CA3169402A1

CA3169402A1 - Diagnosis of congenital cytomegalovirus infection

Info

Publication number: CA3169402A1
Application number: CA3169402A
Authority: CA
Inventors: Dana WOLF
Original assignee: Hadasit Medical Research Services and Development Co
Current assignee: Hadasit Medical Research Services and Development Co
Priority date: 2020-02-03
Filing date: 2021-02-03
Publication date: 2021-08-12
Also published as: IL295233A; EP4100744A1; EP4100744A4; WO2021156860A1; AU2021217240A1

Abstract

The present invention provides biomarkers allowing the diagnosis of symptomatic congenital cytomegalovirus (CMV) infection and in particular to differentiate between symptomatic and asymptomatic infected fetuses, methods of diagnosis of CMV using said biomarkers, diagnostic kits comprising thereof, and use of the kits and methods of diagnosing fetuses infected with a symptomatic congenital cytomegalovirus (CMV).

Description

DIAGNOSIS OF CONGENITAL CYTOMEGALO VIRUS INFECTION
FIELD OF THE INVENTION
[0001] The present invention relates to biomarkers allowing the diagnosis of symptomatic congenital cytomegalovirus (CMV) infection and in particular to differentiate between symptomatic and asymptomatic infected fetuses, to methods of diagnosis of CMV
using said biomarkers, diagnostic kits comprising thereof and use of the kits.
BACKGROUND OF THE INVENTION

[0002] Congenital cytomegalovirus (cCMV) infection, resulting from intrauterine transmission, is the most common congenital infection, affecting an average of 0.64% of live births worldwide. Primary human CMV infection during gestation poses ¨30-40%
risk of intrauterine transmission, whereas recurrent infection is associated with a lower transmission rate. Once fetal/congenital infection occurs, cCMV disease develops in ¨25% of the infected children, and can present as sensorineural hearing loss (SNHL) and a wide range of neurodevelopmental disabilities, brain anomalies, and intrauterine growth restriction. About 10% of congenitally infected newborns are already symptomatic at birth, and 10-15% of initially-asymptomatic neonates with cCMV will develop delayed permanent sequelae, usually hearing loss and psychomotor delay, later in life. Despite the immense clinical and sociological burden of cCMV, no licensed vaccines are available. Moreover, no established prenatal antiviral treatments and prenatal biomarkers for fetal/neonatal disease have been identified thus far.
100031 Recent prenatal treatment trials with HCMV-specific hyperimmune globulins (e.g.
Nigro et al., N Engl J Med. 2005; 353(13):1350-62; Revello et al., N Engl J
Med.
2014,370(14):1316-26) yielded ambiguous results. Prenatal treatment with valacyclovir showed promising results in fetuses with non-severe ultrasound symptoms. These studies have stirred great interest in prenatal diagnosis and prevention of cCMV
infection and disease. The growing awareness of cCMV in recent years has triggered widespread CMV
serological screening in pregnant women in several countries, further underscoring the need to identify reliable prenatal markers for fetal infection and disease. Once the maternal infection is diagnosed, amniocentesis with CMV qPCR testing in amniotic fluid is now routinely utilized for the prenatal diagnosis of fetal infection, typically after the 20th week of pregnancy. However, while positive results in amniotic fluid reliably identify fetal infection, they do not distinguish between symptomatic and asymptomatic fetuses/neonate.
Prenatal prediction of fetal disease is currently limited, being mostly based on fetal imaging (by ultrasound or MRI) which is compromised by suboptimal predictive values, especially during early- and mid-gestation, when timely decisions about medical intervention are highly needed.
100041 The search for prenatal predictors of fetal disease has been ongoing.
In this regard, the correlation of CMV DNA load in amniotic fluid with the congenital disease has been extensively studied. Although higher median viral loads were generally shown to be associated with symptomatic congenital infections, high viral load ¨ a common finding in CMV-infected amniotic fluids ¨ is not predictive of fetal disease (e.g. in Gibson L., Lancet Infect Dis 2017; 17:e177¨e188). The prognostic value of fetal blood parameters, including platelet counts, IgM levels, CMV DNA levels, and beta-2-microglobulin has been advocated. However, the procedure of fetal blood sampling by cordocentesis is invasive and associated with 1-3% risk of fetal loss, and thus not routinely utilized.
Recently, examination of the amniotic fluid peptidome, analyzed by capillary electrophoresis coupled to mass spectrometry, has identified a 34-peptide combination classifier in a discovery cohort of 13 symptomatic and 13 asymptomatic neonates. (Desveaux C. et al., PLoS Pathog.
2016;
12(1):e1005395).
100051 Another study has proposed that high level of amniotic fluid soluble HLA-G in maternal serum and amniotic fluid could potentially serve as predictive biomarkers for cCMV disease, with a PPV of 100% and NPV of 71.4%-83.3%, identified in a small cohort of 12 symptomatic and 5 asymptomatic fetuses with cCMV (Rizzo et al., J
Immunol Res.
2016;2016:3890306.). These markers have not demonstrated a clear advantage over currently available measures or introduced into clinical use. It was concluded that larger studies are required to verify their clinical efficacy in predicting clinical outcomes of a CMV-infected fetuses. There is an unsolved need for safe and robust methods for determining fetal CMV infection, predicting symptomatic CMV infection, and in particular for distinguishing between symptomatic and asymptomatic CMV subjects SUMMARY OF THE INVENTION
100061 The present invention provides that based on the measurement of concentration(s) of one or more biomarkers as defined in the present application, it is possible to unambiguously diagnose fetus infected with cytomegalovirus and even more, distinguishing between symptomatic and asymptomatic infected fetuses.

100071 According to one aspect, the present invention provides a method of diagnosing a congenital cytomegalovirus (cCMV) infection, wherein the method comprises (i) determining a level of at least one biomarker in a biological sample; and (ii) comparing the level of the at least one biomarker from the biological sample to its level in a control, wherein a difference in the level of the biomarker in the biological sample and in the control is indicative of a congenital CMV infection, wherein the at least one biomarker is selected from a biomarker of group A, Group B, Group C and Group D, wherein:
Group A comprises biomarkers: Cathepsin B (CTSB), Signal-regulatory protein beta-1(SIRPB1), Galectin-3-binding protein (LGALS3BP), Agrin (AGRN), Vascular cell adhesion protein 1 (VCAMI), Coactosin-like protein (COTL1), CDS antigen-like (CD5L), ATP synthase subunit beta, mitochondrial (ATP5B), Keratin, type II
cytoskeletal 6A
(KRT6A), Secreted and transmembrane protein 1 (SECTMI), Fatty acid-binding protein, heart (FABP3), Calcyclin (S100A6), (SH3 domain-binding glutamic acid-rich-like protein (SH3BGRL), Interferon-stimulated gene 15 (ISGI5), Cartilage acidic protein 1 (CRTAC I), Retinoic acid receptor responder protein 2 (RARRES2), Bone marrow stromal antigen 2 (BST2), Beta-2-microglobulin (B2M), Epiphycan (EPYC), Dickkopf-related protein (DKK I), Major Histocompatibility Complex, Class I, C (HLA-C), Carboxypeptidase Q
(CPQ), Coagulation factor V (F5), Transmembrane glycoprotein NMB (GPNMB), Laminin subunit alpha-5 (LAMAS), Carbonic anhydrase 3 (CA3), Desmin (DES);
Group B comprises biomarker: Multiple epidermal growth factor-like domains protein 8 (MEGF8), N-acetylgalactosaminyltransferase 7 (GALNT7), Protocadherin Fat 4 (FAT4), (Latrophilin-2 (LPHN2), (Afamin (AFM), Mannan-binding lectin serine protease 1 (MASPI), Extracellular superoxide dismutase [Cu-Zn] (50D3), Polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2), Lipolysis-stimulated lipoprotein receptor (LSR), Hepatocyte growth factor (HGF), Bone marrow proteoglycan (PRG2), Sulthydryl oxidase 1 (QS0X1), Endogenous retrovirus group MER34 member 1 Env polyprotein (ERVMER34-1), Pappalysin-2 (PAPPA2), Aspartate aminotransferase, cytoplasmic (GOTI), Radixin (RDX), Insulin-like growth factor-binding protein 5 (IGFBPS), Desmoyokin (AHNAK), Disintegrin and metalloproteinase domain-containing protein 9 (ADAM9), Lysosome-associated membrane glycoprotein 2 (LAMP2), Carboxypeptidase M
(CPM), Suprabasin (SBSN), Calpastatin (CAST), Serine protease inhibitor Kazal-type 5 (SPINK5), Tenomodulin (TNMD), Cadherin-15 (CDH15), Plasma kallikrein (KLKB1), Prostasin (PRSS8), Collagen alpha-1(111) chain (COL3A1), and Collagen alpha-2(I) chain (COLI A2),

3 Group C comprises biomarkers Nesprin-1 (SYNE1), Myosin-14 (MYH14), GPNMB, LGALS3BP, Carboxypeptidase A2 (CPA2), RARRES2, and Secretoglobin family 3A
member 2 (SCGB3A2); and Group D comprises biomarkers CPM, Platelet-derived growth factor receptor beta (PDGFRB), Angiopoietin-related protein 6 (ANGPTL6), Dermokine (DMKN), Secretogranin-1 (CHGB), ADAM9, Growth arrest-specific protein 1 (GAS1), Platelet basic protein (PPBP), Platelet glycoprotein lb alpha chain (GP1BA). According to one embodiment, the biomarkers are protein biomarkers. According to some embodiments, the biological sample is obtained from a subject selected from a fetus, pregnant woman and neonate. According to some embodiments, the biological sample is selected from an amniotic fluid, fetal blood, fetal blood from the umbilical cord, placental biopsy, neonate blood and maternal blood or serum. According to some embodiments, an increase in the level of at least one biomarker of Group A or C, and/or a decrease in the level of at least one biomarker of Group B or D in the biological sample in comparison to their levels in the control or in comparison to a predefined cutoff level is indicative of cCMV
infection.
[0008] According to some embodiments, the present invention provides a method for diagnosing a symptomatic cCMV. According to some embodiments, the method of diagnosing according to the present invention allows differentiating between symptomatic and asymptomatic cCMV infected subjects, such as symptomatic and asymptomatic fetuses and/or neonates. According to some embodiments, an increase in the level of at least one biomarker of Group C and/or a decrease in the level of at least one biomarker of Group D in the biological sample in comparison to their levels in the control or in comparison to a predefined threshold level is indicative of a symptomatic cCMV infection.
Thus, according to some embodiments, the present invention provides a method of diagnosing a symptomatic congenital cytomegalovirus (symptomatic cCMV) in a fetus, wherein the method comprises (i) determining a level of at least one biomarker in a biological sample; and (ii) comparing the level of the at least one biomarker from the biological sample to its level in a control, wherein a difference in the level of the biomarker in the biological sample and in the control is indicative of presence of the symptomatic cCMV infection, wherein the at least one biomarker is selected from the biomarkers of group Group C and Group D, wherein: Group C comprises biomarkers RARRES2, LGALS3BP, GPNMB, Nesprin-1 (SYNE1), Myosin-14 (MYH14), Carboxypeptidase A2 (CPA2), and Secretoglobin family 3A member 2 (SCGB3A2); and Group D comprises biomarkers CPM, Platelet-derived growth factor receptor beta (PDGFRB), Angiopoietin-related protein 6 (ANGPTL6), Dermokine (DMKN),

