 in a liposomal formulation.

[0284] In another embodiment of the invention, the adjuvant comprises or consists of an oil-in-water emulsion. Suitably, the oil-in-water emulsion comprises a metabolizable oil and an emulsifier. A particularly suitable metabolizable oil is squalene. Squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene) is an unsaturated oil found in large amounts in shark liver oil and in smaller amounts in olive oil, wheat germ oil, rice bran oil, and yeast. In one embodiment, the metabolizable oil is present in the immunogenic composition in an amount of 0.5% to 10% (v / v) of the total volume of the composition. A particularly suitable emulsifier is polyoxyethylene sorbitan monooleate (Polysorbate 80 or Tween 80). In one embodiment, the emulsifier is present in the immunogenic composition in an amount of 0.125 to 4% (v / v) of the total volume of the composition. The oil-in-water emulsion may optionally include a tocol. Tocols are well known in the art and are described in EP 0 382 271 B1. Suitably, the tocol may be alpha-tocopherol or a derivative thereof, such as alpha-tocopherol succinate (also known as vitamin E succinate). In one embodiment, the tocol is present in the adjuvant composition in an amount of 0.25% to 10% (v / v) of the total volume of the immunogenic composition. The oil-in-water emulsion may also optionally include sorbitan trioleate (SPAN 85).

[0285] In an oil-in-water emulsion, the oil and emulsifier must be contained in an aqueous carrier, which can be, for example, phosphate buffered saline or citrate.

[0286] In particular, the oil-in-water emulsion systems used in the present invention have small oil droplet sizes in the submicron range. Suitably, the droplet sizes will be in the range of 120-750 nm in diameter, more particularly 120-600 nm. Even more particularly, the oil-in-water emulsions contain oil droplets at least 70% of which by intensity are less than 500 nm in diameter, more particularly at least 80% of which by intensity are less than 300 nm in diameter, and even more particularly at least 90% of which by intensity are in the range of 120-200 nm in diameter.

[0287] It will be appreciated that the viral Fc receptor or fragment thereof and the adjuvant can be stored separately and mixed (ex tempo) prior to administration to a subject. The viral Fc receptor or fragment thereof and the adjuvant can also be administered to a subject separately but simultaneously.

[0288] In one aspect, there is provided a kit comprising or consisting of a viral Fc receptor or immunogenic fragment thereof described herein and an adjuvant.

[0289] Sequence comparison For purposes of comparing the sequences of two closely related polynucleotides or polypeptides, the "sequence identity" or "% identity" between a first sequence and a second sequence can be determined using an alignment program, such as BLAST® (available at blast.ncbi.nlm.nih.gov, last accessed September 12, 2016) using standard settings. Percentage identity is the number of identical residues divided by the length of the alignment, multiplied by 100. An alternative definition of identity is the number of identical residues divided by the number of aligned residues, multiplied by 100. An alternative method includes using the gapped method, which takes into account gaps in the alignment, e.g., deletions in one sequence relative to the other. A polypeptide or polynucleotide sequence is said to be identical to another polypeptide or polynucleotide sequence if they share 100% sequence identity over their entire length.

[0290] A "difference" between two sequences refers, for example, to the insertion, deletion or substitution of a single amino acid residue at a position in one sequence compared to the other sequence.

[0291] For purposes of comparing a first reference polypeptide sequence with a second comparison polypeptide sequence, the number of additions, substitutions, and / or deletions made to the first sequence to create the second sequence can be ascertained. An addition is the addition of a single amino acid residue to the sequence of the first polypeptide (including additions at either end of the first polypeptide). A substitution is the replacement of a single amino acid residue in the sequence of the first polypeptide with a different single amino acid residue. A deletion is the removal of a single amino acid residue from the sequence of the first polypeptide (including deletions at either end of the first polypeptide).

[0292] Suitably, substitutions in the sequences of the present invention can be conservative substitutions. Conservative substitutions include the replacement of an amino acid with another amino acid that has similar physicochemical properties to the amino acid being replaced (see, for example, Stryer et al., Biochemistry, 5th Edition 2002, pages 44-49). Preferably, conservative substitutions are selected from the group consisting of: (i) a basic amino acid with another different basic amino acid, (ii) an acidic amino acid with another different acidic amino acid, (iii) an aromatic amino acid with another different aromatic amino acid, (iv) a nonpolar aliphatic amino acid with another different nonpolar aliphatic amino acid, and (v) a polar uncharged amino acid with another different polar uncharged amino acid. Preferably, basic amino acids are selected from the group consisting of arginine, histidine, and lysine. Preferably, acidic amino acids are aspartic acid or glutamic acid. Preferably, aromatic amino acids are selected from the group consisting of phenylalanine, tyrosine, and tryptophan. Preferably, the non-polar aliphatic amino acid is selected from the group consisting of alanine, valine, leucine, methionine, and isoleucine. Preferably, the polar, uncharged amino acid is selected from the group consisting of serine, threonine, cysteine, proline, asparagine, and glutamine. In contrast to conservative amino acid substitutions, non-conservative amino acid substitutions are the replacement of one amino acid with any amino acid that does not fall within the conservative substitutions (i) to (v) outlined above.

[0293] term "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide to serve as a template for the synthesis of other polymers and macromolecules in biological processes, such as peptide or protein synthesis. Both the coding strand (the sequence of which is usually provided in a sequence listing) and the non-coding strand (used as a template for transcription of a gene or cDNA) of a double-stranded nucleotide molecule may be said to encode a peptide or protein. Unless otherwise specified, as used herein, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.

[0294] The term "expression" or "expressing" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its operably linked promoter.

[0295] Unless otherwise explained in the context of this disclosure, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of general terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0296] The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. The term "plurality" refers to two or more. It should be further understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for illustrative purposes. In addition, numerical limitations given regarding concentrations or levels of a substance, e.g., an antigen, are intended to be approximate. Thus, where a concentration is indicated to be at least (e.g.,) 200 pg, it is intended that the concentration should be understood to be at least approximately (or "about" or "approximately") 200 pg.

[0297] The term "comprises" means "includes." Thus, unless the context clearly indicates otherwise, the word "comprises" and variations thereof, such as "comprise" and "comprising," will be understood to mean the inclusion of a stated compound or composition (e.g., nucleic acid, polypeptide, antigen) or step, or group of compounds or steps, but not the exclusion of any other compound, composition, step, or group thereof.

[0298] The amino acid sequences provided herein are designated by either one-letter or three-letter nomenclature, as known in the art (see, e.g., Eur. J. Biochem. 138:9-37 (1984)).

[0299] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

[0300] The present invention will now be further illustrated by the following non-limiting examples.

[0301] [Example] [Example 1] Immunogenicity of adjuvanted HSV2 gE and gEgI heterodimeric proteins in mice Materials and Methods Investigational drugs and preparations tested The HSV2 gE tested herein had the amino acid sequence shown in SEQ ID NO: 7 (ectodomain).

[0302] The HSV2 gEgI heterodimer tested herein consisted of HSV2 gE having the amino acid sequence set forth in SEQ ID NO:7 (ectodomain) associated in a noncovalent complex with HSV2 gI having the amino acid sequence set forth in SEQ ID NO:8 (ectodomain).

[0303] HSV2 gE (464 μg / mL) and gEgI heterodimer (824 μg / mL) proteins were produced in human embryonic kidney 293F cells (HEK293F) using the Expi293F expression system and formulated in 20 mM Hepes-150 mM NaCl-5% glycerol solution at pH 7.5.

[0304] AS01 is an adjuvant system containing MPL, QS-21 and liposomes (5 μg MPL and 5 μg QS-21 in 50 μL).

[0305] Research model CB6F1 mice (a hybrid of C57B1 / 6 and Balb / C mice) were used in this study. CB6F1 mice have been shown to generate strong CD4+ / CD8+ T cell and humoral immune responses after vaccination with various types of immunogens, including adjuvanted proteins and viral vectors. The ability to induce CD4+ T cell and antibody responses showed comparable trends between these mice and humans.

[0306] Immunological readout Total gE and gI-specific IgG binding antibodies measured by ELISA Quantification of total gE or gI-specific IgG antibodies was performed using indirect ELISA. Recombinant gE (approximately 51 kDa) or gI protein (approximately 46 kDa) from HSV2 was used as the coating antigen. These proteins were produced in HEK293F cells using the Expi293F expression system.

[0307] Polystyrene 96-well ELISA plates (Nunc F96 Maxisorp catalog 439454) were coated with 100 μL / well of antigen diluted to 2 μg / mL (gE) and 1 μg / mL (gI) in 50 mM carbonate / bicarbonate pH 9.5 buffer (GSK in-house) and incubated overnight at 4°C. After incubation, the coating solution was removed and the plates were blocked for 1 h at 37°C with 200 μL / well of Difkomilk 10% diluted in PBS (blocking buffer) (ref 232100, Becton Dickinson, USA). The blocking solution was removed and 3x serum dilutions (PBS + 0.1% Tween 20 + 1% BSA buffer, GSK in-house) were added to the coated plates and incubated for 1 h at 37°C. The plate was washed four times with PBS 0.1% Tween 20 (wash buffer), and peroxidase-conjugated AffiniPure goat anti-mouse IgG (H+L) (ref 115-035-003, Jackson, USA) was used as the secondary antibody. 100 microliters per well of secondary antibody diluted 1:500 in PBS + 0.1% Tween 20 + 1% BSA buffer was added to each well, and the plate was incubated at 37°C for 45 minutes.

[0308] Plates were then washed four times with wash buffer and twice with deionized water and incubated for 15 min at RT with 100 μL / well of a solution of 75% single-component TMB peroxidase ELISA substrate (ref 172-1072, Bio-Rad, USA) diluted in 0.1 M sodium citrate pH 5.5 buffer (GSK in-house). The enzymatic color development was stopped with 100 μL per well of 1 M 0.4 N sulfuric acid (H2SO4), and plates were read at an absorbance of 450 / 620 nm using a Versamax ELISA reader.

[0309] Optical density (OD) readings were captured and analyzed using SoftMaxPro GxP v5.3 software. A standard curve was constructed by applying a four-parameter logistic regression fit to the reference standard. The antibody titer in the sample was calculated by interpolation of the standard curve. The antibody titer of the sample was obtained by averaging the values ​​obtained from dilutions that fell within the 20-80% dynamic range of the standard curve. Titers were expressed as EU / mL (ELISA units per mL).

[0310] HSV2 gE- and gI-specific CD4+ / CD8+ T cell immune responses measured by ICS assay The frequencies of gE-specific CD4+ and CD8+ T cells producing IL-2 and / or IFN-γ and / or TNF-α were assessed in splenocytes harvested 14, 28, and 42 days after prime immunization after ex-vivo stimulation with HSV2 gE or gI peptide pools.

[0311] Splenocyte isolation - Spleens were harvested from individual mice either 14, 28, or 42 days after immunization and placed in RPMI 1640 medium (RPMI / supplement) supplemented with RPMI supplements (glutamine, penicillin / streptomycin, sodium pyruvate, non-essential amino acids, and 2-mercaptoethanol). A cell suspension was prepared from each spleen using a tissue grinder. The spleen cell suspension was filtered (cell strainer, 100 μm), and the filter was then rinsed with 40 mL of cold PBS-EDTA 2 mM. After centrifugation (335 g, 4°C for 10 min), the cells were resuspended in 7 mL of cold PBS-EDTA 2 mM. A second washing step was performed as previously described, and finally, the cells were resuspended in 2 mL of RPMI / supplement supplemented with 5% FCS. The cell suspension was then diluted 20x (10 μL) with PBS buffer (190 μL) for cell counting (using a MACSQuant analyzer). After counting, the cells were centrifuged (335 g, 10 min at RT) and resuspended in 10 μL RPMI / supplements supplemented with 5% FCS. 7 The cells were resuspended at 100 cells / mL.

[0312] Cell preparation - Fresh splenocytes were cultured in a round-bottom 96-well plate at 10 6 The cells were seeded at 100 μL per well. - a 15-mer, overlapping peptide pool spanning the sequence of the gE protein from HSV2 (1 μg / mL per peptide per well); - a 15-mer, overlapping peptide pool spanning the sequence of the gI protein from HSV2 (1 μg / mL per peptide per well); - a 15-mer, overlapping peptide pool spanning the sequence of the human β-actin protein (1 μg / mL per peptide per well) (irrelevant stimulus), - RPMI / supplemented medium (as a negative control for the assay), - PMA- ionomycin solution at working concentrations of 0.25 μg / mL and 2.5 μg / mL, respectively (as positive controls for the assay) Cells were stimulated with 1 μg / mL of anti-CD28 (clone 37.51) and anti-CD49d antibody (clone 9C10(MFR4.B)) per well containing 100 μL of either HCl or HCl for 6 hours (37° C., 5% CO ).

[0313] After 2 hours of ex vivo stimulation, brefeldin A (Golgi plug ref 555029, BD Bioscience) diluted 1 / 200 in RPMI / additives supplemented with 5% FCS was added for an additional 4 hours to inhibit cytokine secretion. Plates were then transferred to 4°C for overnight incubation.

[0314] Intracellular cytokine staining—After overnight incubation at 4°C, cells were transferred to a V-bottom 96-well plate, centrifuged (189 g, 4°C for 2 min), and washed with 250 μL of cold PBS + 1% FCS (flow buffer). After a second centrifugation (189 g, 4°C for 2 min), cells were resuspended in 50 μL of flow buffer containing a 1 / 50 dilution of anti-CD16 / 32 antibody (clone 2.4G2) to block nonspecific antibody binding (4°C for 10 min). Then, 50 μL of flow buffer containing mouse anti-CD4-V450 antibody (clone RM4-5 diluted 1 / 100), anti-CD8-PerCp-Cy5.5 antibody (clone 53-6.7 diluted 1 / 50), and Live / Dead™ Fixable Yellow dead cell stain (1 / 500) was added for 30 min at 4°C in the dark. After incubation, 100 μL of flow buffer was added to each well, and the cells were then centrifuged (189 g, 4° C., 5 min). A second washing step was performed with 200 μL of flow buffer, and after centrifugation, the cells were fixed and permeabilized by adding 200 μL of Cytofix-Cytoperm solution for 20 min in the dark at 4° C. After plate centrifugation (500 g, 4° C. for 5 min), cells were washed with 200 μL of Perm / Wash buffer, centrifuged (500 g, 4° C. for 5 min), and resuspended in 50 μL of Perm / Wash buffer containing mouse anti-IL2-FITC (clone JES6-5H4, 1 / 400 dilution), anti-IFN-γ-APC (clone XMG1.2, 1 / 200 dilution), and anti-TNF-α-PE (clone MP6-XT22, 1 / 700 dilution) antibodies for 1 h at 4° C. in the dark. After incubation, 100 μL of flow buffer was added to each well, and the cells were then washed a final time with 200 μL of Perm / Wash buffer (centrifuged 500 g, 4° C. for 5 min) and resuspended in 220 μL of PBS.

