WO2025119252A1

WO2025119252A1 - Methods for detecting an anti-hsv antibody in a sample

Info

Publication number: WO2025119252A1
Application number: PCT/CN2024/136965
Authority: WO
Inventors: Xusha ZHOU; Haifang JIANG; Xiaoqing Chen; Grace Guoying ZHOU
Original assignee: Immvira Biopharmaceuticals Co Ltd
Current assignee: Immvira Biopharmaceuticals Co Ltd
Priority date: 2023-12-06
Filing date: 2024-12-05
Publication date: 2025-06-12
Anticipated expiration: 2026-06-06

Abstract

Disclosed is a method for detecting an anti-HSV antibody in a sample, comprising contacting, in vitro, an HSV genetically engineered to express a reporter gene with the sample and a HSV susceptible cell in a condition allowing the HSV to infect the HSV susceptible cell. Also disclosed is a kit comprising the genetically engineered HSV and HSV susceptible cell, and the genetically engineered HSV itself.

Description

METHODS FOR DETECTING AN ANTI-HSV ANTIBODY IN A SAMPLE

Technical Field

The disclosure relates to a method for detecting an anti-HSV antibody, and in particular, to a method for detecting an anti-HSV-1 antibody in a sample, a kit used for the method, and a genetically engineered HSV used in the method.

Background

Herpes simplex virus 1 (HSV-1) and HSV-2 cause human diseases, ranging from benign cold sores to life-threatening infections such as encephalitis and neonatal HSV disease. Currently, 47%of the U.S. population is seropositive for HSV-1 and 16%are seropositive for HSV-2. Protection correlates well with virus neutralization. Viral glycoproteins that are critical for entry can stimulate production of neutralizing antibodies (NAbs) . These proteins include the cell receptor-binding glycoprotein D (gD) , along with the heterodimer gH/gL and the fusion protein gB. Both gH/gL and gB are the “core fusion machinery” of all herpesviruses, and both crystallographic data and electron microscopy show that these proteins are highly homologous across herpesviruses in structure, implying a common mechanism by which they function. In animal models, gC, gD, gH/gL, and gB stimulate a NAb response.

HSV based oncolytic virus, which usually contain attenuated modifications and expresses multiple therapeutic genes, had been proved to be good therapeutic gene therapy vectors for treating central nervous disease and multiple cancers. NAbs against HSV are one of the barriers oncolytic viruses encounter during viral replication in vivo. The circulating NAbs in cancer patients may restrict the efficacy of HSV based virotherapy and are preferably determined prior to the virotherapy. Conventional assays previously employed to quantitatively determine the neutralizing Ab titers against herpes virus include the cytopathic effect-based neutralization test (CPE-NT) and the plaque reduction neutralization test (PRNT) , which are widely accepted and considered the laboratory gold standard test. However, these assays are arduous and time consuming, requiring long time of infections and plenty of manual counting jobs. Thus, these tests may not be feasible to screen large cohorts from clinical trials or epidemiology studies.

Summary

An aspect of the disclosure provides a method for detecting an anti-herpes simplex virus (anti-HSV) antibody in a sample, comprising (a) providing a herpes simplex virus (HSV) genetically engineered to express a reporter gene; (b) contacting, in vitro, the HSV with the sample and a HSV susceptible cell in a condition allowing the HSV to infect the HSV susceptible cell; (c) determining an expression level of the reporter gene; (d) comparing the expression level of the reporter gene with a reference expression level obtained with a negative control without the anti-HSV antibody; and (e) determining presence of the anti-HSV antibody in the sample when a decrease of the expression level of the reporter gene compared to the reference expression level is greater than a predetermined threshold.

In some embodiments, the HSV comprises an insertion of a polynucleotide of the reporter gene between two adjacent UL genes, for example, UL3 and UL4 genes.

In some embodiments, an internal inverted repeat region of the HSV is replaced with a polynucleotide consisting of a promoter and a stop codon, for example, a CMV promoter followed by three stop codons.

In some embodiments, the HSV is engineered to inactivate at least one copy of γ34.5 gene.

In some embodiments, the reporter gene is a gene encoding luciferase, GFP or eGFP, preferably luciferase.

In some embodiments, when the reporter gene is a gene encoding luciferase, step (c) comprises adding a substrate for luciferase.

In some embodiments, step (c) comprises detecting a light intensity emitted from the sample to determine the expression level of the reporter gene.

In some embodiments, the HSV susceptible cell is Vero cell.

In some embodiments, the sample is a serum or plasma sample, preferably isolated from a human subject.

In some embodiments, the predetermined threshold is about 30%to about 50%, preferably about 35%to about 50%, more preferably about 40%to 50%, and most preferably about 45%.

In some embodiments, the anti-HSV antibody is an anti-HSV gD antibody, an anti-HSV gB antibody, an anti-HSV gC antibody, an anti-HSV gH antibody, an anti-HSV gL antibody, or any combination thereof.

In some embodiments, the anti-HSV antibody is a neutralizing antibody. In some embodiments, the HSV is HSV-1, and the anti-HSV antibody is an anti-HSV-1 antibody. In some embodiments, the anti-HSV antibody is an anti-HSV-1 neutralizing antibody. In some embodiments, the method is for non-diagnosis purpose.

Another aspect of the disclosure provides a kit for detection of an anti-herpes simplex virus (anti-HSV) antibody in a sample, comprising (a) a herpes simplex virus (HSV) genetically engineered to express a reporter gene; and (b) a positive control.

In some embodiments, the anti-HSV antibody is a neutralizing antibody. In some embodiments, the HSV is HSV-1, and the anti-HSV antibody is an anti-HSV-1 antibody. In some embodiments, the anti-HSV antibody is an anti-HSV-1 neutralizing antibody. In some embodiments, the positive control is polyclonal anti-HSV antibodies, preferably polyclonal anti-HSV-1 antibodies.

In some embodiments, the kit further comprises a HSV susceptible cell, preferably Vero cell. In some embodiments, the kit further comprises a substrate for a reporter enzyme encoded by the reporter gene. In some embodiments, the kit further comprises a negative control sample without any anti-HSV antibody. In some embodiments, the kit comprises an HSV susceptible cell, a substrate for a reporter enzyme encoded by the reporter gene and/or a negative control sample without any anti-HSV antibody.