4 Secretogranin-1 (CHGB), ADAM9, Growth arrest-specific protein 1 (GAS1), Platelet basic protein (PPBP), and Platelet glycoprotein lb alpha chain (GP1BA). According to some embodiments, the biomarkers are protein biomarkers. According to some embodiments, the wherein the biological sample is obtained from a subject selected from a fetus and pregnant woman. According to other embodiments, the biological sample is selected from an amniotic fluid, fetal blood, plasma or serum, fetal blood, plasma or serum from the umbilical cord, placental biopsy, and maternal blood or serum. According to a certain embodiment, the biological sample is an amniotic fluid.
100091 According to some embodiments, an increase in the level of at least one biomarker of Group C in the biological sample in comparison to their levels in the control; (ii) a decrease in the level of at least one biomarker of Group D in the biological sample in comparison to their levels in the control; or (iii) both (i) and (ii) is indicative of a symptomatic cCMV
infection. According to some embodiments, an increase in the level of RARRES2 in the biological sample in comparison to its level in the control or in comparison to a predefined threshold level is indicative of symptomatic cCMV infection. According to some embodiments, an increase in the level of LGALS3BP in the biological sample in comparison to its level in the control or in comparison to a predefined threshold level is indicative of a symptomatic cCMV infection. According to some embodiments, an increase in the level of GPNMB in the biological sample in comparison to its level in the control or in comparison to a predefined threshold level is indicative of a symptomatic cCMV infection.
According to some embodiments, the method of diagnosis comprises determining levels of at least two, at least 3, at least 4 or at least 5 biomarkers in the biological sample.
According to some embodiments, the method of diagnosis comprises determining levels of 2, 3, 4,

5 or 6 biomarkers in the biological sample. According to some embodiments, an increase in the levels of (i) RARRES2, (ii) LGALS3BP or (iii) both RARRES2 and LGALS3BP in the biological sample in comparison to their levels in the control or in comparison to their predefined threshold levels is indicative of a symptomatic cCMV infection.
According to some embodiments, an increase in the levels of RARRES2 and of LGALS3BP, and (i) an increase in the level of at least one biomarker selected from SYNE], MYH14, GPNMB, CPA2, and SCGB3A2 or (ii) a decrease in the level of at least one biomarker selected from CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS1, PPBP, and GP1BA in comparison to their levels in the control or in comparison to their predefined threshold levels is indicative of a symptomatic cCMV infection. According to some embodiments, an increase in the levels of RARRES2, LGALS3BP, and GPNMB in the biological sample in comparison to their levels in the control is indicative of a symptomatic cCMV
infection.
According to some embodiments, the biological sample is an amniotic fluid.
According to other embodiments, the biological sample is a maternal serum. According to some embodiments, the diagnosis is performed before week 36 of the pregnancy.
According to some embodiments, the method further comprises a step of diagnosing whether the subject, e.g. a fetus, is infected with CMV. According to some other embodiments, the method further comprises differentiating between symptomatic and asymptomatic CMV infection.
100101 According to another aspect, the present invention provides a method of diagnosing an asymptomatic congenital cytomegalovirus in a fetus, wherein the method comprises (i) diagnosing whether the fetus is infected with CMV and (ii) diagnosing whether the fetus has a symptomatic CMV by the method of the present invention, wherein lack of symptomatic CMV in the infected fetus is indicative of presence of asymptomatic cCMV.
NOM According to another aspect, the present invention provides a method for recommending a medical intervention, the method comprises diagnosing a congenital cytomegalovirus using the methods of diagnosis according to the present invention, and recommending the medical intervention based on the obtained results. According to some embodiments, the congenital cytomegalovirus is a symptomatic cCMV. According to other embodiments, the congenital cytomegalovirus is an asymptomatic cCMV. Thus, according to some embodiments, the present invention provides a method for recommending a medical intervention comprising detecting a symptomatic or asymptomatic congenital cytomegalovirus in fetus according to the methods of the present invention and recommending the medical intervention based on the obtained results. According to some embodiments, the present invention provides a method of executing a medical intervention in case of existence of a congenital CMV such symptomatic cCMV comprising diagnosing the cCMV according to the methods of the present invention and effecting the medical intervention. According to some embodiments, the medical intervention is a treatment with an anti-CMV active agent. According to other embodiments, the medical intervention is a termination of pregnancy.
100121 According to some embodiments, the present invention provides a method of treating a congenital cytomegalovirus in a fetus comprising diagnosing the symptomatic or asymptomatic cCMV in the fetus according to the methods of the present invention and administering to the fetus or the mother an anti-CMV active agent. According to some embodiments, the congenital cytomegalovirus is a symptomatic CMV. According to some embodiments, the congenital cytomegalovirus is an asymptomatic CMV.

6 100131 According to another aspect, the present invention provides a kit comprising means for determining a level of at least one biomarker in a biological sample, wherein the at least one biomarker is selected from a biomarker of Group A, Group B, Group C and Group D, and instructions for use. According to some embodiments, the kit comprises instructions for diagnosing a congenital cytomegalovirus (cCMV) infection. According to other embodiments, the kit comprises instructions for diagnosing a symptomatic cCMV.

According to another embodiment, the kit comprises instructions for diagnosing an asymptomatic cCMV. According to some embodiments, the kit provides means for determining the levels of a biomarker selected from RARRES2, LGALS3BP, GPNMB, and a combination thereof. According to some embodiments, the present invention provides a kit comprising means for determining levels of at least two biomarker in a biological sample, wherein the at least two biomarkers are selected from biomarkers of Group C
and Group D, and instructions for use of said kit. According to some embodiments, the kit comprises means for determining the levels of RARRES2 and LGALS3BP, RARRES2 and GPNMB or LGALS3BP and GPNMB or of RARRES2, LGALS3BP and GPNMB. According to some embodiments, the kit further comprises means for determining the level of at least one biomarker selected from SYNE1, MYH14, GPNMB, CPA2, SCGB3A2, CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS1, PPBP, and GP1BA. According to certain embodiments, the means of the kit allow detecting levels of a plurality of biomarkers.
According to some embodiments, the kit comprises all required means and information for detecting the levels of the biomarkers in the biological samples and for comparing the detected levels to their levels in the control or to their threshold levels for diagnosing the cCMV and/or symptomatic cCMV. According to some embodiment, the kit of the present invention is for diagnosing cCMV. According to other embodiments, the kit of the present invention is for diagnosing symptomatic cCMV.
100141 According to another aspect, the present invention provides use of a kit in diagnosis a congenital CMV, wherein the kit comprises means for determining a level of at least one biomarker in a biological sample, wherein the at least one biomarker is selected from a biomarker of Group A, Group B, Group C and Group D. According to some embodiments, the use comprises diagnosing a symptomatic cCMV. According to other embodiments, the use comprises diagnosing an asymptomatic cCMV. According to some embodiments, the use comprises determining the levels of RARRES2 and/or LGALS3BP in a biological sample, and optionally further determining the levels of at least one biomarker selected from SYNE1, MYH14, GPNMB, CPA2, SCGB3A2, CPM, PDGFRB, ANGPTL6, DMKN,

7 CHGB, ADAM9, GAS1, PPBP, GP1BA, and comparing the determined levels to the threshold levels, thereby diagnosing the cCMV and/or symptomatic or asymptomatic cCMV.
BRIEF DESCRIPTION OF DRAWINGS
.. [0015] Fig. 1 shows a heatmap showing the proteins differing between CMV-positive (yes) and CMV-negative (no) amniotic fluid samples. The heatmap was drawn using normalized values, after scaling per protein over all drawn samples.
[0016] Fig. 2A shows a heatmap showing amniotic fluid proteins differing between severely symptomatic (sick) and asymptomatic (not sick). Fig. 2B shows a heatmap showing the same set of proteins shown in Fig. 2A in severely symptomatic (sick) asymptomatic (not sick) and CMV-negative amniotic fluid samples. Heatrnaps were drawn using normalized values, after scaling per protein over all drawn samples.
[0017] Fig. 3A shows the distribution and median values of RARRES2 protein concentrations, as measured by ELISA immunoassay, in amniotic fluid samples from symptomatic (sick), asymptomatic (not sick), and CMV-negative (uninfected) cCMV cases.
Individual amniotic fluid RARRES2 concentration values are shown in Fig. 3B
shows RARRES2 protein concentrations, as measured by ELISA, in individual amniotic fluid samples from symptomatic (sick; black), asymptomatic (not sick; grey), and CMV-negative (uninfected; white) cases. Samples which had been pre-analyzed by proteome analysis (the discovery panel) are indicated by + sign.
[0018] Fig. 4A shows the distribution and median values of LGALS3BP protein concentrations, as measured by ELISA, in amniotic fluid samples from symptomatic (sick), asymptomatic (not sick), and CMV-negative (uninfected) cCMV cases. Fig. 4B
shows LGALS3BP protein concentrations, as measured by ELISA, shown in individual amniotic fluid samples from symptomatic (sick; black), asymptomatic (not sick; grey), and CMV-negative (uninfected; white) cases. Samples which had been pre-analyzed by proteome analysis (the discovery panel) are indicated by "+" sign.
DETAILED DESCRIPTION OF THE INVENTION
1.00191 The present invention is based on an unexpected observation that a set of biomarkers, i.e. proteins, secreted into the amniotic fluid, may be useful to unambiguously diagnose fetal infected with cytomegalovirus. Even more surprising was the finding that several specific biomarkers allow determining whether the fetus has/will develop a symptomatic congenital