[0315] Cell Acquisition and Analysis - Stained cells were analyzed by flow cytometry using an LSRII flow cytometer and FlowJo software. Live cells were identified using Live / Dead staining, and lymphocytes were then isolated based on forward / side scatter (FSC / SSC) gating. Acquisition was performed with approximately 5,000 CD4+ / CD8+ T cell events per acquisition from three time points. The percentages of IFN-γ+ / -IL-2+ / - and TNF-α+ / - producing cells were calculated in the CD4+ and CD8+ T cell populations. For each sample, the nonspecific signal detected after medium stimulation was subtracted from the specific signal detected after peptide pool stimulation.

[0316] Follicular B helper CD4+ T cells and activated B cells measured in draining lymph nodes by immunofluorescent assay In the DLN (left iliac) of mice 10 days after immunization, T fh The percentages of CD4+ T and activated B cells were examined. AS01 and NaCl immunized mice were used as negative control groups.

[0317] Cell isolation from draining lymph nodes—Left iliac lymph nodes were harvested from individual mice immunized with AS01-adjuvanted gE and gE / gI proteins 10 days after immunization. Due to the low number of isolated cells, left and right iliac lymph nodes were pooled along with inguinal and popliteal lymph nodes for both control groups (NaCl- and AS01-injected mice) to increase the number of immune cells available for immunofluorescence staining and flow cytometry acquisition.

[0318] Lymph nodes were placed in 600 μL of RPMI / additive, and a cell suspension was prepared using a tissue grinder, filtered (cell stainer 100 μm), and rinsed with 0.5 mL of cold PBS-EDTA 2 mM. After centrifugation (335 g, 4°C for 5 min), the cells were resuspended in 0.5 mL of cold PBS-EDTA 2 mM and placed on ice for 5 min. A second washing step was performed as previously described, and the cells were resuspended in 0.5 mL of RPMI / additive supplemented with 5% FCS. Finally, the cell suspension was diluted 20x (10 μL) with PBS buffer (190 μL) for cell counting (using a MACSQuant analyzer).

[0319] After counting, cells were centrifuged (335 g, 5 min at RT) and diluted to 2.5 × 10 in RPMI / supplements supplemented with 5% FCS. 7 The cells were resuspended at 100 cells / mL.

[0320] Immunostaining - Fresh cells (2.5 x 10 in 100 μL) 6 The cells (100 cells / well) were transferred to a V-bottom 96-well plate, centrifuged (400 g, 4°C for 5 min), and washed in 200 μL of PBS buffer. After a second centrifugation (400 g, 4°C for 5 min), the cells were resuspended in 200 μL of PBS buffer, and a final washing step was performed (400 g, 4°C for 5 min). The cells were then resuspended in 100 μL of FixableViability dye eFluor 780 diluted 1 / 1000 in PBS buffer and incubated at RT in the dark for 15 min.

[0321] After incubation, the cells were centrifuged (400 g, 5 min at 4°C) and 50 μL of flow buffer (PBS + 1% FCS) containing anti-CD16 / 32 antibody (clone 2.4G2 diluted 1 / 50), rat anti-CD4-PECy7 (clone RM4-5 diluted 1 / 100), rat anti-mouse IgG2a CD19 FITC (clone 1D3 / CD19 diluted 1 / 200), rat anti-mouse CXCR5 biotin (clone 2G8 diluted 1 / 50), hamster anti-mouse CD279 (PD-1) BV421 (clone J43 diluted 1 / 250), and rat anti-mouse IgG2a F4 / 80 APC / cy7 (clone BM8 diluted 1 / 50) was added for 45 min at 4°C in the dark.

[0322] After incubation, 100 μL of flow buffer was added to each well, and the cells were then centrifuged (400 g, 4° C., for 5 minutes). A second washing step was performed with 200 μL of flow buffer, and after centrifugation, 50 μL of flow buffer containing streptavidin-APC (diluted 1 / 200) was added for 30 minutes at 4° C. in the dark.

[0323] After incubation, 100 μL of flow buffer was added to each well, and the cells were then centrifuged (400 g, 4° C., 5 min). A second washing step was performed with 200 μL of flow buffer. After centrifugation, the cells were fixed and permeabilized by adding 200 μL of eBioscience™ Fixation / Permeabilization (Thermofisher, ref. 00-5523-00) solution in the dark for 30 min at 4° C. After plate centrifugation (400 g, 4° C., 5 min), the cells were washed with 200 μL of permeabilization buffer 1× (eBioscience™), centrifuged (400 g, 4° C., 5 min), and resuspended in 100 μL of permeabilization buffer 1× (eBioscience™) containing mouse anti-BCL6-PE (clone K112-91) antibody diluted 1 / 50 for 45 min at 4° C.

[0324] After incubation, 100 μL of permeabilization buffer 1× (eBioscience™) was added to each well and centrifuged (400 g, 4° C. for 5 min). The cells were then washed twice with 200 μL of permeabilization buffer 1× (eBioscience™) (centrifugation 400 g, 4° C. for 5 min) and resuspended in 220 μL of PBS for flow cytometry acquisition.

[0325] Cell Acquisition and Analysis—Stained cells were analyzed by flow cytometry using an LSRII flow cytometer and FlowJo software. Live cells were identified using Live / Dead staining, and lymphocytes were then isolated based on forward / side scatter (FSC / SSC) gating.

[0326] T fh To isolate CD4+ T cells, acquisition was performed on total viable CD4+ T cells and the percentage of PD-1 / CXCR5 positive cells was calculated.

[0327] To isolate activated B cells, acquisition was performed on total viable CD19+ B cells and the percentage of PD-1 / CXCR5 / BCL6 positive cells was calculated.

[0328] Measurement of vaccine-induced antibodies that bind and activate mFcγRIV (Mouse FcγRIV ADCC Reporter Bioassay - Promega) Developed by Promega Laboratories, mouse F c The γRIV Antibody-Dependent Cellular Cytotoxicity (ADCC) Reporter Bioassay (Cat. No. M1201) is a bioluminescent cell-based assay that can be used to measure the potency and stability of antibodies and other biologics that contain an Fc domain that specifically binds and activates mouse FcγRIV (mFcγRIV). mFcγRIV is the receptor responsible for mouse ADCC, as well as human FcγRIV, the primary Fc receptor responsible for ADCC in humans. c Associated with γRIIIa.

[0329] Briefly, 3T3 cells (clone A31, ATCC ref. CCL-163) derived from BALB / c mice purchased from ATCC Laboratories were first prepared in HSV infection medium (DMEM + 10% non-complementary FBS + 2 mM L-glutamine + 1% penicillin / streptomycin) and seeded at 10,000 cells / well (100 μL) in flat-bottom white 96-well plates (Corning, ref. CLS3917). Next, 100 microliters (100 μL) of HSV2 MS strain (ATCC, ref. VR-540) was added to each well at a multiplicity of infection (MOI) of 2, and the cells were incubated at 37°C with 5% CO for 14 hours and 30 minutes. The edges of the plates were not used to avoid edge side effects.

[0330] After incubation, HSV2-infected 3T3 cells (target cells (T)) were washed with 200 μL of PBS, and 25 μL of Promega assay buffer (96% RPMI 1640 medium (36 mL) + 4% low IgG serum (1.5 mL)) was added to each well. Individual mouse sera were serially diluted 2-fold (starting at 1 / 1) with Promega assay buffer in a round-bottom 96-well plate (Nunc, ref. 168136), and 70 μL of each serum dilution was transferred to the corresponding well. Next, 25 μL of engineered luciferase reporter Jurkat cells expressing mouse FcγRIV (effector cells (E)) was added to each well (E / T 6.6 / 1), and the plate was incubated at 37°C with 5% CO for 6 hours.

[0331] After incubation, the plate was left at room temperature for 15 minutes, and 75 μL of Bio-Glow reagent was added to each well. Finally, the plate was incubated at room temperature for 20 minutes and read using a Synergy H1 microplate reader (bioTek™). The area under the curve (AUC) for each mouse was calculated using GraphPad Prism software. AUC was calculated using a 3-fold STD deviation from the mean of the NaCl samples as the positive threshold. In the NaCl control group, a value of 1 was arbitrarily set for all negative values ​​of AUC.

[0332] A cell-based assay for measuring neutralizing antibodies against HSV2 MS strains A neutralization assay was developed to detect and quantify neutralizing antibody titers in serum samples obtained from different animal species. Serum (50 μL / well) was diluted in HSV medium (DMEM supplemented with 1% neomycin and 1% gentamicin) in a flat-bottom 96-well plate (Nunclon Delta Surface, Nunc, Denmark, ref. 167008) by two-fold serial dilutions (starting at 1 / 10). Serum was then incubated with 400 TCID / 50 μL / well of HSV2 MS strain (ref. ATCC VR-540) prediluted in HSV medium supplemented with 2% guinea pig serum complement (Harlan, ref. C-0006E) for 2 h at 37°C (5% CO). The edges of the plates were not used, and one column of each plate was left free of virus and serum (TC) or contained virus but without serum (TV) to serve as a negative or positive control for infection, respectively. Positive control sera for the assay were pooled serum samples obtained from mice immunized with different doses (0.22, 0.66, 2, and 6 μg / dose) of HSV2 gD / AS01 (2.5 μg) and collected 14 days after the second (14PII) or third (14PIII) immunization.

[0333] After incubation of the antibody-virus mixture, 10,000 Vero cells / 100 μL were added to each well of each plate, and the plates were incubated at 37°C (5% CO2) for 4 days. Four days after infection, the supernatant was removed from the plates, and the cells were incubated for 8 hours at 37°C (5% CO2) with WST-1 solution (a reagent for measuring cell viability, Roche, ref. 11644807001) diluted 15x in HSV development medium (DMEM supplemented with 1% neomycin and 1% gentamicin + 2% FBS). To calculate the neutralizing antibody titer, the data sets were normalized to 0 and 100% cytopathic effect (CPE) based on the average WST-1 OD in the "virus-free cells" and "serum-free cells" wells, respectively. The percentage of inhibition of CPE at dilution i was then given by: % Inhibition = (ODi - mean OD serum-free cells) / (mean OD virus-free cells - mean OD serum-free cells)

[0334] The reciprocal of the dilution that resulted in a 50% reduction in CPE was then extrapolated using nonlinear regression with Softmaxpro software.

[0335] ·Statistical methods For IgG antibody responses, a two-way analysis of variance (ANOVA) model is fitted to the log10 titers by including group (HSV2 gE, HSV2 gE / gI, and NaCl), time point, and their interaction as fixed effects, and by considering repeated measures of time point (animal identified).

[0336] For CD4+ T cell responses, a two-way analysis of variance (ANOVA) model is fitted to the log10 frequencies by including group (HSV2 get, HSV2 gE / gi and NaCl), time point and their interaction as fixed effects.

[0337] Geometric means and their 95% CIs and geometric mean ratios of gE to NaCl (or gE / gI) and their 90% CIs are derived from these models for all time points.

[0338] For time point comparisons, geometric mean ratios * and their 95% CIs are also derived from these models. * gE (or gE / gI) after dose II (or I) versus gE (or gE / gI) after dose III (or II).

[0339] NaCl thresholds are based on P95 of NaCl data over several days and are set at 0.19% for HSV2 gE-specific CD4+ T cell responses, 0.32% for HSV2 gI-specific CD4+ T cell responses, and 0.30% for β-actin CD4+ T cell responses.

[0340] Study design Naive female CB6F1 mice, 6-8 weeks old (n = 20 / groups 1-2), were injected intramuscularly (i.m.) into the gastrocnemius muscle with 5 μg / dose of recombinant HSV2 gE (group 1) or HSV2 gE / gI heterodimer (group 2) protein adjuvanted with 50 μL of AS01 on days 0, 14, and 28. As a negative control, mice were injected i.m. with 50 μL of 150 mM NaCl solution (group 4) on days 0, 14, and 28. An additional group of mice (n = 4 / group 3) was injected i.m. with AS01 alone on day 0 only and served as a negative control to assess induction of follicular B helper CD4+ T cell activation and B cell responses in the draining lymph nodes (DLNs).

[0341] Ten days after the first immunization, eight mice in the gE and gEgI / AS01 immunized groups (groups 1-2) and four mice in the control immunized group (groups 3-4) were selected to examine follicular B helper CD4+ T cell activation and B cell responses in the DLN (left iliac node).

[0342] On day 14 after the first (14PI), second (14PII), and third (14PIII) immunizations, four mice in groups 1-2 and 4 were selected to evaluate gE- and gI-specific CD4+ / CD8+ T cell responses in the spleen.

[0343] Finally, sera were collected in each group (4 mice / group) 14 days after the first (14PI), second (14PII), and third (14PIII) immunizations to examine total anti-gE and gI-specific IgG antibody responses and their potential cytotoxic activity by using a mouse ADCC reporter bioassay.

[0344] For CD4+ T cell results (at D28 and D42 time points) and for IgG antibody responses (at D14, D28, and D42 time points), data from this study (Experiment A) were pooled with data from a previous experiment (Experiment B).

[0345] result Recombinant HSV2 gE and gE / gI proteins induce gE- and gI-specific CD4+ / CD8+ T cell responses.