A further aspect of the disclosure provides a herpes simplex virus type 1 (HSV-1) genetically engineered to express a reporter gene, wherein the HSV-1 comprises an insertion of a polynucleotide of the reporter gene between two adjacent UL genes, and wherein an internal inverted repeat region of the HSV-1 is replaced with a polynucleotide consisting of a promoter and a stop codon.

In some embodiments, the two adjacent UL genes are UL3 and UL4 genes.

In some embodiments, the polynucleotide consisting of a promoter and a stop codon is a CMV promoter followed by three stop codons.

In some embodiments, the HSV is engineered to inactivate the other copy of γ34.5 gene.

A further aspect of the disclosure provides a computer readable medium on which computer executable instructions are stored, wherein the execution of the instructions performs any of the methods described herein.

Brief Description of the Drawings

Fig. 1 is a schematic diagram of exemplary viral reporter vectors constructed for use in the methods described herein.

Fig. 2 depicts the assay principle of the methods for detection of anti-HSV antibodies described herein.

Fig. 3 shows the analytic procedure of an exemplary method according to one embodiment of the invention.

Fig. 4 shows the results of positive control dose response.

Fig. 5 shows the selectivity of the assay in the presence of other matrix components.

Detailed Description

Definition

It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “an anti-HSV antibody, ” is understood to represent one or more anti-HSV antibodies. As such, the terms “a” (or “an” ) , “one or more, ” and “at least one” can be used interchangeably herein.

By “inactivate” is meant any mutation in the genomic DNA sequence of a target gene that results in the target gene inactive, non-functional, or absent, including but not limited to insertions, deletions, or substitutions of one or more nucleic acids in the genomic DNA sequence of the target gene, in particular, the coding sequence of the gene of interest. In some embodiments, the inactivating mutation reduces or eliminates mRNA transcription, thereby reducing or eliminating the expression level of the encoded mRNA transcript and protein. In some embodiments, the inactivating reduces or inhibits mRNA translation, thereby reducing the expression level of the encoded protein. In some embodiments, the inactivating encodes a modified protein with reduced or altered function compared to the unmodified (i.e., wild-type) version of the protein. For example, inactivate γ34.5 gene may include a deletion of the coding sequence (CDS) of the γ34.5 gene, or a substitution of one or more nucleic acids in the CDS of the γ34.5 gene that leads to the production of a non-functional ICP34.5 protein. For example, an inactivation of γ34.5 gene may include a deletion of the coding sequence (CDS) of the γ34.5 gene, a deletion of the whole sequence of the γ34.5 gene, or a substitution of one or more nucleic acids in the CDS of the γ34.5 gene that leads to the production of a non-functional ICP34.5 protein.

“HSV-1 glycoprotein” as used herein is meant the 12-13 virally encoded glycoproteins on the HSV-1 viral envelope which help the virus to interact with target cells. Of the 12 or more glycoproteins present on the HSV-1 viral envelope, coordinated action of five glycoproteins: gC, gD, gB and the heterodimer gH and gL, is required for viral entry into the target cell. gC and gB independently interact with cell surface heparan sulphate proteoglycan and mediate initial viral binding. In the absence of both gB and gC, virus binding to the cell surface is severely reduced.

By “specifically binds” or “has specificity to, ” it is generally meant that an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody “A” may be deemed to have a higher specificity for a given epitope than antibody “B, ” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D. ”

As used herein, “cancer” or “tumor” as used interchangeably herein is meant to a group of diseases which can be treated according to the disclosure and involve abnormal cell growth with the potential to invade or spread to other parts of the body. Not all tumors are cancerous; benign tumors do not spread to other parts of the body. Possible signs and symptoms include: a new lump, abnormal bleeding, a prolonged cough, unexplained weight loss, and a change in bowel movements among others. There are over 100 different known cancers that affect humans. The present disclosure is preferably applicable to solid tumors. Non-limiting examples of cancer or tumor are bladder cancer, basal cell carcinoma, cholangiocarcinoma, colon cancer, endometrial cancer, esophageal cancer, Ewing’s sarcoma, prostate cancer, gastric cancer, glioma, hepatocellular carcinoma, Hodgkin lymphoma, laryngeal carcinoma, liver cancer, lung cancer, melanoma, mesothelioma, pancreatic cancer, rectal cancer, renal cancer, thyroid cancer, malignant peripheral nerve cell tumors, malignant peripheral nerve sheath tumors (MPNST) , cutaneous and plexiform neurofibromas, leiomyoadenomatoid tumor, fibroids, uterine fibroids, leiomyosarcoma, papillary thyroid cancer, anaplastic thyroid cancer, medullary thyroid cancer, follicular thyroid cancer, hurthle cell carcinoma, thyroid cancer, ascites, malignant ascites, mesothelioma, salivary gland tumors, mucoepidermoid carcinoma of the salivary gland, acinic cell carcinoma of the salivary gland, gastrointestinal stromal tumors (GIST) , tumors that cause effusions in potential spaces of the body, pleural effusions, pericardial effusions, peritoneal effusions aka ascites, giant cell tumors (GCT) , GCT of bone, pigmented villonodular synovitis (PVNS) , tenosynovial giant cell tumor (TGCT) , TCGT of tendon sheath (TGCT-TS) , and other sarcomas. In preferable embodiment, the present disclosure is used to treat esophageal cancer, lung cancer, prostate cancer, or bladder cancer.

As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total) , whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal, ” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.

Genetically Engineered HSV

A first aspect of the disclosure relates to a herpes simplex virus (HSV) genetically engineered to express a reporter gene.

It is contemplated that the polynucleotide of the reporter gene may be inserted between any adjacent genes of the HSV genome. It is desirous that the insertion of the polynucleotide of the reporter gene does not affect or disturb the expression of any of the genes in the UL and US component of the HSV genome. In some embodiments, the reporter gene is inserted between two adjacent UL genes of the HSV genome, for example between UL3 and UL4 genes. In another embodiment, the reporter gene may be inserted between, e.g., UL37 and UL38 genes.