8 CMV. In other word, the methods of the present invention allow differentiating between symptomatic and asymptomatic infected fetuses. Currently this differentiation is often made by imaging at a later stage and usually does not provide a robust differentiation.
Determination whether the fetus is infected by CMV is usually made by measuring viral load in amniotic fluid and usually is unambiguous. However, diagnosing whether the fetus is expected to develop symptoms of CMV will allow to provide much more specific recommendations and will reduce the number of non-necessary terminations of pregnancies.
As shown in the results, the present invention allows differentiating fetus having symptomatic and asymptomatic CMV with sensitivity, specificity, and positive and predictive values of above 95.0% each (referring to RARRES2 and LGALS3BP
proteins).
Specifically, it was shown that detecting RARRES2 in amniotic fluids in concentration above 40 ng/ml allows diagnosing a symptomatic cCMV with sensitivity of 95.2%, specificity of 92.9%, positive predictive value of 90.9%, and negative predictive value of 96.3% %. In additional it was shown that detection of LGALS3BP in amniotic fluid above 2475 nWm1 allowed a reliable prenatal disease prediction with sensitivity of 90.5%, specificity of 96.4%, positive predictive value of 95.0%, and negative predictive value of 93.1%. These are outstanding results that allow providing a well-supported recommendation on whether to continue the pregnancy or not, on closer monitoring, and on the potential use of antiviral treatments.
100201 In one aspect the present invention provides a method of diagnosing a congenital cytomegalovirus (cCMV) infection, wherein the method comprises (i) determining a level of at least one biomarker in a biological sample; and (ii) comparing the level of the at least one biomarker from the sample to its level in a control, wherein a difference in the level of the biomarker in the biological sample and in the control is indicative of a congenital CMV
infection, wherein the at least one biomarker is selected from Cathepsin B
(CTSB), Signal-regulatory protein beta-1(SIRPB1), Galectin-3-binding protein (LGALS3BP), Agrin (AGRN), Vascular cell adhesion protein 1 (VCAM1), Coactosin-like protein (COTL1), CD5 antigen-like (CD5L), ATP synthase subunit beta, mitochondrial (ATP5B), Keratin, type II
cytoskeletal 6A (KRT6A), Secreted and transmembrane protein 1 (SECTM1), Fatty acid-.. binding protein, heart (FABP3), Calcyclin (S100A6), SH3 domain-binding glutamic acid-rich-like protein (SH3BGRL), Interferon-stimulated gene 15 (ISG15), Cartilage acidic protein 1 (CRTAC1), Retinoic acid receptor responder protein 2 (RARRES2), Bone marrow stromal antigen 2 (BST2), Beta-2-microglobulin (B2M), Epiphycan (EPYC), Dickkopf-related protein 1 (DKK1), Major Histocompatibility Complex, Class I, C (HLA-C),

9 Carboxypeptidase Q (CPQ), Coagulation factor V (F5), Transmembrane glycoprotein NMB
(GPNMB), Laminin subunit alpha-5 (LA1v1A5), Carbonic anhydrase 3 (CA3), and Desmin (DES), Multiple epidermal growth factor-like domains protein 8 (MEGF8), N-acetylgalactosaminyltransferase 7 (GALNT7), Protocadherin Fat 4 (FAT4), Latrophilin-2 (LPHN2), Afamin (AFM), Mannan-binding lectin serine protease 1 (MASP1), Extracellular superoxide dismutase [Cu-Zn] (SOD3), Polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2), Lipolysis-stimulated lipoprotein receptor (LSR), Hepatocyte growth factor (HGF), Bone marrow proteoglycan (PRG2), Sulthydryl oxidase 1 (QS0X1), Endogenous retrovirus group MER34 member 1 Env polyprotein (ERVMER34-1), Pappalysin-2 (PAPPA2), Aspartate aminotransferase, cytoplasmic (GOT1), Radixin (RDX), Insulin-like growth factor-binding protein 5 (IGFBP5), Desmoyokin (AHNAK), Disintegrin and metalloproteinase domain-containing protein 9 (ADAM9), Lysosome-associated membrane glycoprotein 2 (LAMP2), Carboxypeptidase M (CPM), Suprabasin (SBSN), Calpastatin (CAST), Serine protease inhibitor Kazal-type 5 (SPINK5), Tenomodulin (TNMD), Cadherin-15 (CDH15), Plasma kallikrein (KLKB1), Prostasin (PRSS8), Collagen alpha-1(111) chain (COL3A 1 ), and Collagen alpha-2(I) chain (COL1A2), Nesprin-1 (SYNE1), Myosin-14 (MYH14), GPNMB, LGALS3BP, Carboxypeptidase A2 (CPA2), RARRES2, and Secretoglobin family 3A member 2 (SCGB3A2), CPM, Platelet-derived growth factor receptor beta (PDGFRB), Angiopoietin-related protein 6 (ANGPTL6), Dermokine (DMKN), .. Secretogranin-1 (CHGB), ADAM9, Growth arrest-specific protein 1 (GAS1), Platelet basic protein (PPBP), and Platelet glycoprotein lb alpha chain (GP1BA).
100211 The biomarkers according to the present invention are divided into groups solely for the purpose of easing the reading of the invention. Thus, Group A of biomarkers comprises biomarkers CTSB, SIRPB1, LGALS3BP, AGRN, VCAM1, COTL1, CD5L, ATP5B, KRT6A, SECTM1, FABP3, S100A6, SH3BGRL, ISG15, CRTAC1, RARRES2, BST2, B2M, EPYC, DKK1, HLA-C, CPQ, F5, GPNMB, LAMAS, CA3, and DES. Group B of biomarkers comprises biomarkers MEGF8, GALNT7, FAT4, LPHN2, AFM, MASP1, 50D3, GALNT2, LSR, HGF, PRG2, QS0X1, ERVMER34-1, PAPPA2, GOT1, RDX, IGFBP5, AHNAK, ADAM9, LAMP2, CPM, SBSN, CAST, SPINK5, TNMD, CDH15, KLKB1, PRSS8, COL3A1, and COL1A2.
100221 Group B of biomarkers comprises biomarkers Disintegrin and metalloproteinase domain-containing protein 9 (ADAM9), Multiple epidermal growth factor-like domains protein 8 (MEGF8), N-acetylgalactosaminyltransferase 7 (GALNT7), Protocadherin Fat 4 (FAT4), (Latrophilin-2 (LPHN2), (Afamin (AFM), Mannan-binding lectin setine protease 1 (MASP1), Extracellular superoxide dismutase [Cu-Zn] (SOD3), Polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2), Lipolysis-stimulated lipoprotein receptor (LSR), Hepatocyte growth factor (HGF), Bone marrow proteoglycan (PRG2), Sulfhydryl oxidase 1 (QS0X1), Endogenous retrovirus group MER34 member 1 Env polyprotein (ERVMER34-1), Pappalysin-2 (PAPPA2), Aspartate aminotransferase, cytoplasmic (GOT1), Radixin (RDX), Insulin-like growth factor-binding protein 5 (IGFBP5), Desmoyokin (AHNAK), Lysosome-associated membrane glycoprotein 2 (LAMP2), Carboxypeptidase M (CPM), Suprabasin (SBSN), Calpastatin (CAST), Serine protease inhibitor Kazal-type 5 (SPINK5), Tenomodulin (ENMD), Cadherin-15 (CDH15), Plasma kallikrein (KLKB1), Prostasin (PRSS8), Collagen alpha-1(III) chain (COL3A1), and Collagen alpha-2(I) chain (COL1A2).
Group C of biomarkers comprises biomarkers SYNE1, MYH14, GPNMB, LGALS3BP, CPA2, RARRES2, and SCGB3A2.
Group D of biomarkers comprises biomarkers CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS1, PPBP, and GP1BA. According to some embodiments, the biomarkers are protein biomarkers.
100231 According to some embodiments, the present invention provides a method of diagnosing a congenital cytomegalovirus (cCMV) infection, wherein the method comprises (i) determining a level of at least one biomarker in a biological sample; and (ii) comparing the level of the at least one biomarker from the sample to its level in a control, wherein the difference between the level of the biomarker in the biological sample and in the control is indicative of a congenital CMV infection, wherein the at least one biomarker is selected from the biomarkers of group A, Group B, Group C and Group D, as defined hereinabove.
100241 According to any one of the above embodiments, the biomarkers, i.e. the proteins are human proteins. According to certain embodiments, the proteins are fetal proteins.
100251 According to some embodiments, the biological sample is obtained from a subject.
The term "subject" refers to a human subject. According to some embodiments, the subject is selected from a fetus, pregnant woman and neonate.
100261 As used herein, the term "biological sample" refers to a sample obtained from a subject, including a sample of a biological tissue or fluid origin obtained in vivo or in vitro.
Biological samples can be, without limitation, body fluid (e.g., blood, blood plasma, serum), organs, tissues, fractions and cells isolated from the subject/patient.
Biological samples also may include sections of the biological sample including tissues (e.g., sectional portions of an organ or tissue). Biological samples may be dispersed in solution or may be immobilized on a solid support, such as in blots, assays, arrays, glass slides, microtiter, or ELISA plates.
Biological samples may be processed in any known method before their use in the methods of the present invention.
100271 In one embodiment, the biological sample is an amniotic fluid.
According to some embodiments, the biological sample is selected from an amniotic fluid, fetal blood, fetal blood serum, fetal plasma, blood, serum or plasma from the umbilical cord, placental biopsy, neonate blood, neonate dried blood spot and maternal blood or serum. In some embodiments, the biological sample is selected from the group consisting of breast milk, maternal blood, maternal urine, maternal saliva, fetal blood, fetal blood from the umbilical cord, postnatal infant urine, blood, saliva, a uterine biopsy sample, and a placental biopsy.
According to some embodiments, the biological sample is fetal blood. According to another embodiment, the biological sample is maternal blood. According to yet another embodiment, the biological sample is neonate blood. According to any one of the above embodiments, the term blood encompasses the term "serum" and may be replaced by it. According to some embodiments, the biological sample is a maternal serum. According to other embodiments, the biological sample is a fetal or neonatal serum. According to other embodiments, the biological sample is neonatal dried blood spot. According to some embodiments, the fetal marker is measured in the biological sample obtained from the mother such as maternal blood, serum or milk.
100281 The terms "marker" and "biomarker" are used interchangeably and refer to a molecule (typically a protein, nucleic acid, carbohydrate, or lipid) that is differentially expressed in the cell, differentially expressed on the surface of an infected cell, differentially phosphorylated, or differentially secreted by an infected cell in comparison to a normal cell or in a paracrine fashion by neighboring uninfected cells, and which is useful for the diagnosis of congenital CMV infection or symptomatic congenital CMV infection for providing a prognosis for birth defects, and for preferential targeting of a pharmacological agent to an infected fetus or individual. According to some embodiments, the biomarker is a protein. In several cases, such markers are proteins that are secreted by an infected cell in comparison to a normal cell, for instance, 1-fold secretion, 2-fold secretion, 3-fold secretion or more in comparison to a normal cell. Alternatively, such biomarkers are molecules that are underexpressed in an infected cell in comparison to a normal cell. According to some embodiments, the marker is a nucleic acid molecule. According to some embodiments, the marker is a nucleic acid molecule such as DNA or RNA molecule correlating to the amount of a protein.
Alternately, such biomarkers are produced by uninfected cells or tissues, resulting from local infection or damage and protein fragments are secreted from cells or released by proteolytic processing from the plasma membrane. Further, a marker can be a molecule that is inappropriately synthesized in the infected cell, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal cell. A marker can also be a molecule that is inappropriately processed in infected cells, for instance, a molecule that is secreted, proteolytically processed or subject to post-translational modification (e.g., phosphorylation, glycosylation) in comparison to the molecule expressed on a normal cell.
In some embodiments, the term "biomarker" as used herein refers to a protein that is differentially present in a biological sample, such as a protein that is differentially excreted into amniotic fluid. According to some embodiments, the biomarkers, such as proteins, are present in biological samples, whereas the presence of the marker above or below a particular amount, referred also as a threshold or cutoff value, is indicative of a diagnosed condition.
[0029] The terms "cytomegalovirus" and "CMV" are uses herein interchangeably and refer to a genus of viruses of herpes virus as known in the art, more specifically to CMV infecting humans.
[0030] As used herein, the terms "congenital cytomegalovirus", "congenital CMV", "congenital CMV infection" and "cCMV" are used herein interchangeably and refer to in utero transmitted CMV infection. The term refers also to an infection in fetuses, neonates and children that were infected in utero. The congenital CMV may be symptomatic and asymptomatic. The terms "symptomatic cCMV" and "symptomatic congenital CMV", as used in the present invention and claims, refer to the condition in which the infected subject demonstrates a moderate to severe cCMV disease in utero and/or after birth.
This term contemplates the expectancy that the fetus will develop a moderate to severe cCMV disease in utero and/or after birth. The terms "asymptomatic cCMV", "asymptomatic congenital CMV" and "non-symptomatic cCMV" as used in the present invention and claims refer to the condition in which the infected subject does not demonstrate or is not expected to demonstrate signs of cCMV disease in utero and does not develop or is not expected to develop symptoms/signs of a disease caused by CMV for at least 1 year after birth. The term "infected" refers to subject that is infected with CMV virus and may be symptomatic and asymptomatic. Typically, symptomatic cCMV results is sensorineural hearing loss (SNHL), a wide range of neurodevelopmental disabilities, brain anomalies, and intrauterine growth restriction. The definition of moderate to severely symptomatic cCMV disease is well known in the art and includes:

Multiple manifestations attributable to cCMV infection, such as:
thrombocytopenia, petechiae, hepatomegaly, splenomegaly, intrauterine growth restriction, hepatitis (raised transaminases or bilirubin), and/or Central nervous system involvement, such as: microcephaly, radiographic abnormalities .. consistent with cytomegalovirus central nervous system disease (ventriculomegaly, intracerebral calcifications, periventricular echogenicity, cortical or cerebellar malformations), abnormal cerebrospinal fluid indices for age, chorioretinitis, sensorineural hearing loss, and the detection of cytomegalovirus DNA in cerebrospinal fluid (Rawlinson et al., Lancet Infect Di s 2017; 17:e177¨e188.) [0031] As used herein, the term "level" refers to the amount or concentration of a biomarker (marker), e.g. protein, in the biological sample or in control. Any known method to determine the level of the biomarker may be used, such as ELISA, as long as the level of the biomarker in the biological sample and in control are measured in comparable units.
Methods for determining the levels of the biomarker correspond to the type of biomarkers.
Subsequently, nucleic acid biomarkers, such as DNA or RNA may be determined by any known appropriate method.
[0032] The term "control" as used herein refers to biological samples from a particular predefined group of subjects. According to some embodiments, control refers to the biological samples obtained from non-infected subjects, from infected non-symptomatic or to combination thereof. Therefore, the term "level in the control" with respect to a biomarker has the meaning of the level, e.g. amount or concentration, of the biomarker in biological samples of the predefined group of subjects, as defined above. For the purposes of diagnosing prenatal symptomatic cCMV, the control comprises samples of non-infected fetuses, infected non-symptomatic fetuses, or both.
[0033] The terms "diagnosing" and "detecting" are used herein interchangeably and mean assessing whether a subject has a specific condition, specifically whether the subject is infected by the cytomegalovirus or not, more specifically whether the subject has or expected to have a symptomatic congenital CMV. This term also refers to providing a prognosis, i.e.
providing expectancy of developing the condition, i.e. whether the subject is expected to have or develop symptomatic congenital CMV. As will be understood by those skilled in the art, such an assessment is usually not intended to be correct for all (i.e.
100%) of the subjects to be identified. The term, however, requires that a statistically significant portion of subjects can be identified. The term diagnosis also refers, in some embodiments, to screening.
Screening for infection, in some embodiments, can lead to earlier diagnosis in specific cases.

The term refers also to detecting congenital CMV infection. The term in some embodiments refers to distinguishing between symptomatic and asymptomatic congenital CMV
infection.
The term encompasses assessing whether the subject has a symptomatic cCMV
infection.
The term also encompasses assessing whether the subject will develop a symptomatic cCMV
infection. As used herein, the term "providing a prognosis" refers to providing a prediction of the probable course and outcome of the diagnosis. Thus, the term "diagnosing cCMV"
have the meaning of predicting whether the subject has or will be infected by cCMV. The term "diagnosing symptomatic cCMV" have the meaning of predicting whether the subject has or will be infected by symptomatic cCMV. As stated above, the prediction is not always 100%. Thus, the term "diagnosing symptomatic cCMV" may have the meaning predicting that the subject has or will have symptomatic cCMV with a probability of 95%, 90%, 85%, 80%, 75%, 70%, or from 70 to 95%. According to some embodiments, the sensitivity of diagnosing symptomatic cCMV is from 85 to 99%, from 90 to 98% or from 92 to 96%.
According to some embodiments, the specificity in diagnosing symptomatic cCMV
is from 85 to 99%, from 90 to 98% or from 92 to 96%. According to some embodiments, the positive predictive value of diagnosing symptomatic cCMV is from 85 to 99%, from 90 to 98% or from 92 to 96%. According to some embodiments, the negative predictive value of diagnosing symptomatic cCMV is from 85 to 99%, from 90 to 98% or from 92 to 96%.
100341 The term "sensitivity", as used herein, refers to the ratio of positive samples correctly recognized as such by carrying out the assay of interest and the total number of samples examined. In other words, an assay has 100% sensitivity if all samples from infected patients give a positive result, i.e. there are no false negative results.
100351 The term "specificity", as used herein, refers to the ratio of negative samples correctly recognized as such by carrying out the assay of interest and the total number of samples examined. In other words, an assay has 100% specificity if all samples from healthy subjects give a negative result, i.e. there are no false positive results.
100361 The term "positive predictive value" as used herein refers to the probability of the disease being present, among those with positive diagnostic test results. The term "negative predictive value" as used herein refers to the probability that the disease was absent, among those whose diagnostic test results were negative.
100371 According to any one of the above embodiments, the diagnosed subject is a fetus.
Therefore, the present invention provides a method of prenatal diagnosis of a congenital CMV in fetus. According to some embodiments, the present invention provides a method of prenatal diagnosis of a symptomatic congenital CMV in fetus.