[0346] Female inbred CB6F1 mice were injected im with 5 μg of either HSV2 gE (n = 20 / group 1) or HSV2 gE / gI protein (n = 20 / group 2) adjuvanted with 50 μL of AS01 on days 0, 14, and 28. Using the same immunization schedule, an additional group of mice was injected im with saline solution (150 mM NaCl) and used as a negative control (n = 16 / group 4). Fourteen days after the first, second, and third immunizations, four animals from each group were selected for endpoint analysis. After ex vivo stimulation with HSV2 gE or gI peptide pools, spleens were individually harvested and processed to identify vaccine-specific CD4+ and CD8+ T cells expressing IL-2+ / -, IFN-γ+ / -, and / or TFN-α+ / -.

[0347] As illustrated in Figure 5, for Experiment A, both the AS01-adjuvanted HSV2 gE and HSV2 gE / gI proteins induced strong CD4+ T cell responses toward the HSV2 gE antigen after the first, second, and third immunizations compared to the NaCl control group. The geometric mean ratios (GMRs) of gE-specific CD4+ T cell responses calculated between the gE- and gE / gI-immunized groups relative to the NaCl group were all greater than 10-fold (Table 3). A two-fold increase in the third dose (PIII / P1) compared to the first appeared to be observed in the AS01-adjuvanted HSV2 gE-immunized group, and to a lesser extent, in the AS01-adjuvanted HSV2 gE / gI-immunized group (GMRs of 2.43 and 1.79) (Table 4). Regarding gE-specific CD4+ T cell responses, identical results are observed for the pools of experiments (Experiment A and Experiment B) after the second (day 28) and third immunization (day 42) (Figure 6) (Table 4).

[0348] For Experiment A, in mice immunized with AS01-adjuvanted HSV2 gE / gI protein, gI-specific CD4+ T cell responses were detected after the first, second, and third immunizations. The GMRs of gI-specific CD4+ T cell responses calculated between the gE / gI-immunized group and the NaCl group at different time points were all greater than 8-fold (Figure 7) (Table 5). The results suggest a two-fold increase in the third dose compared to the first (Table 6).

[0349] Finally, in experiment A, gE-specific, but not gI-specific, CD8+ T cells were detected after two or three immunizations with AS01-adjuvanted HSV2 gE and gE / gI proteins (Figures 8-9).

[0350] [Table 3]

[0351] [Table 4]

[0352] [Table 5]

[0353] [Table 6]

[0354] Recombinant HSV2 gE and gE / gI proteins promote follicular B helper CD4+ T cell expansion and activated B cells in draining lymph nodes. Female inbred CB6F1 mice were immunized im in the left gastrocnemius muscle with 5 μg of HSV2 gE (n=20 / group 1) or gE / gI (n=20 / group 2) protein adjuvanted with 50 μL of AS01 on day 0. Using the same schedule of immunization, two additional groups of mice (n=4 / group) were injected im with saline solution (NaCl 150 mM) (n=16 / group 4) or with 50 μL of AS01 alone (n=4 / group 3) and served as negative controls.

[0355] Ten days after immunization, eight mice in the gE and gE / gI-AS01 immunized groups and four mice in both negative control groups were selected for endpoint analysis. Left iliac draining lymph nodes were harvested and analyzed for follicular B helper CD4+ T (T fh The frequencies of CD4+ / CXCR5+ / PD-1+ cells and activated B cells (CD19+ / CXCR5+ / Bcl6+) were assessed. Due to the low number of isolated cells, left and right iliac lymph nodes, along with inguinal and popliteal lymph nodes, were pooled for both control groups (NaCl- and AS01-injected mice) to increase the number of immune cells available for immunofluorescence staining and flow cytometry acquisition.

[0356] Compared with the AS01 and NaCl-treated group of mice, both AS01-adjuvanted HSV2 gE or gE / gI immunized groups showed T fh and higher frequencies of activated B cells were detected (Figures 10 and 11). fhThe levels of activated B cells were similar between the AS01- and NaCl-treated groups of mice, suggesting that nonspecific activation of both populations of cells did not occur with the adjuvant alone.

[0357] Follicular B helper CD4+ T cells are CD4 + They are a specialized subset of T cells that play a key role in protective immunity and help B cells generate antibody-producing plasma cells and long-lived memory B cells. The detection of both these cell types in the draining lymph nodes suggests that both AS01-adjuvanted gE or gEgI heterodimeric proteins can induce high-quality antigen-specific antibodies.

[0358] Recombinant HSV2 gE and gE / gI proteins induced gE- and / or gI-specific IgG antibodies. Female inbred CB6F1 mice were immunized im with 5 μg of HSV2 gE (n=20 / group 1) or gE / gI (n=20 / group 2) protein adjuvanted with 50 μL of AS01 on days 0, 14, and 28. Using the same immunization schedule, an additional group of mice was injected im with saline solution (NaCl 150 mM) and used as a negative control (n=16 / group 4). 14 days after the first, second, and third immunizations, four animals in each group were bled for serum collection, and total HSV2 gE- and gI-specific IgG antibody responses were assessed by indirect ELISA.

[0359] For Experiment A, both AS01-adjuvanted HSV2 gE and HSV2 gE / gI proteins induced high titers of total gE-specific IgG antibodies after the first (day 14), second (day 28), and third immunizations (day 42) (Figure 12). The GMRs of gE-specific IgG titers calculated between the gE- and gE / gI-immunized groups relative to the NaCl group were all greater than 1780-fold (see Table 7). The levels of gE-specific antibodies were increased by more than 19-fold after the second immunization compared to the first immunization for both groups of immunized mice (GMRs of 63.68 and 19.29 for the gE and gE / gI groups, respectively) (Table 8). Similar results were observed in pooled experiments (Experiment A and Experiment B) (Figure 13). The GMRs of gE-specific IgG titers calculated between the gE- and gE / gI-immunized groups relative to the NaCl group were all greater than 1963-fold (see Table 7). Furthermore, we confirmed that the levels of gE-specific antibodies increased after the second immunization compared to the first immunization for both groups of immunized mice (GMR of 21.28; Table 8), and that the strength of gE-specific antibodies increased only between the second and third immunizations in the group of mice immunized with HSV2 gE / gI protein (GMR of 2.15) (Table 8).

[0360] For Experiment A, gI-specific IgG antibody responses were detected after the first (day 14), second (day 28), and third immunizations (day 42) in mice immunized with AS01-adjuvanted HSV2 gE / gI protein (Figure 14). The GMRs of gI-specific IgG titers calculated for the NaCl group were all greater than 161-fold (Table 9). The titers of gI-specific antibodies were increased more than 29-fold after the second immunization compared to the first immunization (Table 10).

[0361] [Table 7]

[0362] [Table 8]

[0363] [Table 9]

[0364] [Table 10]

[0365] Non-neutralizing gE and / or gI-specific antibodies can bind to mouse FcγRIV Neutralizing antibody responses against HSV2 MS virus were assessed by a cell-based assay, and murine FcγRIV binding activity was assessed by using an antibody-dependent cellular cytotoxicity reporter bioassay (Promega).

[0366] Non-neutralizing antibody responses against the HSV2 MS strain were detected in both groups of mice immunized with AS01-adjuvanted recombinant HSV2 gE and HSV2 gE / gI proteins after the first (day 14), second (day 28), and third immunizations (day 42) (Figure 15). Interestingly, compared with the NaCl control group, gE / gI-specific antibodies were able to bind to an FcγRIV-expressing Jurkat cell line at each time point (14P1, 14PII, 14PIII) in both immunization groups (luciferase reporter bioassay) (Figure 16). These results suggest that recombinant HSV2 gE and HSV2 gE / gI proteins induced non-neutralizing antibodies that may be able to drive ADCC (antibody-mediated depletion cell death) of HSV-2-infected cells following activation of FcγRIV-expressing cells after Fc binding.

[0367] [Example 2] Evaluation of the therapeutic efficacy of AS01-adjuvanted HSV2 gE or gE / gI heterodimeric proteins in a guinea pig model of chronic genital HSV2 infection Materials and Methods Investigational drugs and formulations tested The HSV2 gE tested herein had the amino acid sequence shown in SEQ ID NO: 7 (ectodomain).

[0368] HSV2 MS strain (7.38 log TCID 50 / mL) were originally purchased from ATCC laboratories (ATCC reference: VR-540) and stored in Biorich-DMEM medium supplemented with 1% L-glutamine, 1% penicillin / streptomycin, and 20% NBCS.

[0369] The HSV2 gEgI heterodimer tested herein consists of HSV2 gE having the amino acid sequence set forth in SEQ ID NO:7 (ectodomain) associated in a non-covalent complex with HSV2 gI having the amino acid sequence set forth in SEQ ID NO:8 (ectodomain).

[0370] HSV2 gE (920 μg / mL) and gEgI heterodimer (824 μg / mL) proteins were produced in human embryonic kidney 293F cells (HEK293F) using the Expi293F expression system and formulated in 20 mM Hepes-150 mM NaCl-5% glycerol solution at pH 7.5.

[0371] HSV2 recombinant gD protein (gD2t) was stored in PBS buffer (1 mg / mL).

[0372] AS01 is an adjuvant system containing MPL, QS-21 and liposomes (50 μg MPL and 50 μg QS-21 in 500 μL).

[0373] Research model It is widely accepted that small animal models (mice, cotton rats, and guinea pigs) are useful tools for genital herpes vaccine research. The literature has demonstrated that the guinea pig model is an appropriate model for addressing the efficacy of adjuvanted glycoprotein vaccine candidates (Skoberne & al. 2013. An Adjuvanted Herpes Simplex Virus 2 Subunit Vaccine Elicits a T Cell Response in Mice and Is an Effective Therapeutic Vaccine in Guinea Pigs. Journal of Virology. 2013 April;87(7):3930-3942). Genital infection in guinea pigs results in self-limited vulvovaginitis with neurological findings closely resembling those seen in human disease. Virus is transported by retrograde transport to cell bodies in sensory ganglia and autonomic neurons in the spinal cord. During this phase of infection, the virus establishes a latent infection, and like humans, animals are subject to spontaneous, intermittent reactivation of the virus. For all these reasons, a guinea pig model of chronic genital infection was chosen in this study to address the therapeutic efficacy of AS01-adjuvanted recombinant HSV2 gE and gE / gI proteins.

[0374] Immunological readout Measurement of genital skin disease During acute HSV2 infection (D0-D14), animals were assessed daily for external genital skin disease using a severity scale of 0-4: - 0: No disease - 1: erythema and / or swelling, - 2: 1 to up to 3 small blisters, - 3: More than three small blisters or one large, fused blisters - 4: Severe vesicular-ulcerative skin disease of the perineum.

[0375] After recovery from acute infection and administration of the first vaccination dose, animals were then examined daily from days 20 to 70 for evidence of recurrent herpetic disease using the same severity scale.

[0376] Assessment of total gE and gI-specific IgG antibodies measured by ELISA Quantification of total gE or gI-specific IgG antibodies was performed using indirect ELISA as described in Example 1 above.

[0377] HSV2 gE- and gI-specific CD4+ / CD8+ T cell responses measured by vaccine-specific T cell proliferation rates The frequency of HSV-specific CD4+ / CD8+ T cells in the spleen was assessed by measuring the total proliferation rate of CD4+ and CD8+ T cells 4 days after ex vivo stimulation with gE- or gI-specific peptide pools. For logistical reasons, half of the animals in each group were culled on day 70 and half on day 74 after HSV2 challenge.

[0378] The cutoff for identifying specific CD4+ / CD8+ T cell responses in AS01-formulated gE or gE / gI-immunized guinea pigs corresponds to the 95th percentile (P95) of CD4+ / CD8+ T cell responses detected in the NaCl-treated group after ex-vivo stimulation of splenocytes with gE or gI or β-actin peptide pools.

[0379] Splenocyte isolation. Spleens were harvested from individual guinea pigs 70 or 74 days after HSV2 challenge and placed in cold RPMI 1640 medium (RPMI / supplements) supplemented with RPMI supplements (glutamine, penicillin / streptomycin, sodium pyruvate, non-essential amino acids, and 2-mercaptoethanol). Individual spleens were cut into small pieces of tissue, and cell suspensions were prepared using a tissue grinder. Each cell suspension was then filtered (100 μM cell stainer), and the filter was rinsed with 50 mL of cold PBS-EDTA 2 mM. After centrifugation (335 g, 4°C for 8 minutes), the cells were resuspended in 4 mL of BD Pharm Lyse buffer (1x concentrated red blood cell lysis buffer) for 15 seconds, and the reaction was blocked by the addition of 35 mL of cold PBS-EDTA 2 mM. The cell suspension was then transferred to a new Falcon tube, filtered (cell stainer 100 μM), and rinsed with 1 mL of PBS-EDTA 2 mM. After centrifugation (335 g, 4°C for 8 min), the cells were resuspended in 5 mL of PBS-EDTA 2 mM and diluted 20-fold (10 μL) with PBS buffer (190 μL) for cell counting (using a MACSQuant analyzer). After counting, the cell suspension was diluted to 10 μL by adding PBS-EDTA 2 mM. 7 The final concentration was adjusted to 100 cells / mL.

[0380] Ex-vivo labeling and peptide stimulation - Ex-vivo labeling of splenocytes was performed using the CellTrace Violet Proliferation Kit (ThermoFisher Scientific, ref C34557). 20 million cells (10 7Cells (2 mL at 4 mM) were labeled by adding Cell trace Violet solution (2 mL at 4 mM) to the cell suspension and incubating in the dark at 37°C for 15 minutes. During this 15-minute incubation period, cells were mixed every 5 minutes. 8 mL of cold RPMI / additive supplemented with 10% FCS was then added for 5 minutes on ice to quench any free dye in the solution. Cells were washed twice (1400 rpm, 4°C for 10 minutes) and resuspended in 1 mL of cold RPMI / additive supplemented with 5% FCS. Cells were then diluted 20x (10 μL) with PBS buffer (190 μL) for cell counting (using a MACSQuant analyzer) to approximately 500,000 cells (5 × 10) per well. 5 cells / well) in a round-bottom 96-well plate. - 15-mer, overlapping peptide pools spanning the entire amino acid sequence of the HSV2 gE and gE / gI heterodimeric proteins (1 μg / mL per peptide), - Concanavalin A (ConA) solution at a working concentration of 2 μg / mL, used as a positive control for the assay; - a 15-mer, overlapping peptide pool spanning the sequence of the human β-actin protein (1 μg / mL per peptide), which was used as the irrelevant peptide pool for the assay; - Cell culture medium used as a negative control The cells were stimulated with 100 μL of the above for 4 days (37°C, 5% CO2).