The reporter gene can be any gene conventionally used in the art for reporting purposes. Reporter genes or rather the proteins translated from the reporter gene sequence can be assayed by detecting enzymatic activity or spectrophotometric characteristics, or indirectly with antibody-based assays. In general, enzymatic assays are more sensitive due to the small amount of reporter enzyme required to generate detectable levels of reaction products. Examples of commonly used reporter systems include GFP, eGFP, and luciferase, and depending on the purpose of the experiment, these may be used as a transcription tag, a transcriptional fusion or a translational fusion. Reporter genes have been successfully used to characterize native and heterologous gene expression as well as protein trafficking.

In a preferable embodiment, the reporter gene is luciferase. Bioluminescence is a natural phenomenon of living organisms creating their own light. The basis of bioluminescence is the interaction of the enzyme luciferase with a luminogenic substrate (e.g., luciferin) to produce light. The luciferases that are used most widely are beetle luciferases (including firefly luciferase) , Renilla luciferase and a modified deep sea shrimp luciferase (luciferase) . The basis of bioluminescent detection assays is harnessing the efficient light-emitting chemistry of the enzyme and its substrate for use in analytical methods. By holding the concentration of each reaction component constant, except for one component that varies in relation to the process being studied, the resulting light is directly proportional to the variable component. This couples an observable parameter to the reaction outcome. In assays using luciferase, the variable component may be the luciferase itself, its substrates or cofactors. Due to the very low backgrounds in bioluminescence, the proportional linear range can be enormous, typically extending 10⁴-to 10⁸-fold over the concentration of the variable component. For example, by detecting a light intensity emitted from the sample, the expression level of the reporter gene can be determined. The light could be emitted directly by the reporter gene, e.g., GFP or eGFP, or indirectly by the product of the substrate, such as luciferin, due to the catalytic reaction mediated by the enzyme, such as luciferase, expressed from the reporter gene.

The HSV used in the disclosed method may, in addition to the insertion of the reporter gene, be further engineered. In some embodiments, internal inverted repeats of the HSV are replaced with a polynucleotide consisting of a promoter and a stop codon. In these embodiments, the internal inverted repeats of the HSV are deleted and inserted into the deleted region a polynucleotide consisting of a promoter and a stop codon, for example, a CMV promoter followed by three stop codons. Alternative promoters can also be used, including but not limited to SV40 promoter, Egr-1 promoter, U6 promoter, or HSV TK promoter, as commonly used in the art. In an embodiment, the deletion causes an excision of a fragment starting from the stop codon of the encoding sequence of the last gene (e.g. U_L56) in the U_L component to the start nucleotide of a promoter sequence of the first gene (e.g. U_S1) in the U_S component, in case of a prototype (P) genome. It is well-known in the art that the genomes of HSV have four different isomers, i.e., P, I_L, I_S, and I_LS. The exact first and last genes of the U_L and U_S components will depend on the isomers of the HSV. In an embodiment, the deletion causes an excision of nucleotides 117005 to 132096 in the P-isomer genome of wild-type HSV-1 F strain (GenBank Accession No. GU734771.1) . It is understood that the exact fragment of nucleotides to be deleted depends on the strains and genome isomers of the HSV virus and can be readily determined by a skilled person in the art without inventive skills.

HSV genome consists of two covalently linked components, designated L and S.Each component consists of unique sequences (U_L for the L component, U_S for the S component) flanked by inverted repeats. The inverted repeats of the L component are designated as ab and b’a’. The inverted repeats of the S component are designated as a’c’ and ca. Inverted repeats b’a’ and a’c’ constitute an internal inverted repeat region. The inverted repeats of both L and S components are known to contain two copies of five genes encoding proteins designated ICP0, ICP4, ICP34.5, ORF P and ORF O, respectively and large stretches of DNA that are transcribed but do not encode proteins. The deletion of the internal inverted repeat region means that one copy of each of the five genes encoding proteins designated ICP0, ICP4, ICP34.5, ORF P and ORF O, respectively, is deleted from the HSV genome and the HSV genome retains the other copy of each of the five genes.

In some embodiments, the HSV is engineered to inactivate at least one copy of γ34.5 gene which encodes protein ICP34.5. In these embodiments, the HSV may be engineered to inactivate only one copy of the γ34.5 gene. In these embodiments, the HSV may be engineered to inactivate both copies of the γ34.5 gene.

In some embodiments, the HSV is HSV-1. In some embodiments, the HSV is HSV-2. The present disclosure is not intended to be limited to any specific genome isomers or strains of an HSV virus. Strains of HSV-1 or HSV-2 virus are widely available in the art, including but are not limited to HSV-1 strain 17, the genome of which is available by GenBank Accession No. NC_001806.2; HSV-1strain KOS 1.1, the genome of which is available by GenBank Accession No. KT899744; HSV-1strain F, the genome of which is available by GenBank Accession No. GU734771; HSV-2 strain HG52, the genome of which is available by GenBank Accession No. NC_001798.2; HSV-2 strain 333, the genome of which is available by GenBank Accession No. LS480640.1; HSV-2 strain SD90e, the genome of which is available by GenBank Accession No. KF781518.1; HSV-2 strain MS, the genome of which is available by GenBank Accession No. MK855052.1; and HSV-2 strain H12211, the genome of which is available by GenBank Accession No. KY922725.1. In preferable embodiments, the HSV virus used in the method disclosed herein is an HSV-1 virus, F strain.

In preferable embodiments, provided is a herpes simplex virus type 1 (HSV-1) genetically engineered to express a reporter gene, wherein the HSV-1 comprises an insertion of a polynucleotide of the reporter gene between two adjacent UL genes, and wherein an internal inverted repeat region of the HSV-1 is replaced with a polynucleotide consisting of a promoter and a stop codon.

In a particular embodiment, provided is a herpes simplex virus type 1 (HSV-1) genetically engineered to express a reporter gene, wherein the HSV-1 comprises an insertion of a polynucleotide of the reporter gene between UL3 and UL4 genes, and wherein an internal inverted repeat region of the HSV-1 is replaced with a polynucleotide consisting of a promoter and a stop codon.

In a particular embodiment, provided is a herpes simplex virus type 1 (HSV-1) genetically engineered to express a reporter gene, wherein the HSV-1 comprises an insertion of a polynucleotide of the reporter gene between UL3 and UL4 genes, and wherein an internal inverted repeat region of the HSV-1 is replaced with a polynucleotide consisting of a CMV promoter followed by three stop codons.