100381 According to some embodiments, an increase in the level of at least one biomarker of Group A in the biological sample in comparison to its levels in the control is indicative of cCMV infection. According to some embodiments, an increase in the level of at least one biomarker of Group A in the biological sample above a particular ratio is indicative of cCMV
infection. According to other embodiments, a ratio of above 1.1:1 between the level of at least one biomarker of Group A in the biological sample to its levels in the control is indicative of cCMV infection. According to other embodiments, a decrease in the level of at least one biomarker of Group B in the biological sample in comparison to its levels in the control is indicative of cCMV infection. According to other embodiments, a decrease in the level of at least one biomarker of Group B in the biological sample below a particular threshold is indicative of cCMV infection. According to yet another embodiment, a ratio of above 1.1:1 between the level of the at least one biomarker in the control to the level of at least one biomarker of Group B in the biological sample is indicative of cCMV
infection.
According to some embodiments, an increase in the level of at least one biomarker of Group .. A and a decrease in the level of at least one biomarker of Group B in the biological sample in comparison to their levels in the control is indicative of the presence of cCMV infection.
100391 The terms "indicative of cCMV", "indicative of presence of cCMV", and "indicative of existence of cCMV" are used herein interchangeably and mean that the parameter, results of the comparison, etc. indicate(s) that the subject is infected with cCMV.
The term "indicative of symptomatic cCMV", "indicative of presence of symptomatic of cCMV", and "indicative of existence of symptomatic cCMV" are used herein interchangeably and mean that the parameter, results of the comparison, etc. indicate(s) that the subject is infected with cCMV and develops or expected to develop CMV symptoms.
100401 The term "at least X" as used herein have the meaning of "X or more", in which X is .. a number. According to some embodiments, the method of diagnosis comprises determining the level of more than one biomarker. Thus, according to some embodiments, an increase in the levels of 2 or more biomarkers of Group A in the biological sample in comparison to their levels in the control is indicative of cCMV infection. According to other embodiments, an increase in the level of 3 or more biomarkers of Group A in the biological sample in .. comparison to their levels in the control is indicative of cCMV infection.
According to some embodiments, a decrease in the level of 2 or more biomarkers of Group B in the biological sample in comparison to their levels in the control is indicative of cCMV
infection.
According to other embodiments, a decrease in the level of 3 or more biomarkers of Group B in the biological sample in comparison to their levels in the control is indicative of cCMV

infection. According to some embodiments, the biomarker is selected from RARRES2, LGALS3BP, GPNMB and a combination thereof. According to some embodiments, an increase in the level of a biomarker selected from RARRES2, LGALS3BP, GPNMB
and a combination thereof in comparison to their levels in the control is indicative of presence of cCMV infection in the subject. According to some embodiments, the biomarker is selected from SYNE I, ANGPTL6, GAS!, CHGB, ADAM9, and any combination thereof.
According to some embodiments, an increase in the level of SYNE1 and/or a decrease in the level of ANGPTL6, GAS!, CHGB, or ADAM9 in the biological sample in comparison to their levels in the control is indicative of presence of cCMV infection in the subject.
100411 According to some embodiments, an increase in the level of 1, 2, 3, 4, 5, 6, 7, 8, 9, or

10 of the biomarkers of group A in comparison to their levels in the control is indicative of cCMV infection in the subject. According to another embodiment, a decrease in the level of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the biomarkers of group B in comparison to their levels in the control is indicative of cCMV infection. According to yet another embodiment, an increase in the level of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the biomarkers of group A
and a decrease in the level of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the biomarkers of group B in comparison to their levels in the control is indicative of cCMV infection.
100421 According to some embodiments, a difference in the levels of the biomarker in maternal blood and in the control is indicative of a congenital CMV infection in fetus.
According to other embodiments, a difference in the levels of the biomarker in amniotic fluid and in the control is indicative of a congenital CMV infection in fetus.
100431 According to any one of the above embodiments, the control comprises samples from non-infected subjects. Thus, a level of the biomarker in the control refers to a level of the biomarker in biological samples of non-infected subjects. According to some embodiments, the subject is fetus. According to other embodiment, the subject is a pregnant woman. It is clear that the biological sample and the control refer correspond one to another. According to some embodiments, a level of the biomarker in the control refers to a level of the biomarker in amniotic fluid of non-infected fetuses.
100441 According to any one of the above embodiments, diagnosing comprises diagnosing of a symptomatic cCMV. According to other embodiments, diagnosing of the symptomatic cCMV comprises differentiating between symptomatic and asymptomatic cCMV.
100451 Thus, according to some embodiments, the present invention provides a method of diagnosing a symptomatic congenital cytomegalovirus (symptomatic cCMV) in a subject, wherein the method comprises (i) determining a level of at least one biomarker in a biological sample; and (ii) comparing the level of the at least one biomarker from the biological sample to its level in a control, wherein a difference in the level of the biomarker in the biological sample and in the control is indicative of presence of a symptomatic cCMV
infection in the subject, wherein the at least one biomarker is selected from the biomarkers of group Group C and Group D, wherein: Group C comprises biomarkers RARRES2, LGALS3BP, GPNMB, Nesprin-1 (SYNE1), Myosin-14 (MYH14), Carboxypeptidase A2 (CPA2), Secretoglobin family 3A member 2 (SCGB3A2); and Group D comprises biomarkers CPM, Platelet-derived growth factor receptor beta (PDGFRB), Angiopoietin-related protein 6 (ANGPTL6), Dermokine (DMKN), Secretogranin-1 (CHGB), ADAM9, Growth arrest-specific protein 1 (GAS!), Platelet basic protein (PPBP), Platelet glycoprotein lb alpha chain (GP1BA).
According to some embodiments, the biomarkers are protein biomarkers.
[0046] According to another embodiment, the present invention provides a method of detecting the presence of a symptomatic congenital cytomegalovirus (symptomatic cCMV) in a subject, wherein the method comprises (i) determining a level of at least one biomarker in a biological sample; and (ii) comparing the level of the at least one biomarker from the biological sample to its level in a control, wherein a difference in the level of the biomarker in the biological sample and in the control is indicative of presence of a symptomatic cCMV
infection, wherein the at least one biomarker is selected from the biomarkers of group Group C and Group D. According to some embodiments, the subject is a fetus.
Therefore, according to some embodiments, the diagnosis or detection is a prenatal diagnosis of detection. Thus, in some embodiments, the present invention provides a method of diagnosing a symptomatic congenital cytomegalovirus (symptomatic cCMV) in fetus, wherein the method comprises (i) determining a level of at least one biomarker in a biological sample; and (ii) comparing the level of the at least one biomarker from the biological sample to its level in a control, .. wherein a difference in the level of the biomarker in the biological sample and in the control is indicative of presence of a symptomatic cCMV infection in fetus, wherein the at least one biomarker is selected from the biomarkers of Group C and Group D.
[0047] According to some embodiments, the biological sample is obtained from a subject selected from a fetus, pregnant woman and neonate. According to some embodiments, the biological sample is an amniotic fluid. According to other embodiments, the biological sample is selected from amniotic fluid, fetal blood or serum, fetal blood or serum from the umbilical cord, placental biopsy, neonate blood or serum and maternal blood or serum.
According to one embodiment, the biological sample is an amniotic fluid.
According to some embodiments, the sample is a sample of an infected subject. According to some embodiments, the sample is a sample of an infected fetus. According to some embodiments, diagnosing the presence of CMV infection may be according to any known method, e.g. by PCR. According to one embodiment, diagnosing the presence of CMV infection is performed according to the teaching of the present invention.
100481 The term "RARRES2" refers to human Retinoic Acid Receptor Responder 2 protein having accession number Q99969.
100491 The term "LGALS3BP" refers to a human Galectin-3-binding protein having accession number Q08380.
100501 The term "GPNM:B" refers to a human transmembrane glycoprotein NIVIB
having accession number Q14956.
100511 According to some embodiments, an increase in the level of 1, 2, 3, 4, 5, 6, or 7 of the biomarkers of group C in comparison to their levels in the control is indicative of cCMV
infection in the subject. According to another embodiment, a decrease in the level of 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the biomarkers of group D in comparison to their levels in the control is indicative of symptomatic cCMV infection. According to yet another embodiment, an increase in the level of 1, 2, 3, 4, 5, 6, or 7 of the biomarkers of group C
and a decrease in the level of I, 2, 3, 4, 5, 6, 7, 8, or 9 of the biomarkers of group D in comparison to their levels in the control is indicative of symptomatic cCMV infection.
100521 According to some embodiments, an increase in the level of at least one biomarker selected from SYNE], MY1114, GPNMB, LGALS3BP, CPA2, RARRES2, and SCGB3A2 in the biological sample in comparison to its levels in the control is indicative of a symptomatic cCMV infection. According to some embodiments, an increase in the level of at least one biomarker of Group C in the biological sample above a particular ratio is indicative of a symptomatic cCMV infection. According to other embodiments, a ratio of above 1.1:1 between the level of the at least one biomarker of Group C in the biological sample to its levels in the control is indicative of a symptomatic cCMV
infection. According to some embodiments, the biological sample is an amniotic fluid.
100531 According to other embodiments, a decrease in the level of at least one biomarker selected from CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS1, PPBP, and GP IBA in the biological sample in comparison to its levels in the control is indicative of a symptomatic cCMV infection. According to other embodiments, a decrease in the level of at least one biomarker of Group D in the biological sample below a particular threshold is indicative of a symptomatic cCMV infection. According to yet another embodiment, a ratio of above 1.1:1 between the level of the at least one biomarker in the control to the level of the least one biomarker of D in the biological sample is indicative of a symptomatic cCMV
infection. According to another embodiment, an increase in the level of at least one biomarker of Group C and a decrease in the level of at least one biomarker of Group D in the biological sample in comparison to their levels in the control is indicative of a symptomatic cCMV infection. According to some embodiments, the biological sample is an amniotic fluid.
100541 According to some embodiment, the present invention provides a method of diagnosing a symptomatic congenital cytomegalovirus (cCMV) infection, wherein the method comprises (i) determining a level of at least one biomarker in a biological sample;
.. and (ii) comparing the level of the at least one biomarker from the biological sample to its level in a control, wherein a difference in the level of the biomarker in the biological sample and in the control is indicative of a symptomatic congenital CMV infection, wherein the at least one biomarker is selected from SYNE1, MYH14, GPNMB, LGALS3BP, CPA2, RARRES2, SCGB3A2, CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS1, PPBP, and GP1BA. According to some embodiments, the control comprises samples from the infected non-symptomatic, non-infected subject or both. Thus, a level of the biomarker in the control refers to a level of the biomarker in biological samples of infected non-symptomatic, in the samples of non-infected subjects, or in combination thereof. According to some embodiments, the subject is a fetus. According to other embodiments, the subject is a pregnant woman. According to some embodiments, the level is an amount or a concentration of the biomarker in the sample. According to some embodiments, the biological sample is an amniotic fluid.
100551 According to some embodiments, an increase in the level of at least one biomarker selected from SYNE1, MYH14, GPNMB, LGALS3BP, CPA2, RARRES2, SCGB3A2 in the biological sample in comparison to its level in the control is indicative of a symptomatic cCMV infection. According to some embodiments, an increase of more than 1.5, 2, 2.5 or 3 folds in the level of the biological sample in comparison to its level in the control is indicative of a symptomatic cCMV infection. According to some embodiments, an increase of more than 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% 60%, 70%, 80 or 100% in the level of the biological sample in comparison to its level in the control is indicative of a symptomatic cCMV infection. According to some embodiments, an increase in the level of at least one biomarker selected from SYNE1, MYH14, GPNMB, LGALS3BP, CPA2, RARRES2, SCGB3A2 in the biological sample above a particular value or threshold as defined based on the control is indicative of a symptomatic cCMV infection.