[0381] Extracellular staining to assess CD4+ / CD8+ T cell proliferation—After 4 days of T cell expansion (37°C, 5% CO2), cells were transferred to a V-bottom 96-well plate, centrifuged (2000 rpm, 4°C for 3 minutes), and washed with 250 μL of cold PBS + 1% FCS. After a second centrifugation (2000 rpm, 4°C for 3 minutes), the cells were resuspended in 50 μL of flow buffer (cold PBS 1% FCS) containing a 1 / 50 dilution of anti-CD16 / 32 antibody (clone 2.4G2) to block nonspecific antibody binding (4°C for 10 minutes). Next, 50 μL of flow buffer containing mouse anti-guinea pig CD4-PE antibody (clone CT7-isotype IgG1 diluted 1 / 50) and mouse anti-guinea pig CD8-FITC antibody (clone CT6-isotype IgG1 diluted 1 / 100) was added for 30 minutes in the dark at 4°C. After the incubation period, cells were washed twice with 200 μL of flow buffer, centrifuged (2000 rpm, 4° C. for 3 min), and Fixable Near-IR Dead Cell Staining Solution (diluted 1 / 5000 in cold PBS) was added for 30 min in the dark at 4° C. After 30 min, 100 μL of flow buffer was added to each well, and then cells were centrifuged (2000 rpm, 4° C. for 3 min) and finally resuspended in 200 μL of PBS.

[0382] Cell acquisition and analysis to assess HSV-specific T cell activation—Stained cells were acquired by flow cytometry, and raw data were analyzed using FlowJo software. Live / Dead staining was used to identify live cells, and lymphocytes were then isolated based on FSC / SSC gating. Total proliferation rates were calculated by sequential gating to isolate viable CD4+ and CD8+ T cell populations. For each sample, the nonspecific proliferation rates of CD4+ and CD8+ T cells detected in the medium-treated samples were subtracted from the samples stimulated with vaccine-specific peptide pools.

[0383] A cell-based assay for measuring neutralizing antibodies against HSV2 MS strains Detection and quantification of neutralizing antibody titers in serum was performed as described in Example 1 above.

[0384] ·Statistical methods Randomization 14 days after vaginal HSV2 challenge Of the 110 guinea pigs used for HSV2 challenge, 64 survivors were randomized and assigned to one of four groups based on cumulative lesion scores over days 0 to 14. The mean and variability of cumulative scores over days 0 to 14 for each group after randomization are presented in Table 11.

[0385] [Table 11]

[0386] statistical methodology Two animals in AS01 / gE (group 1), one in AS01 / gE / gI (group 2), and one in AS01 / gD2t (group 3) were euthanized for ethical reasons after randomization but before spleen collection and were excluded from all analyses.

[0387] Group comparison of standardized cumulative lesion scores - Daily scores of lesion severity (scored 0-4) were summed across all animals for different intervals of interest, corresponding to the cumulative lesion score. The intervals of interest for which these cumulative scores were computed were [0-14], [34-47], [48-70], and [34-70].

[0388] With vaccinations on days 20, 34 and 48, the vaccination efficacy tested was as follows: [34-70]: Second and third vaccination effects [34-47]: Second vaccination effect only [48-70]: Only the third vaccination effect.

[0389] The interval of days [0-14] corresponds to the baseline (acute phase) of the disease before randomization.

[0390] Because the number of days in each interval varies, the individual cumulative scores obtained are divided by the number of days in the interval to provide a standardized cumulative score.

[0391] To assess the impact of the vaccine on lesion scores (standardized cumulative scores), different analysis of covariance (ANCOVA) models are performed: Model 1 tests the combined effect of the second and third vaccinations using the standardized cumulative score on days [34–70] as the response variable and group (4 groups HSV2 infection) as the predictor variable, while adjusting for the baseline covariate (standardized cumulative score on days [0–14]). Indeed, if the baseline covariate is moderately correlated with the response, differences between response values ​​that may be attributable to differences in the covariate can be excluded, leading to a more accurate estimation of the group effect. Model 2 tests the effect of the second vaccination dose only. This model tests the effect of group on the standardized cumulative score at days [34-47] while adjusting for baseline covariates. Model 3 tests the effect of the third vaccination dose only. This model tests the effect of group on the standardized cumulative score at days [48-70] while adjusting for baseline covariates.

[0392] The mean for each group, the difference between vaccinated and unvaccinated, and their respective 90% CIs and p-values ​​(as a one-sided evaluation of the inferiority test) are derived from these models. Different variances between groups are assumed in each model. The % reduction for vaccinated compared to unvaccinated is also calculated by computer.

[0393] Group comparison of days with lesions - The number of days with lesions (regardless of severity) was summed across all animals for the intervals [0-14], [34-47], [48-70], and [34-70] days. The three models above were run using this new variable instead of the standardized cumulative lesion score.

[0394] Group comparison of lesion recurrence - A recurrence occurred for every score equal to 0 on the previous day and greater than 0 on the current day. The number of recurrences for all animals is reported for each group for the interval [34-70] days.

[0395] CD8+ / CD4+ T cell proliferation ratio - Analysis is performed separately for CD4+ and CD8+ T cell responses collected in spleen samples. Medium is used as the reference in the ratio calculation (ratio = stimulation / medium). Cutoffs are determined for CD8+ and CD4+ T results based on data in the NaCl group.

[0396] Elisa Titers - For each IgG antibody response (gE- or gI-specific), a two-way analysis of variance (ANOVA) model is fitted to the log10 titer by including group (HSV2 gE, HSV2 gE / gI, unvaccinated, and NaCl), time point, and their interaction as fixed effects, and by considering repeated measures of time point (animal identification). From these models, geometric means and their 95% CIs are derived.

[0397] For comparison of vaccinated versus unvaccinated groups, the geometric mean ratio of gE (or gE / gI) relative to the unvaccinated group and its 95% CI are derived from these models for all time points.

[0398] For time point comparisons, the geometric mean ratio (gE (or gE / gI) after dose III (or II) to gE (or gE / gI) after dose II (or I)) and its 95% CI are also derived from these models.

[0399] Neutralization titers - Only the PIII (days 70-74) time point was analyzed. A one-way analysis of variance (ANOVA) model was fitted to the log10 titers by including group (HSV2 gE, HSV2 gE / gI, unvaccinated) as a fixed effect and assuming different variability between groups. From this model, geometric means and their 95% CIs were derived. The NaCl group was not included in the model as no variability was observed in this group.

[0400] For comparison of vaccinated versus unvaccinated groups, the geometric mean ratios of gE, gE / gI, or gD2t relative to the unvaccinated group and their 95% CIs are derived from this model.

[0401] Study design Female outbred Hartley guinea pigs, 9-12 weeks old, were obtained from Charles River Laboratories (Crl:HA). Animals were maintained in an in-house animal facility under defined pathogen-free conditions. Guinea pigs (n=110) were infected with HSV2 MS strain (10 5 Mice were infected intravaginally (IVAg) with 100 μL of HSV2 pfu on day 0 and randomized into four different groups based on the cumulative lesion score, which was assessed daily for 14 days (days 0–14 post-infection) using a severity scale. On this scale, lesions range from 0, representing no disease, to 4, representing severe vesicular-ulcerative skin disease at the level of the perineum. Animals that were not infected with HSV2 or developed clinical symptoms that were too severe were either excluded from the study before randomization or euthanized for ethical reasons.

[0402] On days 20, 34, and 48 postinfection, three groups of guinea pigs were injected intramuscularly (i.m.) with 500 μL of either AS01 (50 μg MPL and 50 μg QS-21)-adjuvanted recombinant HSV2 gE (20 μg / dose—n=15 / group 1), AS01 (50 μg MPL and 50 μg QS-21)-adjuvanted recombinant HSV2 gE / gI (40 μg / dose—n=16 / group 2), or AS01 (50 μg MPL and 50 μg QS-21)-adjuvanted recombinant HSV2 gD2t protein (20 μg / dose—n=12 / group 3; positive control for clinical therapeutic efficacy). Guinea pigs in the unvaccinated, HSV2-infected group (n=17 / group 4) were injected with saline solution (150 mM NaCl). Unvaccinated and uninfected guinea pigs served as negative controls for immunological readout (n = 5 / group 5). All animals were scored daily from days 20 to 70 postinfection to assess the severity of recurrent clinical lesions using a severity scale. For ethical reasons, body weight assessments were performed daily during the acute phase of infection (days 5 to 14) and weekly during the chronic phase of infection. Serum samples were collected individually in groups 1-2 and 4 on days 33 (13 PI), 46 (12 PI), and 70 / 74 (22 / 26 PI) postinfection with HSV2, while those from group 3 were collected only on days 70 / 74 (22 / 26 PI). Finally, all animals were culled on days 70 or 74 postinfection to assess vaccine-specific CD4+ / CD8+ T cell responses in the spleen (groups 1-5).

[0403] result AS01-formulated gE or gE / gI heterodimer proteins induced systemic vaccine-specific T cell responses in HSV2-infected guinea pigs. Female outbred guinea pigs (n = 110) were infected with the HSV2 MS strain (10 5Guinea pigs were infected intravaginally (Ivag) on ​​day 0 with 100 μL of AS01-adjuvanted recombinant HSV2 gE (pfu-100 μL) and randomized into four different groups. At days 20, 34, and 48 post-infection, two groups of guinea pigs were intramuscularly (im) injected with 500 μL of either AS01-adjuvanted recombinant HSV2 gE (20 μg / dose—n=15 / group 1) or AS01-adjuvanted recombinant HSV2 gE / gI (40 μg / dose—n=16 / group 2). Guinea pigs in the unvaccinated, HSV2-infected group (n=17 / group 4) were injected with saline solution (150 mM NaCl). Unvaccinated and uninfected guinea pigs served as negative controls for immunological readout (n=5 / group 5). At 70 and 74 days post-HSV2 infection, animals were culled to assess gE- and gI-specific CD4+ / CD8+ T cell responses. Spleens were harvested and the total proliferation of gE / gI-specific CD4+ and CD8+ T cells was assessed 4 days after ex-vivo peptide pool stimulation.

[0404] From an illustrative perspective, the overall proliferation rate of CD4+ T cells detected specifically against gE or gI antigens was slightly increased in the groups of guinea pigs immunized with AS01-gE or AS01-gE / gI protein compared with the unvaccinated guinea pig groups (NaCl-treated or HSV2-infected groups) (Figure 17A). Only three animals in the unvaccinated HSV2-infected group showed some gI- and gE-specific CD4+ T cell responses, suggesting that HSV2 virus does not naturally induce consistent CD4+ T cell responses against gE and gI antigens in guinea pigs (Figure 17B).

[0405] AS01-formulated gE or gE / gI heterodimer protein increased the levels of non-neutralizing vaccine-specific IgG antibodies in HSV2-infected guinea pigs. Female guinea pigs (n = 110) were infected with HSV2 MS strain (10 5Guinea pigs were infected intravaginally (Ivag) with 100 μL of AS01-adjuvanted recombinant HSV2 gE (pfu - 100 μL) on day 0 and randomized into four different groups. On days 20, 34, and 48 post-infection, three groups of guinea pigs were injected intramuscularly (im) with 500 μL of either AS01-adjuvanted recombinant HSV2 gE (20 μg / dose - n = 15 / group 1), AS01-adjuvanted recombinant HSV2 gE / gI (40 μg / dose - n = 16 / group 2), or AS01-adjuvanted recombinant HSV2 gD2t protein (20 μg / dose - n = 12 / group 3; positive control in terms of clinical therapeutic efficacy). Guinea pigs in the unvaccinated HSV2-infected group (n = 17 / group 4) were injected with saline solution (NaCl 150 mM), and unvaccinated and uninfected guinea pigs served as negative controls (n = 5 / group 5). Serum samples were collected from individual animals within all groups at days 33 (13 PI), 46 (12 PI), and 70 / 74 (22 / 26 PI) post-HSV2 infection to assess total gE and gI-specific IgG antibody responses by ELISA, and the neutralizing activity of these antibodies against HSV2 MS strains was assessed only at days 70 / 74 post-infection (22 / 26 PI). Serum from one animal in the unvaccinated, HSV2-infected group at time 13 PI (4.7) was not properly collected and was not evaluated in this analysis.

[0406] Analysis of the geometric means (GM) in each group reveals that the titers of gE-specific IgG antibodies detected after 13PI immunizations were approximately 25-31 times higher in the AS01-gE and AS01-gE / gI vaccinated groups compared with the unvaccinated HSV2-infected group (Figures 18A and 19A). Interestingly, the gE-specific IgG antibody responses were significantly boosted after the second immunization in both the AS01-gE and AS01-gE / gI vaccinated groups (antibody titers increased 7.91-fold for gE and 3.85-fold for gE / gI). However, a third immunization did not increase the levels of gE-specific antibodies in both groups of vaccinated guinea pigs (Figures 18A and 19B).

[0407] The GM of the AS01-gE / gI immunized group increased approximately 21-fold compared to the unvaccinated HSV2-infected group 13 days after the first immunization (13 PI) (Figures 18B and 19C). In this case, gI-specific IgG antibody titers significantly increased after the second (3.93-fold PI vs. PII) and third immunizations (2.31-fold PII vs. PIII) in HSV2-infected guinea pigs immunized with AS01-gE / gI protein (Figures 18 and 19D).