In a particular embodiment, provided is a herpes simplex virus type 1 (HSV-1) genetically engineered to express a reporter gene selected from a group consisting of genes encoding luciferase, GFP and eGFP, wherein the HSV-1 comprises an insertion of a polynucleotide of the reporter gene between UL3 and UL4 genes, and wherein an internal inverted repeat region of the HSV-1 is replaced with a polynucleotide consisting of a CMV promoter followed by three stop codons.

In a particular embodiment, provided is a herpes simplex virus type 1 (HSV-1) genetically engineered to express luciferase, wherein the HSV-1 comprises an insertion of a polynucleotide encoding luciferase between UL3 and UL4 genes, and wherein an internal inverted repeat region of the HSV-1 is replaced with a polynucleotide consisting of a CMV promoter followed by three stop codons.

In a particular embodiment, provided is a herpes simplex virus type 1 (HSV-1) genetically engineered to express a reporter gene, wherein the HSV-1 comprises an insertion of a polynucleotide of the reporter gene between UL3 and UL4 genes, wherein an internal inverted repeat region of the HSV-1 is replaced with a polynucleotide consisting of a CMV promoter followed by three stop codons, and wherein the HSV-1 is further engineered to inactivate the other copy of γ34.5 gene (i.e., both copies of γ34.5 gene are not functional in the HSV-1) .

In a particular embodiment, provided is a herpes simplex virus type 1 (HSV-1) genetically engineered to express a reporter gene selected from a group consisting of genes encoding luciferase, GFP and eGFP, wherein the HSV-1 comprises an insertion of a polynucleotide of the reporter gene between UL3 and UL4 genes, wherein an internal inverted repeat region of the HSV-1 is replaced with a polynucleotide consisting of a CMV promoter followed by three stop codons, and wherein the HSV-1 is further engineered to inactivate the other copy of γ34.5 gene (i.e., both copies of γ34.5 gene are not functional) .

In a particular embodiment, provided is a herpes simplex virus type 1 (HSV-1) genetically engineered to express luciferase, wherein the HSV-1 comprises an insertion of a polynucleotide encoding luciferase between UL3 and UL4 genes, wherein an internal inverted repeat region of the HSV-1 is replaced with a polynucleotide consisting of a CMV promoter followed by three stop codons, and wherein the HSV-1 is further engineered to inactivate the other copy of γ34.5 gene (i.e., both copies of γ34.5 gene are not functional) .

Methods

An second aspect of the disclosure provides a method for detecting an anti-herpes simplex virus (anti-HSV) antibody in a sample, comprising (a) providing a herpes simplex virus (HSV) genetically engineered to express a reporter gene; (b) contacting, in vitro, the HSV with the sample and a HSV susceptible cell in a condition allowing the HSV to infect the HSV susceptible cell; (c) determining an expression level of the reporter gene; (d) comparing the expression level of the reporter gene with a reference expression level obtained with a negative control without the anti-HSV antibody; and € determining presence of the anti-HSV antibody in the sample when a decrease of the expression level of the reporter gene compared to the reference expression level is greater than a predetermined threshold.

In the methods disclosed herein, the HSV genetically engineered to express a reporter gene provided in step (a) can be any HSV described in the above under section titled Genetically Engineered HSV. In a preferable embodiment, the HSV is an HSV-1 genetically engineered to express luciferase, wherein the HSV-1 comprises an insertion of a polynucleotide encoding luciferase between UL3 and UL4 genes, and wherein an internal inverted repeat region of the HSV-1 is replaced with a polynucleotide consisting of a CMV promoter followed by three stop codons.

HSV-1 and HSV-2 have evolved numerous strategies to infect a wide range of hosts and cell types. HSV entry into host cell is a multistep process that involves the interaction of the viral glycoproteins with various cell surface receptors. Based on the cell type, HSV enters host cell using different modes of entry. The combination of various receptors and entry modes has resulted in a virus that is capable of infecting virtually all cell types. It has the unparalleled ability to infect human and nonhuman cells alike. The reason behind this successful story of infection is an accumulation of multiple supporting factors. These include a) involvement of several multifunctional HSV glycoproteins in entry; b) existence of multiple alternative receptors: an array of HSV entry receptors for HSV glycoproteins already exists, and evidence suggests even more unidentified HSV receptors; c) multiple entry modes: HSV has the ability to enter into host cells by direct fusion with the plasma membrane, or via endocytic pathways, and the latter can be pH dependent or independent; and d) Multiple spread strategies of HSVs, including transmission of free virions, movement of HSV along filopodia-like cellular membrane protrusions (surfing) towards the cell body, and lateral cell-to-cell spread. Therefore, the present disclosure contemplates that the HSV susceptible cell used in the method disclosed herein could be any cell type. Preferably, the HSV susceptible cell is an immortalized cell, such as Vero cell, 293T cell, CHO cell or HEK293 cell. In preferable embodiments, the HSV susceptible cell used in the present disclosure is Vero cell.

In preferable embodiments, the predetermined threshold in the method disclosed herein is about 30%to about 50%. In preferable embodiments, the predetermined threshold is about 35%to about 50%, more preferably about 40%to 50%, and most preferably about 45%. In some embodiments, the decrease of the expression level of the reporter gene in the sample is calculated according to a formula: %decrease (or inhibition) = 100%* (RLUnc-RLUsample) /RLUnc, wherein RLUnc indicates Relative Luminescence Unit of the negative control sample, and RLUsample indicates Relative Luminescence Unit of the sample. For example, the predetermined threshold is set to about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, or any value therebetween. In a specific example, the predetermined threshold is set to 44.73%.

Therefore, in some embodiments, when a decrease of the expression level of the reporter gene compared to the reference expression level is greater than about 30%, about 40%, about 35%or about 50%, the sample is determined as presence of an anti-HSV antibody. In some embodiments, when a decrease of the expression level of the reporter gene compared to the reference expression level is not greater than about 30%, about 40%, about 35%or about 50%, the sample is determined as absence of an anti-HSV antibody. In a particular embodiment, when a decrease of the expression level of the reporter gene compared to the reference expression level is greater than about 45%, e.g., about 60%, the sample is determined as presence of an anti-HSV antibody. In a particular embodiment, when a decrease of the expression level of the reporter gene compared to the reference expression level is not greater than about 45%, e.g., about 20%, the sample is determined as absence of an anti-HSV antibody.