[0056] According to other embodiments, a decrease in the level of at least one biomarker selected from CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS1, PPBP, and GP1BA in the biological sample in comparison to its level in the control is indicative of a symptomatic cCMV infection. According to some embodiments, a decrease in the level of at least one biomarker selected from CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS!, PPBP, and GP1BA more than 10%, more than 15%, more than 20% or more than % in the biological sample in comparison to its level in the control is indicative of a symptomatic cCMV infection. According to some embodiments, a decrease in the level of at least one biomarker selected from CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS!, PPBP, and GP IBA in the biological sample below a particular value or threshold as defined based on the control is indicative of a symptomatic cCMV infection.
[0057] As known in the art, comparison to the level in a control may be performed by defining a particular threshold above which or below which the level of the marker is considered as indicative to cCMV, and in particular indicative to symptomatic cCMV. Thus, in some embodiments, the term "comparing the level of the at least one biomarker from the biological sample to its level in a control" and alike may be replaced by "comparing the level of the at least one biomarker to a threshold". Subsequently, the term "an increase in the level of a biomarker X in the biological sample in comparison to their levels in the control" and alike may be replaced by the term "an increase in the level of a biomarker X
in the biological sample above a threshold". The term "a decrease in the level of a biomarker X
in the biological sample in comparison to their levels in the control" and alike may be replaced by the term "a decrease in the level of a biomarker X in the biological sample below a threshold". As also clear, the threshold may be dependent on the method of detection of the markers and thus may vary.
[0058] According to some embodiments, the method comprises determining the level of RARRES2. According to certain embodiments, an increase in the level of RARRES2 in the biological sample in comparison to its level in the control is indicative of a symptomatic cCMV infection. According to some embodiments, an increase of more than 1.5, 2, 2.5 or 3 folds in the level of RARRES2 in the biological sample in comparison to its level in the control is indicative of a symptomatic cCMV infection. According to some embodiments, an increase of from 2 to 20 folds in the level of RARRES2 in the biological sample in comparison to its level in the control is indicative of a symptomatic cCMV
infection.
According to certain embodiments, an increase of from 4 to 16, 5 to 15, 8 to 12 folds is indicative of a symptomatic cCMV infection. According to some embodiment, an increase of 10% or more, 15% or more, 20% or more, 25 % or more, 50% or more, 75% or more, 100%, of above 150%, above 200%, above 250%, above 300% in the level of RARRES2 in the biological sample in comparison to its level in the control is indicative of a symptomatic cCMV infection. According to some embodiments, an increase in the level of RARRES2 in the biological sample above a particular value as define based on the control is indicative of a symptomatic cCMV infection. According to some embodiments, an increase in the level of RARRES2 of 10% or more, 15% or more, 20% or more, 25 % or more, 50% or more, 75%
or more, 100% or more, or from 2 to 20 times above a threshold is indicative of a symptomatic cCMV infection. According to some embodiments, the threshold is 40 ng/ml of RARRES2 in amniotic fluid, as determined by the methods of the present invention.
According to another embodiment, the threshold is 51 ng/ml. According to some embodiments, an increase in the level of RARRES2 in the biological sample above 30 ng/ml, above 35 ng/ml or above 40 ng/ml or above 45 ng/ml or above 50 ng/ml or above 51 ng/ml or above 55 ng/ml is indicative of a symptomatic cCMV infection. According to some embodiments, the threshold for RARRES2 in amniotic fluid is from 20 to 80 ng/ml.
According to some embodiments, the threshold for RARRES2 in amniotic fluid is from 25 to 75, from 30 to 70, or from 35 to 65 ng/ml. According to some embodiments, the sensitivity of diagnosing symptomatic cCMV is from 85 to 99%, from 90 to 98% or from 92 to 96%.
According to some embodiments, the specificity in diagnosing symptomatic cCMV
is from 85 to 99%, from 90 to 98% or from 92 to 96%. According to some embodiments, the positive predictive value of diagnosing symptomatic cCMV is from 85 to 99%, from 90 to 98% or from 92 to 96%. According to some embodiments, the negative predictive value of diagnosing symptomatic cCMV is from 85 to 99%, from 90 to 98% or from 92 to 96%.
100591 According to some embodiments, the method comprises determining the level of .. LGALS3BP. According to certain embodiments, an increase in the level of LGALS3BP in the biological sample in comparison its level to the control is indicative of a symptomatic cCMV infection. According to some embodiment, the increase in the level of in the biological sample in comparison to its level in the control is indicative of a symptomatic cCMV infection. According to some embodiments, an increase in the level of LGALS3BP in the biological sample above a particular value as defined based on the control is indicative of a symptomatic cCMV infection. According to some embodiments, increase of more than 1.5, 2, 2.5 or 3 folds in the level of LGALS3BP in the biological sample in comparison to its level in the control is indicative of a symptomatic cCMV
infection.
According to some embodiments, an increase of from 2 to 20 folds in the level of LGALS3BP

in the biological sample in comparison to its level in the control is indicative of a symptomatic cCMV infection. According to certain embodiments, an increase of from 4 to 16, 5 to 15, 8 to 12 folds is indicative of a symptomatic cCMV infection.
According to some embodiment, an increase of 10% or more, 15% or more, 20% or more, 25 % or more, 50%
.. or more, 75% or more, 100%, of above 150%, above 200%, above 250%, above 300% of LGALS3BP in the biological sample in comparison to its level in the control is indicative of a symptomatic cCMV infection. According to some embodiments, an increase in the level of LGALS3BP of 10% or more, 15% or more, 20% or more, 25 % or more, 50% or more, 75%
or more, 100% or more, or from 2 to 20 times above a threshold is indicative of a symptomatic cCMV infection. According to some embodiments, the threshold for is 2475 ng/ml of LGALS3BP in amniotic fluid, as determined by the methods of the present invention. According to some embodiments, an increase in the level of LGALS3BP
in the biological sample above 1500 ng/ml, above 2000 ng/ml or above 2200 ng/ml or above 2400 ng/ml or above 2475 ng/ml or above 2500 ng/ml or above 2700 ng/ml is indicative of a symptomatic cCMV infection. According to some embodiments, the threshold for LGALS3BP in amniotic fluid is from 1000 to 4000 ng/ml. According to some embodiments, the threshold for LGALS3BP in amniotic fluid is from 1200 to 3800 ng/ml, from 1500 to 3500 ng/ml, from 1700 to 3200 ng/ml, from 2000 to 3000 ng/ml, or from 2200 to 2800 ng/ml.
According to some embodiments, the sensitivity of diagnosing symptomatic cCMV
is from 85 to 99%, from 90 to 98% or from 92 to 96%. According to some embodiments, the specificity in diagnosing symptomatic cCMV is from 85 to 99%, from 90 to 98%
or from 92 to 96%. According to some embodiments, the positive predictive value of diagnosing symptomatic cCMV is from 85 to 99%, from 90 to 98% or from 92 to 96%.
According to some embodiments, the negative predictive value of diagnosing symptomatic cCMV
is from 85 to 99%, from 90 to 98% or from 92 to 96%.
100601 According to some embodiments, the method comprises determining the level of GPNMB. According to certain embodiments, an increase in the level of GPNMB in the biological sample in comparison to its level in the control is indicative of symptomatic cCMV. According to some embodiments, an increase of more than 1.5, 2, 2.5 or 3 folds in the level of GPNMB in the biological sample in comparison to its level in the control is indicative of a symptomatic cCMV infection. According to some embodiments, an increase of from 2 to 20 folds in the level of GPNMB in the biological sample in comparison to its level in the control is indicative of a symptomatic cCMV infection. According to certain embodiments, increase of from 4 to 16, 5 to 15, 8 to 12 folds is indicative of a symptomatic cCMV infection. According to some embodiment, an increase of 10% or more, 15%
or more, 20% or more, 25 % or more, 50% or more, 75% or more, above 100%, above 150%, above 200%, above 250%, above 300% of GPNMB in the biological sample in comparison to its level in the control is indicative of a symptomatic cCMV infection. According to some embodiments, an increase in the level of GPNMB of 10% or more, 15% or more, 20% or more, 25 % or more, 50% or more, 75% or more, 100% or more, or from 2 to 20 times above a threshold is indicative of a symptomatic cCMV infection.
100611 According to some embodiments, the method comprises determining the level of SYNE1. According to certain embodiments, an increase in the level of SYNE] in the biological sample in comparison to its level in the control is indicative of symptomatic cCMV. According to some embodiments, an increase in the level of SYNE1 of 10%
or more, 15% or more, 20% or more, 25 % or more, 50% or more, 75% or more, 100% or more, or from 2 to 20 times above a threshold is indicative of a symptomatic cCMV
infection.
100621 According to some embodiments, the method comprises determining the level of ANGPTL6. According to certain embodiments, a decrease in the level of ANGPTL6 in the biological sample in comparison to its level in the control is indicative of symptomatic cCMV. According to some embodiments, a decrease in the level of ANGPTL6 of 10%
or more, 15% or more, 20% or more, 25 % or more, 50% or more, 75% or more in comparison to a threshold is indicative of a symptomatic cCMV infection.
100631 According to some embodiments, the method comprises determining the level of GAS1. According to certain embodiments, a decrease in the level of GAS1 in the biological sample in comparison to its level in the control is indicative of symptomatic cCMV.
According to some embodiments, a decrease in the level of GAS1 of 10')/0 or more, 15% or more, 20% or more, 25 % or more, 50% or more, 75% or more in comparison to a threshold is indicative of a symptomatic cCMV infection.
100641 According to some embodiments, the method comprises determining the level of CHGB. According to certain embodiments, a decrease in the level of CHGB in the biological sample in comparison to its level in the control is indicative of symptomatic cCMV.
According to certain embodiments, a decrease in the level of CHGB in the biological sample of 10% or more, 15% or more, 20% or more, 25 % or more, 50% or more, 75% or more in comparison to a threshold is indicative of symptomatic cCMV.
100651 According to some embodiments, the method comprises determining the level of ADAM9. According to certain embodiments, a decrease in the level of ADAM9 in the biological sample in comparison to its level in the control is indicative of symptomatic cCMV. According to certain embodiments, a decrease in the level of ADAM9in the biological sample of 10% or more, 15% or more, 20% or more, 25 % or more, 50%
or more, 75% or more in comparison to a threshold is indicative of symptomatic cCMV.
[0066] According to some embodiments, the method comprises determining the level of at least two biomarkers selected from SYNE], MYH14, GPNMB, LGALS3BP, CPA2, RARRES2, SCGB3A2, CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS!, PPBP, and GP1BA in the biological sample.
[0067] According to some embodiments, the method comprises determining the levels of RARRES2 and LGALS3BP. According to some embodiments, an increase in the levels of RARRES2 and of LGALS3BP in the biological sample in comparison to their levels in the control is indicative of symptomatic cCMV infection. According to some embodiments, an increase of 10% or more, 15% or more, 20% or more, 25 % or more, 50% or more, 75% or more, 100% or more, or from 2 to 20 times above thresholds in the levels of RARRES2 and of LGALS3BP is indicative of a symptomatic cCMV infection. According to some embodiments, an increase in the levels of RARRES2 above 35 ng/ml, 40 ng/ml or above 50 ng/ml and an increase in the levels of LGALS3BP above 2400 ng/ml, above 2450 ng/ml or above 2475 ng/ml is indicative of a symptomatic cCMV infection. According to some embodiments, the method comprises determining the levels of RARRES2 and and an increase in the levels of RARRES2 or LGALS3BP in the biological sample in comparison to their levels in the control is indicative of symptomatic cCMV
infection.
[0068] According to some embodiments, the method comprises determining the levels of RARRES2, LGALS3BP and/or GPNMB. According to some embodiments, an increase in the levels of RARRES2 and of GPNMB in the biological sample in comparison to their levels in the control is indicative of a symptomatic cCMV infection. According to other embodiments, an increase in the levels of LGALS3BP and GPNMB in the biological sample in comparison to their levels in the control is indicative of a symptomatic cCMV infection According to some embodiments, an increase in the levels of RARRES2, LGALS3BP
or GPNMB is indicative of symptomatic cCMV infection. According to some embodiments, an increase in the levels of RARRES2, LGALS3BP and GPNMB is indicative of symptomatic cCMV infection.
[0069] According to some embodiments, the method comprises determining levels of at least three biomarkers selected from SYNE1, MYH14, GPNMB, LGALS3BP, CPA2, RARRES2, SCGB3A2, CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS1, PPBP, and GP1BA in the biological sample.