[0408] Finally, evaluation of the functionality of gE- and gI-specific antibody responses by neutralization assays revealed similar levels of neutralizing antibody titers between the HSV2-infected AS01-gE or AS01-gE / gI-vaccinated and unvaccinated HSV2-infected groups. This suggests that the gE or gE / gI vaccine candidates do not enhance natural neutralizing antibody responses, suggesting that the AS01-gE and AS01-gE / gI vaccine candidates do not induce neutralizing antibody responses. Finally, as expected, the AS01-gD2t formulation was able to induce higher levels of neutralizing antibody titers (11.69-fold increase) compared to the unvaccinated HSV2-infected group (Figures 20A and 20B).

[0409] AS01-formulated gE or gE / gI heterodimer protein shows therapeutic effects on the frequency of genital recurrent HSV2 lesions Female guinea pigs (n = 110) were infected with HSV2 MS strain (10 5Guinea pigs were infected intravaginally (Ivag) with 100 μL of AS01-adjuvanted recombinant HSV2 gE (pfu - 100 μL) on day 0 and randomized into four different groups. On days 20, 34, and 48 post-infection, three groups of guinea pigs were injected intramuscularly (im) with 500 μL of either AS01-adjuvanted recombinant HSV2 gE (20 μg / dose - n = 15 / group 1), AS01-adjuvanted recombinant HSV2 gE / gI (40 μg / dose - n = 16 / group 2), or AS01-adjuvanted recombinant HSV2 gD2t protein (20 μg / dose - n = 12 / group 3; positive control in terms of clinical therapeutic efficacy). Guinea pigs in the unvaccinated HSV2-infected group were injected with saline solution (150 mM NaCl) and used as a negative control (n = 17 / group 4). Clinical evaluation of genital HSV2 reactivation in guinea pigs (groups 1-4) was performed daily from days 20 to 70 using a scoring system to assess the severity of genital lesions at the level of the vulva. Vaccine efficacy was tested for the period beginning on the second vaccination day (day 34) and ending at the end of the study (day 70). Daily lesion scores (ranging from 0 to 4) for each individual animal were accumulated for this period (Figure 21). Individual accumulated scores were divided by the number of days in the period to provide a standardized cumulative score.

[0410] A positive correlation was observed between the baseline interval (0–14, prerandomization) and the scores accumulated between days 34 and 70 (Figure 22), indicating that guinea pigs exhibiting severe and frequent lesions before vaccination tended to exhibit similarly severe and frequent lesions after vaccination. The effect of vaccination on the standardized cumulative score between days 34 and 70 was examined, adjusting for this baseline. The results showed a clear therapeutic effect of vaccination for both the AS01-gE and AS01-gE / gI vaccine candidates in terms of clinical manifestations of genital herpes (Figures 23A and 23B). Compared with the unvaccinated HSV2-infected group, therapeutic immunization with AS01-gE, AS01-gE / gI, and AS01-gD2t significantly reduced the mean standardized cumulative lesion score between days 34 and 70 by 56%, 45%, and 53%, respectively (Figure 23C). No significant differences in cumulative lesion scores were observed among all vaccinated groups, which may suggest similar therapeutic efficacy of the AS01-gE and AS01-gE / gI vaccine candidates and the AS01-gD2t positive control group (Figure 24).

[0411] Because the standardized cumulative lesion score combined the frequency and severity of lesion days, the total number of genital lesion days over the [34-70] period was calculated for each group to assess the vaccine's ability to affect the duration and / or number of herpes reactivations. As expected, the frequency of lesion days was also significantly reduced in all vaccinated groups compared with unvaccinated groups (Figures 25A and 25B). Compared with unvaccinated HSV2-infected groups, the total number of lesion days was reduced by 47%, 37%, and 52% in the AS01-gE, AS01-gE / gI, and AS01-gD2t groups, respectively (Figure 25B). Furthermore, the number of reactivation episodes appeared to be lower in unvaccinated HSV2-infected guinea pigs compared with vaccinated groups (Figure 26). These data suggest that the HSV2 vaccine candidate can significantly reduce the duration and / or number of genital herpes reactivations in this guinea pig model.

[0412] The second vaccination dose was evaluated over the period [34-47] days, and the third over the period [48-70] days. The results show that for all tested vaccine candidates, the therapeutic effect of vaccination was observed already after the second vaccination dose. Compared with the unvaccinated HSV2-infected group, therapeutic immunization with AS01-gE, AS01-gE / gI, and AS01-gD2t significantly reduced the mean standardized cumulative lesion score by 51%, 48%, and 51%, respectively, over the period [34-47] days (Figure 27). Similar data were observed after the third immunization in all vaccinated groups. Indeed, compared with the unvaccinated HSV2-infected group, therapeutic immunization with AS01-gE, AS01-gE / gI, and AS01-gD2t significantly reduced the mean standardized cumulative lesion score by 61%, 44%, and 55%, respectively, over the period [48-70] days (Figure 27). This may indicate that the third vaccination dose still influences the therapeutic effect of the vaccine.

[0413] [Example 3] HSV1 and HSV2 gEgI mutants Design of HSV1 and HSV2 gE mutants to prevent or limit the ability of gE to bind to the IgG Fc domain Peptide insertion mutants - HSV1 gE peptide insertion mutants that result in loss of gE Fc binding function while retaining the gE / gI complex are known from Polcicova K. et al., The extracellular domain of Herpes simplex virus gE is indispensable for efficient cell to cell spread: Evidence for gE / gI receptors. 2005. J. Virol., Vol 79(18), pp11990-12001. Suitable peptide insertion mutations in gE from HSV1 strain KOS321 (UniProtKB accession number Q703E9) include the following: LDIGE inserted between amino acid residues Y277 and E278 of SEQ ID NO: 3 (277_insert_LDIGE), ADIGL inserted between amino acid residues S291 and P292 of SEQ ID NO: 3 (291_insert_ADIGL), an ARAA inserted between amino acid residues A339 and A340 of SEQ ID NO: 3 (339_inset_ARAA), an ARAA inserted between amino acid residues A340 and S341 of SEQ ID NO: 3 (340_inset_ARAA); and ADIT inserted between amino acid residues D348 and A349 of SEQ ID NO: 3 (348_insert_ADIT).

[0414] The first approach for the generation of HSV2 gE mutants was based on the insertion of peptides after the corresponding residues in HSV2 gE based on the alignment shown in Figure 1: LDIGE inserted between amino acid residues Y275 and E276 of SEQ ID NO: 1 (275_insert_LDIGE), ADIGL inserted between amino acid residues S289 and P290 of SEQ ID NO: 1 (289_insert ADIGL), an ARAA inserted between amino acid residues A337 and S338 of SEQ ID NO: 1 (337_insert_ARAA), an ARAA inserted between amino acid residues S338 and T339 of SEQ ID NO: 1 (338_insert_ARAA), and an ADIT inserted between amino acid residues H346 and A347 of SEQ ID NO: 1 (346_insert_ADIT).

[0415] A single point mutation—the histidine residue at position 435 of human IgG (hIgG)—has been identified as essential for the binding of the hIgG Fc domain to the HSV1 gEgI complex (Chapman TL et al., Characterization of the interaction between the Herpes simplex virus type I Fc receptor and immunoglobulin G. 1999. JBC., Vol. 274 (11), pp. 6911-6919). Using the crystal structure of the HSV-1 gEgI / Fc complex (PDB 2GJ7) and MOE (Molecular Operating Environment) software (Chemical Computing Group), three positions (H247, P319, and P321 in SEQ ID NO: 3) in the HSV1 gE FcR within the area where binding to hIgG residue H435 occurs were identified, which may affect gE binding to the hIgG Fc domain while preserving the overall folding of gE. Based on the alignment shown in Figure 1, these positions correspond to residues H245, P317 and P319 of the HSV2 gE sequence shown in SEQ ID NO:1.

[0416] Eight HSV1 gE single point mutants (H247A, H247K, P319R, P321A, P321R, P321G, P321K, and P321T) and five HSV1 gE double point mutants (H247A-P321A, H247A-P321R, H247A-P321G, H247A-P321K, and H247A-P321T) were computationally validated to have no effect on gE stability but a negative effect on the gE / Fc binding interface. The corresponding HSV2 gE single point mutants (H245A, H245K, P317R, P319A, P319R, P319G, P319K, and P319T) and double point mutants (H245A-P319A, H245A-P319R, H245A-P319G, H245A-P319K, and H245A-P319T) were also designed.

[0417] The gE / Fc interface (32 positions identified) was identified using the crystal structure of the HSV-1 gEgI / Fc complex (PDB 2GJ7) and the PDBePISA website (http: / / www.ebi.ac.uk / msd-srv / prot_int / pistart.html). Structural analysis was performed using Rosetta macromolecular modeling software (http: / / www.rosettacommons.org), keeping only positions not involved in the 2D structure and with minimal potential impact on gE folding. Six single point mutations (A339G, P321D, P321S, A340D, N243A, and R322D) and two double point mutations (N243A-R322D and N243A-P321D) were computationally verified (computed using Rosetta) to have no negative effect on the overall gE fold or on the gE-Fc interface. Corresponding HSV2 gE single point mutations (A337G, P319D, P319S, S338D, N241A, and R320D) and double point mutations (N241A / R320D and N241A / P319D) were also designed.

[0418] The HSV-2 gE protein (alone or complexed with IgG Fc) was modeled using MOE software, see Figure 28. The mutants identified above (using the crystal structure of the HSV1 gEgI / Fc complex) were validated in silico with this new model.

[0419] A thorough analysis of the HSV-2 gE / Fc binding interface was performed using MOE, and a novel position for mutation was identified as interesting due to its potential interaction with one of the three loops identified in Fc as involved in gE binding (the Fc loop). Exhaustive mutational scanning (using the residue scanning tool in MOE) was performed at all previously identified and newly identified positions, and the following 77 additional single point mutations were computationally validated as having no effect on gE stability and a negative effect on the gE / Fc binding interface: H245E, H245V, H245R, H245D, H245Q, H245G, H245I, H245K, H245S, H245T, A246W, A248K, A248T, A248G, R314A, R314N, R314D, R314Q, R314E, R314G, R314I, R314L, R314K, R314M, R314F, R314P, R314S , R314T, R314Y, R314V, P317N, P317G, P317I, P317L, P317K, P317F, P317S, P318R, P318D, P 318Q, P318I, P318S, P318T, P318Y, P319L, R320A, R320S, R320N, R320Q, R320E, R320G, R32 0H, R320I, R320L, R320M, R320P, R320T, R320V, F322A, F322N, F322I, F322K, F322P, F322T, S338G, S338E, S338L, S338T, V340A, V340R, V340D, V340Q, V340M, V340F, V340P and V340W.

[0420] Amino acid positions affecting at least two Fc loops were selected for the design of double mutants: A246 / P317, A246 / R320, A248 / V340, A248 / F322, H245 / R320, H245 / P319, R314 / P318, R314 / V340, R314 / F322, P317 / V340, P317 / S338, P317 / F322, P318 / S338, and P319 / V340.

[0421] The following additional double point mutations were computationally validated as having no effect on gE stability and a negative effect on the gE / Fc binding interface: A246W / P317K; A246W / P317F; A246W / P317S; A246W / R320D; A246W / R320G; A246W / R320T; A248K / V340R; A248K / V340M; A248K / V340W; A248T / V340R; A248T / V340M; A248T / V340W; A248G / V340R; A248G / V340M; A248G / V340W; A 248K / F322A;A248K / F322I;A248K / F322P;A248T / F322A;A248T / F322I;A24 8T / F322P;A248G / F322A;A248G / F322I;A248G / F322P;H245A / R320D;H245A / R320G;H245A / R320T;H245G / R320D;H245G / R320G;H245G / R320T;H245S / R 320D;H245S / R320G;H245S / R320T;H245A / P319G;H245A / P319L;H245G / P31 9G;H245G / P319L;H245S / P319G;H245S / P319L;R314G / P318R;R314G / P318 D;R314G / P318I;R314L / P318R;R314L / P318D;R314L / P318I;R314P / P318R; R314P / P318D;R314P / P318I;R314G / F322A;R314G / F322I;R314G / F322P;R3 14L / F322A;R314L / F322I;R314L / F322P;R314P / F322A;R314P / F322I;R314 P / F322P;R314G / V340R;R314G / V340M;R314G / V340W;R314L / V340R;R314L / V340M;R314L / V340W;R314P / V340R;R314P / V340M;R314P / V340W;P317K / V3 40R;P317K / V340M;P317K / V340W;P317F / V340R;P317F / V340M;P317F / V340 W;P317S / V340R;P317S / V340M;P317S / V340W;P317K / S338G;P317K / S338H;P317K / S338L;P317F / S338G;P317F / S338H;P317F / S338L;P317S / S338G;P317S / S338H;P317S / S338L;P318R / S338G;P318R / S338H;P318R / S338L;P318D / S338G;P 318D / S338H;P318D / S338L;P318I / S338G;P318I / S338H;P318I / S338L;P319G / V340R;P319G / V340M;P319G / V340W;P319L / V340R;P319L / V340M;and P319L / V340W. ;

[0422] Recombinant expression of HSV2 gEgI mutants Cloning - The genes listed in Table 12 were codon-optimized for human protein expression, synthesized, and cloned into the pmaxCloning™ vector by GENEWIZ (Lonza, catalog VDC-1040) using EcoRI / NotI restriction sites. The pmaxCloning™ vector backbone contains the cytomegalovirus immediate early promoter (PCMV IE) for protein expression, a chimeric intron for enhanced gene expression, and a pUC origin of replication for propagation in Escherichia coli (E. coli). A bacterial promoter (P) provides kanamycin resistance gene expression in E. coli. A multiple cloning site (MCS) is located between the CMV promoter and the SV40 polyadenylation signal (SV40 polyA).

[0423] Each construct contained a sequence encoding an HSV2 gE ectodomain (SEQ ID NO: 7) with mutations as shown in Table 12 and a sequence encoding an HSV2 gI ectodomain (SEQ ID NO: 8) separated by an IRES sequence. All constructs contained a 6xHis-tag at the C-terminus of the gI ectodomain.