In preferable embodiments, the sample is a serum sample. In some embodiments, the sample is a plasma sample. In preferable embodiments, the serum or plasma sample is isolated from a human. In preferable embodiments, the human is a patient diagnosed with a cancer. In preferable embodiments, the human is a patient to be treated by an oncolytic HSV therapy.

In this disclosure, the expression of the reporter gene is indicative of the infection of the HSV in the HSV susceptible cells (e.g., Vero cells) . The expression level of the reporter gene is indicative of the extent or amount of the infection in the, e.g., Vero cells. A less expression level is indicative of less infection and a higher concentration of the anti-HSV antibody in the sample.

In the present disclosure, the anti-HSV antibody to be detected in a sample could be any antibody elicited by the immune system of a subject due to the infection of an HSV virus, including HSV-1 and HSV-2. The anti-HSV antibody is most likely present in the circulating blood of a subject in various forms. The predominant forms of the anti-HSV antibody in the circulating blood of a subject may be an anti-HSV gD antibody, an anti-HSV gB antibody, an anti-HSV gC antibody, an anti-HSV gH antibody, an anti-HSV gL antibody, or any combination thereof. Some of the anti-HSV antibody may be an antibody that specifically binds to a homodimer or a heterodimer, such as a gD/gB heterodimer. In some embodiments, the anti-HSV antibody in a sample is a mixture of anti-HSV antibodies, for example, a mixture of an anti-HSV gD antibody and an anti-HSV gB antibody. It is understood that the anti-HSV antibody pool in the circulating blood varies from individual to individual due to V (D) J gene rearrangement and somatic hypermutation. However, all anti-HSV antibody in the circulating blood binds to HSV, in particular to the glycoproteins expressed on the viral envelope.

In some embodiments, the anti-HSV antibody is an anti-HSV-1 antibody. In some embodiments, the anti-HSV antibody is an anti-HSV-2 antibody. An anti-HSV-1 antibody could be any antibody elicited by the immune system of a subject due to the infection of an HSV-1 virus. Similarly, an anti-HSV-2 antibody could be any antibody elicited by the immune system of a subject due to the infection of an HSV-2 virus. It is contemplated that the method disclosed here is applicable to detect either an anti-HSV-1 antibody or an anti-HSV-2 antibody or both in a sample by using either a genetically engineered HSV-1 or a genetically engineered HSV-2 or both, as described herein. For example, in some embodiments, a method for detecting an anti-HSV-1 antibody in a sample is provided, which comprises, among others, providing an HSV-1 genetically engineered to express a reporter gene. In some embodiments, a method for detecting an anti-HSV-1 antibody in a sample is provided, which comprises, among others, providing an HSV-2 genetically engineered to express a reporter gene. In some embodiments, a method for detecting an anti-HSV-2 antibody in a sample is provided, which comprises, among others, providing an HSV-1 genetically engineered to express a reporter gene. In some embodiments, a method for detecting an anti-HSV-2 antibody in a sample is provided, which comprises, among others, providing an HSV-2 genetically engineered to express a reporter gene.

In some embodiments, the anti-HSV antibody described above is a neutralizing anti-HSV (HSV-1 and/or HSV-2) antibody. A neutralizing anti-HSV antibody is an anti-drug antibody (ADA) that binds to HSV-1 and/or HSV-2 based drug and diminishes or eliminates (i.e., neutralizes) associated biological activity of the drug. A neutralizing anti-HSV antibody may neutralize both HSV-1 and HSV-2 based drug due to the shared genomes and proteomics between the two types. A neutralizing anti-HSV antibody may also be HSV-1 or HSV-2 based drug specific. For example, a neutralizing anti-HSV antibody may be HSV-1 based drug specific that does not neutralize HSV-2 based drug. In preferable embodiments, the HSV-1 and/or HSV-2 based drug is an oncolytic HSV-1 and/or HSV-2 viruses, which are genetically modified variants compared to wild-type HSV-1 and/or HSV-2 viruses. Exemplary oncolytic HSV-1/2 viruses include, but are not limited to, T-vec, G47Δ, T3011, R3616, DM33, R7020, G207, HF10, and BS001. A systematic review of HSV-based oncolytic viruses is available in the art (see, e.g., Aldrak, Norah et al. “Oncolytic Herpes Simplex Virus-Based Therapies for Cancer. ” Cells vol. 10, 6 1541.18 Jun. 2021, doi: 10.3390/cells10061541) . In some embodiments, the existence of the anti-HSV antibody inhibits, precludes, or eliminates the infection of an HSV oncolytic virus into a target cell (e.g., a tumor cell) . In some embodiments, an HSV oncolytic virus is less effective in terms of inhibiting tumor growth compared to that in the absence of the anti-HSV neutralizing antibody. In some embodiments, an HSV oncolytic virus is not effective in terms of inhibiting tumor growth due to the presence of the anti-HSV neutralizing antibody.

In some embodiments, provided is a readable computer medium on which is stored computer executable instructions, wherein the execution of the instructions performs any of the methods disclosed herein.

In some embodiments, provided is a method for treatment of a cancer in a subject by administering an oncolytic HSV virus therapy, comprising, prior to delivering the oncolytic HSV virus therapy, detecting an anti-HSV antibody in a sample isolated from the subject. The detecting step could be any of the methods disclosed above for detecting an anti-HSV antibody in a sample. In some embodiments, the method of treatment further comprises delivering the oncolytic HSV virus therapy after determining that the decrease of the expression level of the reporter gene compared to the reference expression level is not greater than a predetermined threshold, as described in the above.

Kits

A third aspect of the disclosure provides a kit for detection of an anti-herpes simplex virus (anti-HSV) antibody in a sample, comprising (a) a herpes simplex virus (HSV) genetically engineered to express a reporter gene; and (b) a positive control.

In this disclosure, the HSV genetically engineered to express a reporter gene, as provided in the kit, can be any HSV described in the above under section titled Genetically Engineered HSV. In a preferable embodiment, the HSV is an HSV-1 genetically engineered to express luciferase, wherein the HSV-1 comprises an insertion of a polynucleotide encoding luciferase between UL3 and UL4 genes, and wherein an internal inverted repeat region of the HSV-1 is replaced with a polynucleotide consisting of a CMV promoter followed by three stop codons.