100701 According to some embodiments, the method comprises determining the level of RARRES2, LGALS3BP and at least one biomarker selected from SYNE1, MYH14, GPNMB, CPA2, SCGB3A2, CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS1, PPBP, and GP1BA. According to some embodiments, an increase in the levels of RARRES2 and of LGALS3BP, and in the level of at least one biomarker selected from SYNE1, MYH14, GPNMB, CPA2, and SCGB3A2 in the biological sample in comparison to their levels in the control is indicative of a symptomatic cCMV infection.
According to other embodiments, an increase in the levels of RARRES2 and of LGALS3BP, and a decrease in the level of at least one biomarker selected from CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS1, PPBP, and GP1BA in comparison to their levels in the control is indicative of a symptomatic cCMV infection. According to some embodiments, an increase in the levels of RARRES2, LGALS3BP and GPNMB in the biological sample in comparison to their levels in the control is indicative of a symptomatic cCMV
infection.
According to some embodiments, an increase in the levels of RARRES2, LGALS3BP
and SYNE1 in the biological sample in comparison to their levels in the control is indicative of symptomatic cCMV infection. According to some embodiments, an increase in the levels of RARRES2, LGALS3BP, GPNMB and SYNE1 in the biological sample in comparison to their levels in the control is indicative of symptomatic cCMV infection.
100711 According to some embodiments, an increase in the levels of RARRES2 and LGALS3BP and a decrease in the level of a biomarker selected from ANGPTL6, GAS], CHGB, ADAM9 or a combination thereof in the biological sample in comparison to their levels in the control is indicative of symptomatic cCMV infection. According to some embodiments, the biological sample is amniotic fluid. According to some embodiments, an increase in the levels of RARRES2 and LGALS3BP and a decrease in the level of in the biological sample in comparison to their levels in the control is indicative of symptomatic cCMV infection.
100721 According to some embodiments, an increase in the levels of RARRES2, GPNMB
and LGALS3BP and a decrease in the level of a biomarker selected from ANGPTL6, GAS!, CHGB, ADAM9 or a combination thereof in the biological sample in comparison to their levels in the control is indicative of symptomatic cCMV infection. According to some embodiments, the biological sample is amniotic fluid. According to some embodiments, an increase in the levels of RARRES2, GPNMB and LGALS3BP and a decrease in the level of ADAM9 in the biological sample in comparison to their levels in the control is indicative of symptomatic cCMV infection.

100731 Thus, according to some embodiments, an increase in the levels of at least one or at least two or at least three biomarkers selected from SYNE1, MYH14, GPNMB, LGALS3BP, CPA2, RARRES2, and SCGB3A2 in amniotic fluid in comparison to their levels in the control is indicative of a symptomatic cCMV infection in fetus. According to certain embodiments, an increase in the levels of RARRES2 and/or LGALS3BP, and optionally in the level of at least one biomarker selected from SYNE1, MYH14, GPNMB, CPA2, and SCGB3A2 in amniotic fluid in comparison to their levels in the control is indicative of a symptomatic cCMV infection in fetus.
100741 According to some embodiment, the present invention provides method of diagnosing a symptomatic congenital cytomegalovirus (symptomatic cCMV) in a fetus, wherein the method comprises (i) determining a levels of biomarkers RARRES2, LGALS3BP or both in amniotic fluid; and (ii) comparing the levels determined in (i) to their levels in a control, wherein an increase in the level of the biomarker in the biological sample in comparison to their levels in the control is indicative of presence of a symptomatic congenital cCMV infection in fetus. According to some embodiment, the present invention provides a method of diagnosing a symptomatic congenital cytomegalovirus (symptomatic cCMV) in a fetus, wherein the method comprises (i) determining a levels of biomarkers RARRES2, LGALS3BP or both in amniotic fluid; and (ii) comparing the levels determined in (i) predefined thresholds, wherein an increase in the level of the biomarker in the biological sample in comparison to the thresholds is indicative of presence of a symptomatic congenital cCMV infection in fetus. According to some embodiment, the present invention provides a method of diagnosing a symptomatic congenital cytomegalovirus (symptomatic cCMV) in a fetus, wherein the method comprises (i) determining a levels of biomarkers RARRES2, LGALS3BP, and GPNMB in amniotic fluid; and (ii) comparing the levels determined in (i) to their levels in a control or to thresholds, wherein an increase in the level of the biomarker in the biological sample in comparison to their levels in the control or above the thresholds is indicative of presence of a symptomatic congenital cCMV infection in the fetus. According to some embodiments, the threshold for RARRES2 is from 35 to 55 ng/ml and the threshold for LGALS3BP is from 2400 to 2500 ng/ml, as determined by the methods of the present invention.
100751 It is clear that different methods and assays may result in different values and equivalents of the thresholds. Nevertheless, these values can be easily correlated with the values obtained by the methods used in the present invention and therefore any such values and equivalents are encompassed by the present invention as well.

100761 According to any one of the above embodiments, the method further comprises diagnosing whether the subject, e.g. the fetus is infected with CMV. According to some embodiments, the method comprises measuring a viral load of CMV as known in the art in the biological sample, e.g. in an amniotic fluid.
[0077] According to any one of the above embodiments, the method further comprises a step of obtaining the biological sample. According to some embodiments, the step of obtaining the biological sample is effected before determining the level of at least one biomarker.
According to some embodiments, the biological sample is obtained by any known method.
According to some embodiments, amniotic fluid is collected via amniocentesis.
According to some embodiments, the biological sample is further processed before determining the level of at least one biomarker, such as purification and dilutions.
[0078] According to any one of the above embodiments, the diagnosis is made before week 21 of pregnancy. According to some embodiments, the diagnosis is performed before week 24 of pregnancy. According to some embodiments, the diagnosis is made at week 12, 13, 14, 15, 16, 17, 18, 19 or week 20 of pregnancy. According to any one of the above embodiments, the diagnosis is made at or before week 36 of pregnancy.
[0079] According to any one of the above embodiments, the biological sample is obtained before week 21 of pregnancy. According to some embodiments, the biological sample is obtained before week 24 of pregnancy. According to some embodiments, the biological sample is obtained at week 12, 13, 14, 15, 16, 17, 18, 19 or week 20 of pregnancy. According to any one of the above embodiments, the biological sample is obtained at or before week 36 of pregnancy. According to other embodiments, the biological sample is obtained at week 12 to 36 of pregnancy. According to some embodiments, the biological sample is an amniotic fluid. Thus, according to some embodiments, the amniotic fluid is obtained at week 15, 16, 17 or 18 of the pregnancy. According to some embodiments, the amniotic fluid is obtained before week 24 of pregnancy. According to certain embodiments, the sample is fetal blood or serum. According to a further embodiment, the biological sample is maternal blood or serum. According to certain embodiments, the difference in the level of the at least one biomarker in the maternal blood or serum in comparison to the control is indicative of the presence of cCMV such as symptomatic cCMV infection in fetus. As such, according to some embodiments, the diagnosis is performed on or before week 36 of pregnancy.
According to some embodiments, the diagnosis is made between week 12 to 36 of pregnancy.
[0080] According to any one of the above embodiments, the biomarker is a protein biomarker.