[0424] [Table 12]

[0425] Recombinant Protein Expression—Expi293F™ cells (ThermoFisher, catalog A14528) were used for recombinant protein expression. Cell culture and transfection were performed according to the manufacturer's instructions. Briefly, the day before transfection, cell density and viability were assessed using a TC20™ automated cell counting instrument (Bio-Rad). Cells were seeded at 2 x 10 in fresh, pre-warmed Expi293™ Expression Medium (ThermoFisher, catalog A1435102). 6 Cells were seeded at a density of 10 cells / mL and cultured in a humidified 8% CO2 incubator at 37°C / 110 rpm. On the day of transfection, cell density and viability were assessed (viability ≥ 95%) and cells were transfected at 3 x 10 6 The cells were diluted to a final density of 100 cells / mL. Transfection was performed using the ExpiFectamine™ 293 Transfection Kit (Thermofisher, catalog A14524), which includes a transfection enhancer and ExpiFectamine 293 transfection reagent. Briefly, the plasmid DNA and transfection reagent were separately diluted in OptiMEM medium (Thermofisher, catalog 31985062) and incubated at RT for 5 minutes (1 μg of plasmid DNA was used per mL of cell culture). Both mixtures were then combined and incubated at RT for an additional 20 minutes. The ExpiFectamine™ 293 and plasmid DNA complex solution was carefully added to the cells. The cells were cultured at 37°C / 110 rpm in a humidified 8% CO2 incubator. One day post-transfection (18-22 hours post-transfection), half a volume of ExpiFectamine™ 293 Transfection Enhancer was added. Four days after transfection, cells were harvested by centrifugation at 4°C / 5000 x g for 10 min (cell viability ranged between 45 and 75% for different candidates). The cell pellet was discarded, and cOmplete™ protease inhibitor cocktail (Roche, catalog 11697498001) was added to the supernatant.

[0426] Analysis of Protein Expression by SDS-PAGE and Western Blot—To assess protein expression levels at harvest, cell culture supernatants were analyzed by SDS-PAGE and Western blot. Supernatant samples were mixed (1:3) with NuPAGE™ LDS Sample Buffer (4x)-1M DTT (Invitrogen™, catalog NP0007) and incubated at 95°C for 5 minutes. 10 μL of each sample was loaded onto a 4-20% Criterion™ TGX Stain-Free™ Protein Gel (Bio-Rad, catalog 567-8094). Gels were run at 250V for 25 minutes in TGS (Trys-Glycine-SDS) running buffer. For Western blot analysis, a 1 / 2000 dilution of mouse monoclonal anti-polyhistidine peroxidase antibody (Sigma, catalog A7058-1VL) was used, followed by development using 1-Step™ Ultra TMB-blotting solution (ThermoFisher, catalog 37574). SDS-PAGE image acquisition was performed using the Gel Doc™ EZ Gel system (Bio-Rad) using stain-free technology. Western blot images were acquired with an Amersham™ Imager 600 (GE Healthcare, Life Sciences). See Figure 29. The Western blot pattern of the band of interest (gI, as it is a protein containing a 6xHis-tag) corresponds to that of a heterogeneously glycosylated protein. The MW of gE and gI are 45.5 kDa and 27 kDa, respectively. Protein N-glycosylation predictions by NetNGlyc 1.0 are two sites for gE and four sites for gI. For O-glycosylation, predictions using NetOGlyc 4.0 give 12 sites for gE and 20 sites for gI.

[0427] Purification of HSV2 gEgI mutants The culture was centrifuged at 5000 g for 10 min at 4°C. The supernatant was collected and passed through a 0.22 μM filter (Sartorius) after the addition of 20 mM bicine pH 8.3 / 0.2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (Sigma). The protein was then purified by immobilized metal affinity chromatography (IMAC) followed by size-exclusion chromatography (SEC).

[0428] Purification of mutants expressing HTP (2.5 ml cultures in a 24-deep-well format) was performed using Phytips (PhyNexus) or Thompson filter plates. Culture supernatants were supplemented with 0.2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) (Sigma) and 20 mM bicine, pH 8.3. Phytips containing 80 μl of Nickel Sepharose Excel (GE) was equilibrated with Buffer A (20 mM bicine, 500 mM NaCl, 20 mM imidazole, pH 8.3). The culture supernatant was then aspirated and dispensed into the Phytips to capture the target protein. After capture, the Phytips was washed with Buffer A, and the protein was eluted with 300 μL of Buffer B (20 mM bicine, 500 mM NaCl, 500 mM imidazole, pH 8.3). For filter plate purification, 200 μl of Nickel Sepharose Excel (GE) slurry pre-equilibrated with buffer A (20 mM bicine, 500 mM NaCl, 20 mM imidazole, pH 8.3) was added to the culture supernatant. After 90 min of shaking at 900 rpm, the sample was transferred to a 96 DW Thompson filter plate and washed three times with 1 ml of buffer A under negative pressure. Proteins were eluted with two 110 μl portions of buffer B (20 mM bicine, 500 mM NaCl, 500 mM imidazole, pH 8.3) by centrifugation (800 g for 10 min) and desalted on a PD multitrap G-25 column. Proteins were analyzed by SDS-PAGE and SEC (Superdex 200 Increase 5 / 150 (GE) or BEH200 (Waters)).

[0429] Alternatively, purification of mutants from small-scale expression (100 ml culture) was performed using a gravity-flow column packed with 3 ml of Nickel Sepharose Excel (GE) pre-equilibrated with buffer A (20 mM bicine, 500 mM NaCl, 20 mM imidazole, pH 8.3). After loading the sample, the resin was washed with 15 CV of buffer A, and the protein was eluted with 5 CV of buffer B (20 mM bicine, 500 mM NaCl, 500 mM imidazole, pH 8.3). The protein was then concentrated using a Vivaspin 20 column with a 10 kDa cutoff at 3000 g at 4 °C. The concentrated sample was loaded onto a Superdex 200 increase 10 / 300 (GE) column equilibrated with buffer C (20 mM bicine, 150 mM NaCl, pH 8.3) at a flow rate of 0.75 ml / min. Fractions corresponding to the protein of interest were pooled together, 0.22 μM filtered, and stored at −80°C.

[0430] Purification of wild-type and mutant strains from large-scale expression (1-2 L culture) was performed using an AKTA FPLC chromatography system (GE) with an XK16 / 20 column packed with 20 ml of Nickel Sepharose Excel (GE) pre-equilibrated with buffer A (20 mM bicine, 500 mM NaCl, 20 mM imidazole, pH 8.3). The supernatant was loaded onto the column at a flow rate of 12 ml / min. The resin was washed with 15 CV of buffer A, and the protein was eluted with 10 CV of buffer B (20 mM bicine, 500 mM NaCl, 500 mM imidazole, pH 8.3) at a flow rate of 12 ml / min. The protein was then concentrated using a Vivaspin 20 column with a 10 kDa cutoff at 3000 g at 4 °C. The concentrated sample was loaded onto a HiLoad 26 / 600 Superdex 200pg (GE) column equilibrated with Buffer C (20mM bicine, 150mM NaCl, pH 8.3) at a flow rate of 2.6ml / min. Fractions corresponding to the protein of interest were pooled together, 0.22µM filtered, and stored at -80°C.

[0431] Protein concentration was determined by RCDC assay (Biorad) and purity by SDS PAGE.

[0432] All proteins, except HSV39, were purified as monodisperse samples (Figure 30 and Table 13). Aggregation and yield of purified proteins varied among mutants (Table 13).

[0433] [Table 13]

[0434] Biophysical characterization of HSV-2 gEgI functional knockout mutants Materials and Methods Recombinant Mutant gEgI Proteins - Unless otherwise stated, purified proteins were maintained in 20 mM bicine pH 8.3 150 mM NaCl.

[0435] Reagents and consumables - Human IgG isotype control (ThermoFischer Scientific, ref. 12000C), kinetic buffer (Pall Fortebio, ref. 18-1105), Prometheus NT.Plex nanoDSF grade high sensitivity capillary tip (Nanotemper technologies, ref. PR-AC006), Octet Red Dip&Read Ni-NTA biosensor (Pall Fortebio, ref. 18-5101)

[0436] Experimental procedure Biolayer Interferometry—An Octet Red instrument (Pall-ForteBio, Menlo Park, USA) was used for all IgG binding measurements. All measurements were performed in Kinetic Buffer (KB) (Pall-ForteBio, Menlo Park, USA) 5x, and constant stirring at 1000 rpm was maintained throughout. Mutant proteins were prepared at a fixed concentration of 50 μg / ml in KB 5x and immobilized on a Ni-NTA sensor chip for 120 seconds. Unbound ligand was washed off by incubating the sensor chip in buffer solution for 60 seconds. Binding to IgG was monitored during a 300-second immersion in a 100 μg / ml IgG KB 5x solution. Dissociation was monitored during a 400-second immersion in KB 5x buffer.

[0437] The NanoDSF-protein solution was loaded into a glass capillary and subjected to a linear heating process (20-95 °C at 1 °C / min) in a NanoDSF NT-Plex instrument (Nanotemper Technologies, Munich, Germany). The fluorescence intensity at 330 nm was constantly recorded during the heating process. The first derivative of the fluorescence intensity plotted against temperature was used to determine the melting temperature, Tm.

[0438] Experimental results BLI (BioLayer Interferometry, Pall ForteBio) was used to record the IgG-binding properties of 25 mutant proteins expressed at small scale in HEK cells versus a WT protein control. Proteins were immobilized on Ni-NTA-functionalized sensor chips in an Octet Red BLI system. After washing away unbound material, proteins were incubated in a human IgG solution for a determined period, and the binding kinetics were recorded. The sensor chip was then removed from the IgG solution and placed in buffer, and the dissociation of IgG from the gEgI construct was recorded (see Table 14, Figure 31).

[0439] [Table 14]

[0440] Constructs HSV41, 45, 49, 57, 61 were chosen because they segregated into regions of the graph corresponding to slower binders and faster releasers compared to the control (see Figure 31). HSV44 also exhibits high k off These constructs were selected for their relative affinity. The relative affinities of these six constructs ranged from 1% to 17% of that of the control. These six constructs were expressed on a larger scale and characterized to confirm their biophysical properties. BLI analysis suggested that all, except for HSV44, exhibited significantly altered IgG binding behavior (Figure 32). The six constructs were then analyzed by dynamic scanning fluorometry (using intrinsic Trp fluorescence) (Table 15). The protein melting temperatures were determined and compared with the WT control. The data suggested a slight decrease in the melting temperature of the mutants. This shifted Tm suggests a slightly less stable protein folding than that of the WT, but the shift was not significant enough to indicate a major destabilization of protein folding. Therefore, the six constructs are considered to have sufficient folding stability for further use in preclinical studies.

[0441] [Table 15]

[0442] Further HSV-2 gEgI mutations Based on the characterization results of the 25 HSV-2 gEgI mutants reported above, the inventors considered that among the further mutations designed and described above, the following may reduce the ability of gE to bind to the IgG Fc domain (all positions are with respect to the sequence shown in SEQ ID NO: 1): P317K, R320N, R320S, R320E, R320G, R320D / H245S, R320D / H245G, R320D / H245A, P317K / V340M, P317K / V340R, P317K / S338G, P319G / H245A, P319G / H245S, P319G / V340R, P319G / V340M, P318R, H245G, H245S, R320G / H245A, R320G / H245G, R320G / H245S, R320T / H245A, R320T / H245G, R320T / H245S, P318R / R314G and P318R / S338G.

[0443] Based on these results, the inventors also considered that the following additional HSV2 gE mutations might also be suitable to reduce the ability of gE to bind to the IgG Fc domain (all positions are with respect to the sequence shown in SEQ ID NO: 1): - P317R / P319D, - P317R / R320D, - P319D / R320D, - deletion of amino acid residue P319, - deletion of amino acid residue R320, - deletion of amino acid residues P319 and R320, - deletion of amino acid residues P319 and R320 and point mutations P317G and P318G, - deletion of amino acid residues P319 and R320 and the point mutation P318E, - deletion of amino acid residues P319 and R320 and the point mutation P318G, - deletion of amino acid residues P319 and R320 and the point mutation P318K, - deletion of amino acid residues P319 and R320 and point mutations P317R and P318E, - deletion of amino acid residues P319 and R320 and point mutations P317R and P318G, - deletion of amino acid residues P319 and R320 and point mutations P317R and P318K.

[0444] [Example 4] Expression, purification, and biophysical characterization of HSV2 and HSV1 gEgI mutants Materials and Methods Recombinant Protein Expression - For recombinant protein expression, Expi293F™ cells (ThermoFisher, catalog A14528) and ExpiCHO-S™ (ThermoFisher, catalog A29127) expression system were used.

[0445] ·Expi293F(trademark) cells Cell culture and transfection were performed according to the manufacturer's instructions. For small-scale expression, 0.5 ml was transfected in deep wells, and for medium-scale production (ranging from 30 ml to 1 L), culture was performed in shake flasks with the appropriate volume as recommended by the manufacturer's instructions. Briefly, for Expi293-F™ cells, cell density and viability were assessed the day before transfection using a TC20™ automated cell counter (Bio-Rad). Cells were seeded at 2 × 10 in fresh, pre-warmed Expi293™ Expression Medium (ThermoFisher, catalog A1435102). 6 Cells were seeded at a density of 10 cells / mL and cultured in a humidified 8% CO2 incubator at 37°C / 110 rpm. On the day of transfection, cell density and viability were assessed (viability ≥ 95%) and cells were transfected at 3 x 10 6The cells were diluted to a final density of 100 cells / mL. Transfection was performed using the ExpiFectamine™ 293 Transfection Kit (Thermofisher, catalog A14524), which contains the transfection enhancer and ExpiFectamine 293 transfection reagent. Briefly, the plasmid DNA and transfection reagent were separately diluted in OptiMEM medium (Thermofisher, catalog 31985062) and incubated at RT for 5 minutes (1 μg of plasmid DNA was used per mL of cell culture). The combined mixture was then incubated at RT for an additional 20 minutes. The ExpiFectamine™ 293 and plasmid DNA complex solution was carefully added to the cells. The cells were cultured at 37°C / 110 rpm in a humidified 8% CO2 incubator. One day post-transfection (18-22 hours post-transfection), half a volume of ExpiFectamine™ 293 Transfection Enhancer was added. Four days after transfection, cells were harvested by centrifugation at 4°C / 5000 x g for 10 min (cell viability ranged between 45 and 75% for different candidates). The cell pellet was discarded, and cOmplete™ protease inhibitor cocktail (Roche, catalog 11697498001) was added to the supernatant.