In some embodiments, the positive control is polyclonal anti-HSV antibodies, e.g., polyclonal anti-HSV-1 antibodies. Polyclonal anti-HSV antibodies are commercially available in the art. They can be prepared by immunizing a mammal (e.g., a rodent or rabbit) with HSV-1 or HSV-2 through a standard immunization protocol and collecting and purifying a serum from the mammal. The serum contains polyclonal anti-HSV antibodies and can be used as a positive control in the present invention. The serum may be tested to verify the presence of anti-HSV neutralizing antibody before serving as a positive control.

In some embodiments, the kit may comprise a HSV susceptible cell. The present disclosure contemplates that the HSV susceptible cell used in the kit disclosed herein could be any cell type. Preferably, the HSV susceptible cell is an immortalized cell, such as Vero cell, 293T cell, CHO cell or HEK293 cell. In preferable embodiments, the HSV susceptible cell used in the present disclosure is Vero cell.

In some embodiments, the kit further comprises a substrate for a reporter enzyme encoded by the reporter gene. In these embodiments, the reporter gene is a gene encoding a catalytic enzyme, e.g., a luciferase. The substrate can, e.g., be luciferin.

In some embodiments, the kit further comprises a negative control sample without any anti-HSV antibody. The negative control sample can be a serum or plasma sample, isolated from healthy individuals. By healthy individuals it means the individuals were not infected with HSV including HSV-1 and HSV-2 before the sample was isolated from the individuals.
Examples

Example 1. Constructions of Viral Reporter Vectors

Fig. 1 is a schematic diagram of the recombinant viruses. In MVR-T1013L, 15, 091 base pairs (nucleotides 117005 to 132096) of DNA from the inverted repeats (IR) of the viral genome were deleted and replaced by sequences encoding the CMV. An open reading frame (ORF) encoding the luciferase was inserted between UL3 and UL4 ORFs of the viral genome. In MVR-C1213L, the 15, 091 base pairs of DNA from the inverted repeats of the viral genome and the remaining copy of γ34.5 genes in the terminal repeat (TR) region were deleted. The IR region is replaced with replaced by sequences encoding the CMV. An open reading frame (ORF) encoding the luciferase was inserted between UL3 and UL4 ORFs of the viral genome.

MVR-T1013L (hereinafter “T1013L” or “HSV-1 Luciferase” ) virus was used for neutralization antibody detection. As depicted in the assay principle shown in Fig. 2, when there is no neutralization antibody exists, virus infected cells can express firefly luciferase, which can be detected by adding luciferase substrate. If the antibody is effective in neutralizing the surface glycoproteins of the virus and blocking the virus entry to the cells, a significant reduction in light emission will be measured.

Example 2. Analytical Procedure

Neutralizing antibodies (NAbs) refer to the anti-drug antibody (ADA) which binds to drug products and may diminish or eliminate the associated biological activity. A cell-based immunoassay was developed and validated for the detection of Neutralizing Antibodies (NAbs) against HSV-1 in human serum.

Vero cell culture medium (DMEM with 10%FBS and 1%PS) : Mix 445 ml of DMEM with 50 ml FBS and 5 ml PS. Mix well and sterile filter with 0.2 μm filter. Store at 5 ± 3 ℃ for up to 1 month.

Cell freezing medium (DMEM with 50%FBS and 10%DMSO) : Mix 80 ml of DMEM with 100 ml FBS and 20 ml DMSO. Mix well and sterile filter with 0.2 μm filter. Store at 5 ± 3 ℃ for up to 6 months.

Preparation of Negative Control (NC)

NC is pooled from 30 individual normal human serum (pNHS) , which were pre-screened as anti-HSV-1 NAb negative. NC was ordered from BioIVT (Cat#HUMANSRM, Lot# (HMN744464, HMN744465, HMN744467, HMN744468, HMN744469, HMN744478, HMN744482, HMN744483, HMN744485, HMN744493, HMN744494, HMN744498, HMN744499, HMN744505, HMN744514, HMN744517, HMN744533, HMN744581, HMN744594, HMN744606, HMN744620, HMN744636, HMN744661, HMN744663, HMN744665, HMN744666, HMN744667, HMN744669, HMN744684, HMN744692) ) .

Preparation of Positive Controls (HPC and MPC, collectively “PC” )

Polyclonal Rabbit Anti-Herpes Simplex Virus Type-1 was used as NAb PC. The concentration of HPC and MPC are 300,000 ng/mL and 150,000 ng/mL respectively.

Analytical Procedure

Analytical procedure is illustrated in Fig. 3. Samples were thawed at room temperature. All non-biological reagents, blanks, buffers, frozen controls, and samples were brought to room temperature prior to use.

Day 1

2.1 Vero cells (ATCC #CCL-81, passage number 6-12) from T75 flask (Corning, #431464U) were washed with sterile DPBS (Gibco, #14190-144) and then added with 1-2 ml of Trypsin-EDTA (0.25%, Gibco, #25200-056) .

2.2 Cells were resuspended in Vero cell culture medium and the cell concentration was counted using Vi-Cell XR (Beckman Coulter, #980) .

2.3 Vero cell suspension was diluted with Vero cell culture medium into 200,000 cells/ml. 100 μl/well of cell suspension was seeded in 96-well white solid plate and cultured in a CO₂ incubator overnight.

Day 2

2.4 50 μl/well of controls/samples were added into the cluster tubes. 200 μl/well of Medium 199 (Gibco, #11150067) were added to each cluster tubes and mixed well.

2.5 Virus working solution (20,000 PFU/mL) was made under the Biosafety cabinet. For example, 10 μl of HSV1-luciferase (1.68E9 PFU/mL) was added into 158 μl of Medium 199 and make the stock solution I (1.0E8 PFU/mL) , then added 10 μl of stock solution I to 990 μl of Medium 199 to make solution II (1.0E6 PFU/mL) . Added 300 μl of solution II to 14700 μl of Medium 199 to make the working solution of HSV1-Luciferase virus (20,000 PFU/mL) .

2.6 250 μl of the working solution of HSV1-Luciferase virus (20,000 PFU/mL) was added into cluster tubes from Step 2.4. The cluster tubes were rotated on a mini-rotator at 37℃ for 50 -90 mins.

2.7 After virus incubation was completed, the mixture of controls/samples and virus was added to the cell culture plate from Step 2.3 following the plate map.

2.8 The plate was incubated in CO₂ incubator for 1 -2 hours.

2.9 The medium was aspirated from the plate, refilled with 100 μl/well of fresh media DMEM and incubating the plate in the CO₂ incubator for 18 -24 hours.

Day 3

2.10 Bright-Glo luciferase assay substrate (Promega, Cat#E2620) was added to each well of the cell culture plate and incubating the plate on a shaker at 450 rpm for 10-15 minutes under ambient temperature (keeping from light) .

2.11 Read the plate in GloMax Navigator using Bright-Glo protocol.

Data Analysis

The raw data (RLU) was captured using the GloMax Navigator reader. Microsoft Office 365 was used to calculate the mean, %Signal Inhibition and CV%. JMP software was used for the statistical evaluations of the cut point factor. The RLU mean and CV%was kept as 1 decimal place. %Signal Inhibition and ACP and sensitivity were kept as 2 decimal places.

Plate Control Acceptance Criteria

%Signal Inhibition of HPC >%Signal Inhibition of MPC ≥ ACP ≥ %Signal Inhibition of NC

The CV%of NC, MPC and HPC will be reported, but no CV%criteria will be applied.

Plate NC is the mean of all accepted NCs.

For all the assays, two-thirds of the QC samples on every plate, at least half (50%) at each level, must meet the acceptance criteria described in this section. The run (plate) will be rejected if the above acceptance criteria are not met.

Example 3. Positive control dose response

T1013L virus neutralization by neutralizing antibodies targeting HSV-1. Quantification of T1013L virus infected Vero cell in the presence of the indicated concentrations of Polyclonal Rabbit Anti-Herpes Simplex Virus Type-1 (Agilent/Dako, #B0114) . The infectivity of T1013L was quantified by measuring Relative Luminescence Unit (RLU) . Results are shown in Fig. 4.

Example 4. Individual Matrix Selectivity

Selectivity is the ability of the assay to detect anti-HSV-1 NAb in the presence of other matrix components. NAb at the MPC level (150,000 ng/mL) was spiked into 8 lots of individual normal human serum. Six out of eight unspiked samples were tested negative. All 8 NAb spiked samples at the MPC level were confirmed as positive. See Fig. 5.

Example 5. Assay Cut-Point

Given the prevalence of HSV-1 infectious and the presence of neutralizing antibodies is about 50%across all ages, in this study, the individual human serum samples of healthy volunteers were pre-screened for the neutralizing activities against HSV-1. Two analysts tested 30 individual pre-screened negative human serum in a total of six different panels for the calculation of Assay Cut-Point (ACP) .

To lessen the degree of analytical variability, the common approach is to use the percent signal inhibition to determine the cut-off value. The percent signal inhibition for the detection of neutralizing antibodies are calculated from the formula below.

%Signal Inhibition = 100%* (RLU NC –RLU controls or samples) /RLU NC.

The ACP (%Signal Inhibition) , using a 5%false-positive rate, was calculated using the equation below based on the %Inhibition data distribution.

Assay Cut-Point (ACP) = “Mean %Inhibition + 1.645 *SD%Inhibition” (if data normally distributed) or “95^th” Percentile” (if data not normally distributed)

The final assay cut point was determined as 44.73 using a non-parametric approach based on 169 of %Signal Inhibition data values. Samples will be classified as positive when %Signal Inhibition value ≥ 44.73%

Example 6. Intra-assay precision

Intra-assay precision was evaluated by analyzing multiple samples of each run acceptance control level in one validation run. Intra-assay precision was calculated using %Signal Inhibition of MPC (Medium Positive Control) and HPC (High Positive Control) responses. Positive control preparations were made from six aliquots each of MPC and HPC and analyzed at 4 replicates each. The NC (Negative Control) was analyzed from 6 aliquots and analyzed at 4 replicates each. Intra-assay precision, an evaluation of the within-run performance of the controls, was evaluated in one run using six sets of the HPC (300,000 ng/mL) , MPC (150,000 ng/mL) and NC (0.0 ng/mL) . The results shown in Table 1 demonstrated that the intra-assay precision of the HPC and MPC were 0.5%and 4.7%respectively.

Table 1.

Example 7. Sensitivity

Serial 2.0-fold dilutions of the spiked samples (from 600,000 ng/mL to 9375 ng/mL) in pNHS were analyzed. The assessment of sensitivity in human serum was based on the sample with the lowest concentration of ADA that consistently produced a result below the plate-specific assay cut-point. The assay sensitivity was determined for each sensitivity set using a point-to-point linear regression, by interpolation at the assay cut point. The final assay sensitivity was calculated using concentration among all the qualified curves. The results showed that the assay sensitivity of the assay to detect neutralizing HSV-1 was 123292.1 ng/mL in human serum (Table 2) . The assay sensitivity was evaluated 6 times.

Table 2.

Example 8. Drug Tolerance

The presence of the drug (HSV-1 Luciferase virus) in the samples may interfere with the detection of anti-HSV-1 NAb and hence, the ability of the assay system to correctly detect the NAb must be evaluated. Drug tolerance is defined as the highest level of drug that cannot change the samples/controls from positive to negative or negative to positive.

Potential interference by the drug was evaluated using PCs prepared at HPC level (300,000 ng/mL) , MPC level (150,000 ng/mL) and NC level (0 ng/mL) . All these levels of NAb PC samples were spiked with HSV-1 Luciferase virus at concentrations of 0.0, 625, 1250, 2500, and 5000 PFU/mL.

As shown in Table 3, the addition of up to 5000 PFU/mL of HSV-1 Luciferase virus did not interfere with NAb detection in samples spiked at the HPC level (300,000 ng/mL) , MPC level (150,000 ng/mL) and NC level.

Table 3.