[0446] ·ExpiCHO-S(TM) cells Cell culture and transfection were performed according to the manufacturer's instructions. Briefly, ExpiCHO-S™ cells were assessed for cell density and viability the day before transfection using a TC20™ automated cell counter (Bio-Rad). Cells were seeded at 3-4 x 10 in fresh, pre-warmed ExpiCHO™ Expression Medium (ThermoFisher, catalog A2910001). 6 Cells were seeded at a density of 10 cells / mL and cultured in a humidified 8% CO2 incubator at 37°C / 110 rpm. On the day of transfection, cell density and viability were assessed (viability ≥ 95%) and cells were transfected at 6 x 106 The cells were diluted to a final density of 100 cells / mL. Transfection was performed using the ExpiFectamine™ CHO Transfection Kit (Thermofisher, catalog A29129), which contains transfection enhancer and ExpiFectamine CHO transfection reagent. Briefly, the plasmid DNA and transfection reagent were diluted separately in cold OptiPRO™ medium (Thermofisher, catalog 12309-050) and incubated at RT for no more than 5 minutes (0.8 μg of plasmid DNA was used per mL of cell culture). The combined mixture was then incubated at RT for an additional 1-5 minutes. The ExpiFectamine™ CHO and plasmid DNA complex solution was carefully added to the cells. The cells were cultured at 37°C / 110 rpm in a humidified 8% CO2 incubator. One day after transfection (18-22 hours after transfection), ExpiFectamine™ CHO transfection enhancer and ExpiCHO™ Feed were added. Six days after transfection, cells were harvested by centrifugation at 4°C / 4000-5000 x g for 30 minutes (cell viability ranged between 40-80% for different candidates). The cell pellet was discarded, and cOmplete™ protease inhibitor cocktail (Roche, catalog 11697498001) was added to the supernatant.

[0447] Analysis of Protein Expression by SDS-PAGE and Western Blot—To assess protein expression levels at harvest, cell culture supernatants were analyzed by SDS-PAGE and Western blot. Supernatant samples were mixed (1:3) with NuPAGE™ LDS Sample Buffer (4x)-1M DTT (Invitrogen™, catalog NP0007) and incubated at 95°C for 5 minutes. 10 μL of each sample was loaded onto a 4-20% Criterion™ TGX Stain-Free™ Protein Gel (Bio-Rad, catalog 567-8094). Gels were run at 250V for 25 minutes in TGS (Trys-Glycine-SDS) running buffer. For Western blot analysis, a mouse monoclonal anti-polyhistidine peroxidase antibody (Sigma, catalog A7058-1VL) was used at a 1 / 2000 dilution, followed by development using 1-Step™ Ultra TMB-blotting solution (ThermoFisher, catalog 37574). SDS-PAGE image acquisition was performed using the Gel Doc™ EZ Gel system (Bio-Rad) using stain-free technology. Western blot images were acquired with an Amersham™ Imager 600 (GE Healthcare, Life Sciences). The Western blot pattern of the band of interest (gI, as it is a protein containing a 6xHis-tag) corresponds to that of a heterogeneously glycosylated protein. The MW of gE and gI are 45.5 kDa and 27 kDa, respectively. Protein N-glycosylation predicted sites by NetNGlyc 1.0 are 2 sites for gE and 4 sites for gI. For O-glycosylation, predictions using NetOGlyc 4.0 indicate 12 sites for gE and 20 sites for gI.

[0448] Purification of HSV2 gEgI mutants - see Example 3

[0449] Biolayer Interferometry with Octet - Ni-NTA sensors (Pall, #18-5101) were pre-wetted by incubation in 1x kinetic buffer (Pall, #18-1105) for at least 30 minutes at room temperature before starting measurements using an Octet Red 96e (Pall) instrument. All samples, standards, and controls were diluted to a final volume of 200 μL with 1x kinetic buffer in the wells of a Greiner black 96-w microplate (Greiner, #655076). All measurements were performed at 30°C, and the microplate containing the test samples was maintained under constant 1000 rpm shaking. The Octet uses biosensors in the form of disposable chips, while the Octet Red 96e reads eight chips in parallel. After measurements, the sensors were replaced. The workflow was as follows:

[0450] JPEG2025163028000018.jpg54165

[0451] The analytical software Data Analysis HT version 10.0.3.7 was used to review the experimental data and calculate the analyte content. For ranking purposes, only the response (binding signal at the end of the association period) was used. Affinity values ​​(KD) were generated but were deemed inaccurate given the low signal intensities of the selected candidates.

[0452] The melting profile of the sample was determined using the Nano-DSF-Prometheus NT.Plex instrument (NanoTemper Technologies) by using the intrinsic fluorescence from tryptophan residues. A 10 μL sample was filled into the test capillary and placed on the sample holder. A 1°C / min temperature gradient from 25 to 95°C was applied, and intrinsic protein fluorescence was recorded at 330 and 350 nm. Scattered light was also detected at 350 nm. After the measurement was completed, Tm (temperature of melting) and Ton (onset of melting transition) were automatically determined by calculating the first derivative of the experimental signal. Because it was observed that this readout correlated best with DSF, only the 330 nm signal was used for calculation in this experiment.

[0453] DSF (Dynamic Scanning Fluorescence) - DSF is similar to nanoDSF except that the methodology uses the exogenous dye Sypro Orange. Upon heating the protein sample, the dye, initially buried internal hydrophobic patches, become exposed to the solvent and become fluorescent.

[0454] Sypro Orange (5000x concentrated in DMSO, Thermo) was added to the protein sample (final concentration 2x), and the sample was then subjected to a temperature gradient (ambient to 95°C, 1°C per minute) in a LightCycler 480II (Roche) instrument. Fluorescence (excitation 498 nm, emission 630 nm) was constantly recorded during heating. The second derivative of the fluorescence signal allowed the determination of the Tm.

[0455] DLS (Dynamic Light Scattering) - DLS uses the temporal pattern of fluctuations in light scattered from a protein solution to infer the size distribution of protein particles in a sample. This technique is extremely sensitive to the presence of aggregates and can be used to assess aggregate formation during stress tests.

[0456] Protein solutions were analyzed on a Wyatt DynaPro II instrument and the raw data were converted to particle size distributions by using the software Dynamics (Wyatt).

[0457] The chromatographic system used for the UPLC-SEC-UV measurements was an Agilent 1290 Infinity II instrument equipped with a quaternary pump and a DAD detector. Proteins were injected onto an analytical SEC column (Waters BEH200, 150 x 4.6 mm) equipped with a 50 mm precolumn. The column was eluted (isocratic mode) with a 20 mM bicine pH 8.3 150 mM NaCl mobile phase at 0.3 ml / min at a temperature of 30 °C. The run length was 10 min. The elution profile was established by constant recording of UV at 280 nm.

[0458] result HSV2 gEgI mutant One hundred and seventy-five gEgI mutant HSV2 gEgI constructs were generated and purified. Each construct contained a sequence encoding an HSV2 gE ectodomain (SEQ ID NO:7) with mutations as shown in Table 16 and a sequence encoding an HSV2 gI ectodomain (SEQ ID NO:8) separated by an IRES sequence. All constructs contained a 6xHis tag at the C-terminus of the gI ectodomain.

[0459] After purification, all samples were analyzed by Octet to record the remaining bioactivity of human IgG binding. The mutation-free gEgI (HRV4) and the P317R mutant (HSV45) tested in Example 3 were used as controls. The BLI data and protein concentrations after purification are presented in Table 16.

[0460] [Table 16] JPEG2025163028000020.jpg247167JPEG2025163028000021.jpg249168JPEG2025163028 000022.jpg249168JPEG2025163028000023.jpg249168JPEG2025163028000024.jpg41169

[0461] 48 of the constructs were then analyzed by nanoDSF to assess the conservation of the protein signature observed in the template protein, HSV2 WT. Most of the constructs exhibited Tm values ​​around 67°C, with only a few constructs with Tm below 65°C suggesting conformational changes. Notably, all positive controls inserted into the sample set exhibited highly reproducible Tm (Table 17).

[0462] [Table 17] JPEG2025163028000026.jpg79170

[0463] Ten constructs, among those displaying high yield and low human IgG binding and / or high Tm, were produced in large quantities. The constructs were subjected to further characterization to assess their structural quality. Octet was used to probe the binding of a conformational monoclonal antibody that binds to the gEgI heterodimer and the gE Fc-binding domain. DLS was used in conjunction with stress testing to assess the colloidal stability of the leads. A combined view of the dataset is presented in Table 18.

[0464] [Table 18]

[0465] Additional HSV2 gEgI mutants were generated and purified. Each construct contained a sequence encoding an HSV2 gE ectodomain (SEQ ID NO:7) with mutations as shown in Table 19 and a sequence encoding an HSV2 gI ectodomain (SEQ ID NO:8) separated by an IRES sequence. All constructs contained a 6xHis tag at the C-terminus of the gI ectodomain.

[0466] [Table 19]

[0467] After high-throughput small-scale expression, proteins were purified using two different modalities: either proteins were extracted by Phy-tips (pipette tips containing small volumes of resin with IMAC functionality) or using filter plates with similar IMAC capabilities.

[0468] To determine the protein content, proteins were analyzed by UPLC-SEC-UV. Specifically, proteins were separated into aggregates and monomers. Protein content was based on the observed peak area of ​​the monomer. It was observed that using filter plates instead of Phy-tips allowed for the extraction of more protein from the expression supernatant, as the calculated protein content was on average between 4- and 5-fold higher compared to Phy-Tips (Figure 33). Therefore, all characterization measurements were parallel between filter plates and Phy-tips, and only filter plate data was considered later.

[0469] The effect of the mutations on the ability of the construct to bind human IgG was then assessed by BLI (Octet). The relative binding response of immobilized gEgI protein to human IgG was measured (Figure 34).

[0470] To further evaluate the mutant candidates, nanoDSF was performed to measure protein folding stability. Fluorescence at 330 nm was considered the primary readout because previous experiments showed that this wavelength correlated with other methodologies, such as dye-based DSF. PN94, PN95, and PN100 exhibited the lowest Tm values, suggesting less stable folding relative to the other proteins (Figure 35).

[0471] The information collected for each construct (protein content, BLI response, nanoDSF) is summarized in Table 20.

[0472] [Table 20]

[0473] HSV1 gEgI mutant Thirty-two HSV1 mutant gEgI constructs were generated as described above for the HSV2 gEgI constructs. Each construct contained a sequence encoding an HSV1 gE ectodomain (SEQ ID NO:9) with mutations as shown in Table 21 and a sequence encoding an HSV2 gI ectodomain (SEQ ID NO:10) separated by an IRES sequence. All constructs contained a 6xHis tag at the C-terminus of the gI ectodomain.

[0474] [Table 21]

[0475] At the end of the purification scheme, protein concentration was determined by colorimetric method (see Figure 36).

[0476] After purification, all samples were analyzed by Octet to document the remaining bioactivity of human IgG binding. DSF was then used to further explore the folding quality of the mutant HSV1 gEgI constructs (Figure 37).

[0477] [Example 5] Bicistronic SAM vector for expression of HSV gEgI heterodimer Materials and Methods SAM characterization RNA gel electrophoresis RNA samples were analyzed on a 1% agarose gel. RNA samples were prepared as follows: 100-500 ng of RNA was mixed with 3 μL of loading buffer (50 mM EDTA pH 8, 30% sucrose (w / v), 0.05% bromophenol blue) and water to a final volume of 10 μL. Samples were denatured at 50°C for 20 minutes. Agarose gels were run at 130 V for 45 minutes in Northern Max Gly gel running buffer (Invitrogen™).

[0478] Protein Expression Analysis: Western Blot On day 0, baby hamster kidney (BHK) cells were cultured in a T225 flask at 1 x 10 in Growth Medium (DMEM High Glucose (Gibco™), 1% L-glutamine, 1% Pen-Strep (Corning®), 5% FBS (Gibco™)). 7 The cells were seeded at 1000 x g for 1 hour. For trypsinization, the medium was removed and the cells were washed with 5 mL of PBS. The PBS wash was removed, and 5 mL of pre-warmed trypsin (Gibco™) was added and thoroughly spread throughout the plate. The trypsin was removed, and the plate was kept at 37°C for 1-2 minutes. The cells were then resuspended in 10 mL of growth medium. The cells were counted and seeded at the required concentration into a new flask. The cells were then incubated at 37°C, 5% CO2 for approximately 20 hours.

[0479] On day 1, plates were prepared by adding 2 mL of growth medium (DMEM high glucose, 1% L-glutamine, 1% Pen-Strep, 1% FBS) to each well of a 6-well plate (one well per electroporation). The plates were incubated in a 37°C incubator. The electroporation device (BIO-RAD Gene Pulser Xcell) was set to deliver 120 V, 25 ms pulses, 0.0 pulse interval, and 1 pulse for a 2 mm cuvette. The cuvettes were labeled and kept on ice.

[0480] The proliferating cells were collected in BHK growth medium and counted using a cell counting instrument. The cells were trypsinized according to the same trypsinization protocol as above. The cells were then centrifuged at 462 x g for 3 minutes. The medium was aspirated, and the cells were washed once with 20 mL of cold Opti-MEM medium (Gibco™). The cells were centrifuged again at 462 x g for 5 minutes. The medium was aspirated, and the cells were counted at 1 x 10 per electroporation. 6 The cells were resuspended in Opti-MEM medium at a volume of 0.25 mL per cell. Standard and negative control electroporations were also prepared.