Example 9. Hemolyzed and Lipemia Interference

Matrix interference was evaluated in 300 mg/dL lipemia or 2%hemolyzed samples. Eight individual serum samples with 300 mg/dL triglycerides were assessed unspiked and spiked at MPC level (150,000 ng/mL) . As shown in Table 4, All the unspiked lipemic serum samples were confirmed negative, and all MPC spiked samples are confirmed positive. These results meet the acceptance criteria, indicating that 300 mg/dL lipemia matrix does not interfere with the assay results.

Table 4.

Eight individual serum samples with 2%hemolyzed samples were assessed unspiked and spiked at MPC level (150,000 ng/mL) . As shown in Table 5, all the unspiked hemolyzed serum samples were confirmed negative, and all MPC spiked hemolyzed samples are confirmed positive. These results indicated that 2%hemolyzed matrix does not interfere with the assay results.

Table 5.

Example 10. Robustness

The robustness test includes the incubation time range. The robustness was evaluated by applicably accepted validation runs. Three sets of HPC, LPC and NC were applied for the robustness assay. Only qualified robustness parameters were used in sample analysis.

Acceptance Criteria:

The mean MPC signal and mean HPC signal must be ≤ the assay cut point calculated for the corresponding plate, the mean NC signal > plate assay cut point.

At least 2/3 of robustness samples at each level must meet the above acceptance criteria.

Table 6. Results of Robustness Test

Claims

A method for detecting an anti-herpes simplex virus (anti-HSV) antibody in a sample, comprising

(a) providing a herpes simplex virus (HSV) genetically engineered to express a reporter gene;

(b) contacting, in vitro, the HSV with the sample and a HSV susceptible cell in a condition allowing the HSV to infect the HSV susceptible cell;

(c) determining an expression level of the reporter gene;

(d) comparing the expression level of the reporter gene with a reference expression level obtained with a negative control without the anti-HSV antibody; and

(e) determining presence of the anti-HSV antibody in the sample when a decrease of the expression level of the reporter gene compared to the reference expression level is greater than a predetermined threshold.
The method of claim 1, wherein the HSV comprises an insertion of a polynucleotide of the reporter gene between two adjacent UL genes, for example, UL3 and UL4 genes.
The method of claim 1 or 2, wherein an internal inverted repeat region of the HSV is replaced with a polynucleotide consisting of a promoter and a stop codon, for example, a CMV promoter followed by three stop codons.
The method of any one of claims 1 to 3, wherein the HSV is engineered to inactivate at least one copy of γ34.5 gene.
The method of any one of claims 1 to 4, wherein the reporter gene is a gene encoding luciferase, GFP or eGFP, preferably luciferase.
The method of claim 5, wherein when the reporter gene is a gene encoding luciferase, step (c) comprises adding a substrate for luciferase.
The method of any one of claims 1 to 6, wherein step (c) comprises detecting a light intensity emitted from the sample to determine the expression level of the reporter gene.
The method of any one of claims 1 to 7, wherein the HSV susceptible cell is Vero cell.
The method of any one of claims 1 to 8, wherein the sample is a serum or plasma sample, preferably isolated from a human subject.
The method of any one of claims 1 to 9, wherein the predetermined threshold is about 30%to about 50%, preferably about 35%to about 50%, more preferably about 40%to 50%, and most preferably about 45%.
The method of any one of claims 1 to 10, wherein the anti-HSV antibody is an anti-HSV gD antibody, an anti-HSV gB antibody, an anti-HSV gC antibody, an anti-HSV gH antibody, an anti-HSV gL antibody, or any combination thereof.
The method of any one of claims 1 to 11, wherein:

(i) the anti-HSV antibody is a neutralizing antibody;

(ii) the HSV is HSV-1, and the anti-HSV antibody is an anti-HSV-1 antibody; and/or

(iii) the method is for non-diagnosis purpose.
A kit for detection of an anti-herpes simplex virus (anti-HSV) antibody in a sample, comprising

(a) a herpes simplex virus (HSV) genetically engineered to express a reporter gene; and

(b) a positive control.
The kit of claim 13, wherein the HSV comprises an insertion of a polynucleotide of the reporter gene between two adjacent UL genes, for example, UL3 and UL4 genes.
The kit of claim 13 or 14, wherein an internal inverted repeat region of the HSV is replaced with a polynucleotide consisting of a promoter and a stop codon, for example, a CMV promoter followed by three stop codons.
The kit of any one of claims 13 to 15, wherein the HSV is engineered to inactivate at least one copy of γ34.5 gene.
The kit of any one of claims 13 to 16, wherein the reporter gene is a gene encoding luciferase, GFP or eGFP, preferably luciferase.
The kit of any one of claims 13 to 17, wherein the anti-HSV antibody is an anti-HSV gD antibody, an anti-HSV gB antibody, an anti-HSV gC antibody, an anti-HSV gH antibody, an anti-HSV gL antibody, or any combination thereof.
The kit of any one of claims 13 to 18, wherein:

(i) the anti-HSV antibody is a neutralizing antibody;

(ii) the HSV is HSV-1, and the anti-HSV antibody is an anti-HSV-1 antibody; and/or

(iii) the positive control is a polyclonal anti-HSV antibodies, preferably polyclonal anti-HSV-1 antibodies.
The kit of any one of claims 13 to 19, wherein the kit further comprises:

(i) a HSV susceptible cell, preferably Vero cell;

(ii) a substrate for a reporter enzyme encoded by the reporter gene; and/or

(ii) a negative control sample without any anti-HSV antibody.
A herpes simplex virus type 1 (HSV-1) genetically engineered to express a reporter gene, wherein the HSV-1 comprises an insertion of a polynucleotide of the reporter gene between two adjacent UL genes, and wherein an internal inverted repeat region of the HSV-1 is replaced with a polynucleotide consisting of a promoter and a stop codon.
The HSV-1 of claim 21, wherein:

(i) the two adjacent UL genes are UL3 and UL4 genes;

(ii) the polynucleotide consisting of a promoter and a stop codon is a CMV promoter followed by three stop codons;

(iii) the HSV-1 is engineered to inactivate the other copy of γ34.5 gene;

(iv) the reporter gene is a gene encoding luciferase, GFP or eGFP, preferably luciferase; or

(v) any combination of (i) to (iv) .
A computer readable medium on which computer executable instructions are stored, wherein the execution of the instructions performs the method of any of claims 1 to 12.