[0481] For each sample, 0.1 or 2 μg of RNA was mixed with 250 μL of cells, and the mixture was gently pipetted 4–5 times. The cell and RNA mixture was transferred to a 2 mm cuvette and subjected to one pulse of electroporation using the parameters described above. The cells were allowed to sit at room temperature for 10 minutes. Cells from one cuvette were added to one well of a pre-warmed 6-well plate, and the plate was tilted back and forth, then left and right at a 45° angle to distribute the cells evenly. On day 2 (17 h after electroporation), cell culture supernatants were collected, concentrated 10-fold, and treated with PNGase (NEB) according to the manufacturer's instructions to deglycosylate proteins. The supernatants were analyzed by Western blot at different concentrations. Primary rabbit anti-gE and anti-gI and mouse anti-HA antibodies were used at a 1:1000 dilution, and mouse / rabbit anti-actin was used at a 1:5000 dilution. Secondary Licor antibody was used at 1:15000.

[0482] result The SAM vector VEEV TC-83, as described in WO 2005 / 113782, was used as a background construct for cloning the gEgI heterodimer. This SAM vector contains, from 5' to 3', non-coding sequences, sequences encoding viral nonstructural proteins 1 to 4 (nsP1-4), a subgenomic promoter, a gE ectodomain, regulatory elements, an insertion site containing the construct encoding the gI ectodomain, non-coding sequences, and a poly(A) tail. DNA encoding an empty SAM is shown in SEQ ID NO: 130 and Figure 39, and the corresponding empty SAM is shown in SEQ ID NO: 134. The insertion site is immediately after nucleotide 7561. The gE and gI coding sequences were codon-optimized. An exemplary codon-optimized DNA sequence encoding a gE ectodomain with the P317R mutation is shown in SEQ ID NO: 128. An exemplary codon-optimized DNA sequence encoding a gI ectodomain is shown in SEQ ID NO: 129.

[0483] Selection of regulatory elements Bicistronic SAM vectors were prepared to express gEgI (gE wt and gE_P317R mutant) heterodimers. For each vector, gE expression was driven by a single 26S subgenomic promoter (SEQ ID NO: 126). Four regulatory elements were tested for gI expression: an internal ribosome entry site (IRES EV71) (SEQ ID NO: 127), two 2A "self-cleaving" peptide sequences: GSG-P2A (SEQ ID NO: 124) and F2A (SEQ ID NO: 125), and a second 26S subgenomic promoter (SEQ ID NO: 126) (Figure 38A). Additionally, vectors with HA-tags at the C-terminus of the gE and gI proteins were generated (Figure 38B).

[0484] Effect of regulatory elements on gE and gI expression levels The expression levels of gE and gI from PNGase-treated BHK cell supernatants were visualized using near-infrared Western blot detection (Figure 40, left), from which intensity signals were extracted (Figure 40, right). Furthermore, the IRES outperformed other regulatory elements to produce more gE and gI protein. No significant difference in expression levels was observed between the wt and P317R mutant gEgI.

[0485] Determining gE:gI stoichiometry under different regulatory elements To assess gE:gI stoichiometry, we generated vectors with HA tags at the C-terminus of the gE and gI proteins. These offer the advantage of allowing detection of gE and gI on the same gel using a single antibody (anti-HA), thus enabling relative quantification. The HA-tagged constructs exhibited similar in vitro potency (% J2-positive cells) as the untagged IRES constructs (data not shown). Near-infrared Western blot detection was used to quantify relative protein expression and stoichiometry. WB conditions were identical to those for the untagged constructs (Figure 41A). The signals for the gE-HA and gI-HA bands (Figure 41B) were extracted, and intensity values ​​were normalized by the gE-HA intensity level (Figure 41C). The IRES outperformed other regulatory elements, producing more gI in the supernatants of cells transfected with an equivalent amount of SAM vector, reaching a gE:gI ratio of 1:1 compared to 1:0.5 for the other regulatory elements.

[0486] Mutant gEgI SAM vector Bicistronic SAM vectors encoding the HSV2 gE wt ectodomain (SEQ ID NO:7) or mutant gE ectodomain (ectodmain) and the HSV2 gI wt ectodomain (SEQ ID NO:8) and the HSV1 gE wt ectodomain (SEQ ID NO:9) or mutant gE ectodomain and the HSV1 gI wt ectodomain (SEQ ID NO:10) were prepared as described above. For all vectors, gE expression was driven by the S26 subgenomic promoter (SEQ ID NO:126), and gI expression was driven by an internal ribosome entry site (IRES EV71, SEQ ID NO:127). The HSV2 gE mutations present in each vector are listed in Table 22. The HSV2 gE mutations are relative to SEQ ID NO:7. The HSV1 gE mutations are relative to SEQ ID NO:9.

[0487] [Table 22]

[0488] RNA pattern uniformity assessment To study RNA pattern uniformity, RNA samples were analyzed on a 1% agarose gel. RNA samples were prepared as follows: 100–500 ng of RNA was mixed with 3 μL of loading buffer (50 mM EDTA pH 8, 30% sucrose, 0.05% bromophenol blue) and water to a final volume of 10 μL. Samples were denatured at 50°C for 20 minutes. Agarose gels were run at 130 V for 45 minutes in Northern Max Gly gel running buffer (Invitrogen™).

[0489] Results: All HSV SAM candidates (HSV2 and HSV1) displayed similar homogeneity patterns in agarose gel analysis (Figure 42). Major bands were observed for all constructs without significant degradation.

[0490] Protein expression assessment by Western blot The ability of cells to express a given antigen from different HSV SAM constructs was assessed as follows: On day 0, baby hamster kidney (BHK) cells were cultured in a T225 flask at 1 x 10 cells / ml in growth medium (DMEM high glucose (Gibco™), 1% L-glutamine, 1% Pen-Strep (Corning®), 5% FBS (Gibco™)). 7 The cells were seeded at 1000 x g for 1 hour. For trypsinization, the medium was removed and the cells were washed with 5 mL of PBS. The PBS wash was removed, and 5 mL of pre-warmed trypsin (Gibco™) was added and thoroughly spread throughout the plate. The trypsin was removed, and the plate was kept at 37°C for 1-2 minutes. The cells were then resuspended in 10 mL of growth medium. The cells were counted and seeded at the required concentration into a new flask. The cells were then incubated at 37°C, 5% CO2 for approximately 20 hours.

[0491] On day 1, plates were prepared by adding 2 mL of growth medium (DMEM high glucose, 1% L-glutami...

Claims

1. An Fc receptor or immunogenic fragment thereof derived from a virus for use in treating a subject infected with said virus.

2. 2. The Fc receptor or immunogenic fragment thereof for use according to claim 1, wherein the Fc receptor is derived from a herpesvirus, preferably from HSV2, HSV1 or HCMV.

3. 3. The Fc receptor or immunogenic fragment thereof for use according to claim 2, wherein the Fc receptor is selected from HSV2 gE2, HSV1 gE1, HCMV gp34 and HCMV gp68.

4. The Fc receptor or immunogenic fragment thereof for use according to any one of claims 1 to 3, wherein the Fc receptor or immunogenic fragment thereof is selected from the group consisting of HSV2 gE2 ectodomain, HSV1 gE1 ectodomain, HCMV gp34 ectodomain and HCMV gp68 ectodomain.

5. 5. The Fc receptor or immunogenic fragment thereof for use according to claim 4, wherein the Fc receptor or immunogenic fragment thereof is an HSV2 gE2 ectodomain having the amino acid sequence set forth in SEQ ID NO: 7, or a variant thereof that is at least 90% identical thereto.

6. The Fc receptor or immunogenic fragment thereof is part of a heterodimer with a binding partner or fragment thereof from the virus, preferably - the Fc receptor is HSV2 gE2 or an immunogenic fragment thereof and the binding partner is HSV2 gI2 or a fragment thereof, or - the Fc receptor is HSV1 gE1 or an immunogenic fragment thereof and the binding partner is HSV1 gI1 or a fragment thereof; 6. An Fc receptor or an immunogenic fragment thereof for use according to claim 5.

7. 7. The Fc receptor or immunogenic fragment thereof for use according to claim 6, wherein the binding partner or fragment thereof is selected from the HSV2 gI2 ectodomain and the HSV1 gI1 ectodomain.

8. 8. The Fc receptor or immunogenic fragment thereof for use according to claim 7, wherein the binding partner or fragment thereof is the HSV2 gI2 ectodomain having the amino acid sequence set forth in SEQ ID NO: 8, or a variant thereof that is at least 90% identical thereto.

9. the binding partner or fragment thereof is administered to the subject together with an adjuvant, preferably an adjuvant comprising a TLR4 agonist and an immunologically active saponin, more preferably an adjuvant comprising 3D-MPL and QS21 in a liposomal formulation; 9. An Fc receptor or an immunogenic fragment thereof for use according to any one of claims 1 to 8.

10. said use does not include administration of an immunodominant viral antigen to a subject; In particular, when the Fc receptor is HSV2 gE2 or HSV1 gE1, the Fc receptor or immunogenic fragment thereof is not administered to the subject together with HSV2 gD2 or HSV1 gD1 (respectively) or a fragment thereof comprising an immunodominant region.

10. An Fc receptor or an immunogenic fragment thereof for use according to any one of claims 1 to 9.

11. 11. The Fc receptor or immunogenic fragment thereof for use according to any one of claims 1 to 10, wherein the Fc receptor is not VZV gE.

12. the Fc receptor is selected from HSV2 gE2 and HSV1 gE1; the Fc receptor is administered to the subject together with HSV2 gC2 or an immunodominant fragment thereof or HSV1 gC1 or an immunogenic fragment thereof (respectively); 12. An Fc receptor or an immunogenic fragment thereof for use according to any one of claims 1 to 11.

13. A recombinant viral FcR or an immunogenic fragment thereof, wherein the ability of the recombinant viral FcR or the immunogenic fragment thereof to bind to a human antibody Fc domain is reduced or eliminated compared to the corresponding native viral Fc receptor.

14. HSV2 gE2 or an immunogenic fragment thereof, wherein the HSV2 gE2 or the immunogenic fragment thereof is 289_insert ADIGL; 338_insert ARAA; H245K; P317R; P319R; P319G; P319K; H245A_P319R; H245A_P319G; H245A_P319K; H245A_P319T; P319D; S338D; R320D; N241A_R320D; A248K_V340M; P318Y; A248K_V340R; A248T_V340W; A248K_V340W; A246W_R320G; A246W_P317K; A246W_R320D ;A246W_R320T;V340W;A248G_V340W;H245G_R320D;P318D;A246W_P317F ;P319G_V340W;A248T_V340M;P317K_V340W;V340F;V340D;H245A_R320D; P317F_V340W;A246W_P317S;H245S_R320D;R314G_P318D;A248T;P318S; P317K;P317S_V340W;H245D;R314P_V340W;R314L_318D;P319L_V340W;P3 17F;P318D_S338G;R314G_V340W;P317K_S338H;R314L_V340W;P318R;P3 18Q;P317F_S338G;R314G_P318I;H245G_P319G;P317L;P318I;A248T_F32 2A;H245E;P318T;P318R_S338G;P318D_S338H;P317F_S338H;A248T_V34 0R;A248T_F322I;H245A_R320G;P318R_S338H;H245S_R320G;P317K_S338 G;A248T_F322P;V340R;R314L_P318R;H245S_R320T;R314G_P318R;R320 E;H245G_R320G;H245A_R320T;A246W;P318I_S338G;P317K_V340M;P317I ;R320H;R314P_P318I;P318I_S338H;P317F_V340M;H245A_P319G;H245A _P319L;R320P;H245G_R320T;R314L_V340R;P319G_V340R;R314G_F322I;R314L_P318I;R320A;R314N;P317F_V340R;P318D_S338L;A248G_V340R;R314E;R31 4P_P318D;H245S_P319G;V340Q;A248K_F322I;R320G;H245S_P319L;R314F;P319L;P 317K_S338L;P319L_V340M;P317G;R320S;R320Q;R314P_V340R;V340A;H245G_P319L ;R320T;R314P_P318R;A248G_F322I;R320N;P317N;R314D;R314Y;R314P_F322I;P31 14. The recombinant viral FcR or immunogenic fragment thereof of claim 13, comprising a mutation or combination of mutations with respect to the sequence set forth in SEQ ID NO: 1 selected from: 9G_V340M; P317S_V340R; R314V; P317R_P319D; P317R_R320D; P319D_R320D; Δ319_Δ320; P317G_P318G_Δ319_Δ320; P318E_Δ319_Δ320; P318G_Δ319_Δ320; P318K_Δ319_Δ320; P317R_P318E_Δ319_320; P317R_P318G_Δ319_Δ320 and P317G_P318K_Δ319_Δ320.

15. A heterodimer comprising or consisting of an Fc receptor from an HSV virus or an immunogenic fragment thereof and a binding partner from said HSV virus or a fragment thereof for use in therapy.

16. - the Fc receptor is HSV2 gE2 and the binding partner is HSV2 gI2, or - the Fc receptor is HSV1 gE1 and the binding partner is HSV1 gI1; 16. A heterodimer for use according to claim 15.

17. A pharmaceutical composition comprising an Fc receptor derived from an HSV virus or an immunogenic fragment thereof, a binding partner derived from the HSV virus or a fragment thereof, and a pharmaceutically acceptable carrier.

18. - the Fc receptor is HSV2 gE2 and the binding partner is HSV2 gI2, or - the Fc receptor is HSV1 gE1 and the binding partner is HSV1 gI1; 18. The pharmaceutical composition of claim 17.

19. A nucleic acid encoding a viral Fc receptor or an immunogenic fragment thereof described in any one of claims 1 to 12, a recombinant viral Fc receptor or an immunogenic fragment thereof described in claim 13 or 14, or a heterodimer described in claim 15, for use in therapy.

20. Encoding the heterodimer of claim 15, wherein the sequences encoding the viral FcR or immunogenic fragment thereof and its binding partner or fragment thereof are separated by an internal ribosome entry site (IRES) sequence; 20. The nucleic acid of claim 19.

21. 21. The nucleic acid for use according to claim 19 or 20, wherein the nucleic acid is an RNA molecule.

22. 22. The nucleic acid for use according to claim 21, wherein the RNA molecule is a self-amplifying RNA molecule.

23. 23. The nucleic acid for use according to claim 21 or 22, wherein the RNA molecule or self-amplifying RNA molecule is associated with a non-viral delivery material, for example to form a cationic nanoemulsion (CNE) or a lipid nanoparticle (LNP).

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