100811 According to the teaching of the present invention, any known method may be used for the determination of the level of the at least one biomarker. According to some embodiments, the biomarker is a protein biomarker and any method for determining the level of proteins or peptides may be used. According to some embodiment, the method comprises use of an enzyme-linked immunosorbent assay (ELISA), luminex assay, or any other immune-detection assay. According to other embodiment, the method comprises use of a mass spectroscopy. According to some embodiments, the methods are chromatography methods.
100821 According to some embodiments, an increase in the level of at least one biomarker selected from SYNE1, MYH14, GPNMB, LGALS3BP, CPA2, RARRES2, and SCGB3A2 in the biological sample in comparison to its levels in the control is indicative of a symptomatic cCMV infection. According to other embodiments, a decrease in the level of at least one biomarker selected from CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS1, PPBP, and GP1BA in the biological sample in comparison to their levels in the control is indicative of a symptomatic cCMV infection. According to another embodiment, an increase in the level of at least one biomarker of Group C and a decrease in the level of at least one biomarker of Group D in the biological sample in comparison to their levels in control is indicative of a symptomatic cCMV infection. According to some embodiments, an increase in levels of 2, 3, 4 or 5 biomarkers selected from SYNE1, MYH14, GPNMB, LGALS3BP, CPA2, RARRES2, and SCGB3A2 and/or a decrease in levels of 2, 3, 4 or biomarkers selected from CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS1, PPBP, and GP1BA in the biological sample in comparison to their levels in control is indicative of a symptomatic cCMV infection. According to some embodiments, increase in levels of a biomarker selected from RARRES2, LGALS3BP and the combination thereof in the biological sample in comparison to their levels in control is indicative of a symptomatic cCMV infection. According to some embodiments, the biological sample is amniotic fluid.
According to one embodiment, the amniotic fluid is obtained between weeks 12 to 36, or between weeks 14 to week 24 of pregnancy According to some embodiments, the level of the biomarkers is determined by immunoassay method. According to any one of the above embodiments, the biomarker is a protein biomarker.
100831 According to some embodiments, the present invention provides a method of providing an indication useful in distinguishing between subjects infected with cCMV and uninfected, comprising (i) determining the level of at least one biomarker in a biological sample; and (ii) comparing the level of the at least one biomarker from the sample to its level in a control, wherein a difference in the levels of the biomarker in the biological sample and in the control provides an indication useful in distinguishing between infected and uninfected subjects, wherein at least one biomarker is selected from a biomarker of Group A and Group B. According to some embodiments, the subject is a fetus. According to some embodiments, the biological sample is amniotic fluid.
[0084] According to some embodiments, the present invention provides a method of providing an indication useful in distinguishing between subjects having a symptomatic cCMV and asymptomatic cCMV subjects, comprising (i) determining a level of at least one biomarker in a biological sample; and (ii) comparing the level of the at least one biomarker from the sample to its level in a control, wherein a difference in the level of the biomarker in the biological sample and in the control provides an indication useful in distinguishing between symptomatic and asymptomatic cCMV or uninfected subjects, wherein at least one biomarker is selected from a biomarker of Group C and Group D. According to some embodiments, the subject is a fetus. According to some embodiments, the biological sample is amniotic fluid.
[0085] According to some embodiments, the present invention provides a method or diagnosing an asymptomatic cCMV infection in a subject, such as fetus.
Typically the presence of infection in fetus may be carried out by the methods of the present invention or by any known method. Once it is identified than the fetus has CMV infection, it is possible .. according to the teaching of the present invention to diagnose whether the subject has a symptomatic or asymptomatic CMV. In case the fetus is infected but is not diagnosed as having a symptomatic CMV, said fetus is identified/diagnosed as having asymptomatic CMV. In such a case a preventive treatment may be administered or adjusted.
Thus, according to some embodiments, the present invention provides a method of diagnosing an asymptomatic cCMV infection in a fetus comprising (i) diagnosing whether the fetus is infected with cCMV and (ii) diagnosing whether the fetus has a symptomatic cCMV
according to the methods of the present invention. In case the fetus is infected with cCMV
but is not diagnosed as having symptomatic cCMV said fetus is diagnosed as having an asymptomatic cCMV. Any method for diagnosing whether the fetus is infected with cCMV
can be used. Examples of such methods is as defined by the methods of the present invention or determining viral load e.g. by PCR. Therefore, according to some embodiments, the present invention provides a method of diagnosing an asymptomatic cCMV
infection in fetus comprising (i) determining a level of at least one biomarker in a biological sample of an infected subject; and (ii) comparing the level of the at least one biomarker from the biological sample to its level in a control, wherein a lack of statistically significant difference in the level of the biomarker in the biological sample and in the control is indicative of presence of asymptomatic cCMV infection in the fetus, wherein the at least one biomarker is selected from the biomarkers of Group C and Group D. According to some embodiments, the present invention provides a method of diagnosing an asymptomatic cCMV infection in the infected fetus comprising (i) determining a level of at least one biomarker in the biological sample;
and (ii) comparing the level of the at least one biomarker from the biological sample to its level in a control, wherein a lack of statistically significant difference in the level of the biomarker in the biological sample and in the control is indicative of presence of asymptomatic cCMV infection in the fetus, wherein the at least one biomarker is selected from the biomarkers of Group C and Group D and the biological sample is an amniotic fluid.
[0086] According to another aspect, the present invention provides a method for recommending a medical intervention, the method comprises detecting whether a subject has a congenital cytomegalovirus according to the methods of the present invention and recommending the medical intervention based on the obtained results. Any terms and definition according to the previous aspects and embodiment are valid and implemented in this aspect as well. Thus, according to some embodiments, the present invention provides a method of diagnosing and recommending a medical intervention, wherein the method comprises (i) determining a level of at least one biomarker in a biological sample; (ii) comparing the level of the at least one biomarker from the biological sample to its level in a control, wherein a difference in the level of the biomarker in the biological sample and in the control is indicative of a congenital CMV infection; and (iii) providing a recommendation of a medical intervention based on the obtained results, wherein the biomarker is selected from CTSB, SIRPB1, LGALS3BP, AGRN, VCAM1, COTL1, CD5L, ATP5B, KRT6A, SECTM1, FABP3, S100A6, SH3BGRL, ISG15, CRTAC1, RARRES2, BST2, B2M, EPYC, DKK1, HLA-C, CPQ, F5, GPNMB, LAMAS, CA3, DES, MEGF8, GALNT7, FAT4, LPHN2, AFM, MASP1, SOD3, GALNT2, LSR, HGF, PRG2, QS0X1, ERVMER34-1, PAPPA2, GOT1, RDX, IGFBP5, AHNAK, ADAM9, LAMP2, CPM, SBSN, CAST, SPINK5, 'T'NMD, CDH15, KLKB1, PRSS8, COL3A1, COL1A2, SYNE1, MYH14, GPNMB, LGALS3BP, CPA2, RARRES2, SCGB3A2, CPM, PDGFRB, ANGPTL6, DM:KN, CHGB, ADAM9, GAS1, PPBP, and GP1BA, and any combination thereof.
According to some embodiment, the present invention provides a method of recommending a medical intervention, wherein the method comprises (i) determining a level of at least one biomarker in a biological sample; (ii) comparing the level of the at least one biomarker from the biological sample to its level in a control, wherein a difference in the level of the biomarker in the biological sample and in the control is indicative of a congenital CMV
infection; and (iii) providing a recommendation of a medical intervention based on the obtained results, wherein the biomarker is selected from CTSB, SIRPB1, LGALS3BP, AGRN, VCAM1, COTL1, CD5L, ATP5B, KRT6A, SECTM1, FABP3, S100A6, SH3BGRL, ISG15, CRTAC1, RARRES2, BST2, B2M, EPYC, DKK I, HLA-C, CPQ, F5, GPNMB, LAMM, CA3, DES, MEGF8, GALNT7, FAT4, LPHN2, AFM, MASP1, SOD3, GALNT2, LSR, HGF, PRG2, QSOXI, ERVMER34-1, PAPPA2, GOT!, RDX, IGFBP5, AHNAK, ADAM9, LAMP2, CPM, SBSN, CAST, SPINK5, 'T'NMD, CDH15, KLKB1, PRSS8, C0L3A 1 , COL1A2, SYNE1, MYH14, GPNMB, LGALS3BP, CPA2, RARRES2, SCGB3A2, CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS1, PPBP, and GP1BA, and any combination thereof. According to some embodiments, the cCMV is a symptomatic cCMV. Thus, according to some embodiments, the present invention provides a method of recommending a medical intervention, wherein the method comprises (i) determining a level of at least one biomarker in a biological sample; (ii) comparing the level of the at least one biomarker from the biological sample to its level in a control, wherein a difference in the level of the biomarker in the biological sample and in the control is indicative of a congenital CMV infection; and (iii) providing a recommendation of a medical intervention based on the obtained results, wherein the biomarker is selected from biomarkers of Group C and Group D. According to some embodiments, the cCMV is an asymptomatic CMV. Thus, according to some embodiments, the present invention provides a method of recommending a medical intervention, wherein the method comprises (i) detecting whether the fetus has an asymptomatic CMV according to any one of the above aspects and embodiments and (ii) providing a recommendation of a medical intervention based on the obtained results. According to some embodiments, the intervention comprises monitoring. According to some embodiments, an increase in the level of at least one biomarker selected from SYNE1, MYH14, GPNMB, LGALS3BP, CPA2, RARRES2, and SCGB3A2 in the biological sample in comparison to its levels in the control is indicative of a symptomatic cCMV infection. According to other embodiments, a decrease in the level of at least one biomarker selected from CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS1, PPBP, and GP1BA in the biological sample in comparison to their levels in the control is indicative of a symptomatic cCMV infection. According to another embodiment, an increase in the level of at least one biomarker of Group C and decrease in the level of at least one biomarker of Group D in the biological sample in comparison to their levels in control is indicative of a symptomatic cCMV infection. According to some embodiments, an increase in levels of 2, 3, 4 or 5 biomarkers selected from SYNE1, MYH14, GPNIv1B, LGALS3BP, CPA2, RARRES2, and SCGB3A2 and/or a decrease in levels of 2, 3, 4 or biomarkers selected from CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS1, PPBP, and GP1BA in the biological sample in comparison to their levels in control is indicative of a symptomatic cCMV infection. According to some embodiments, an increase in levels of RARRES2 and LGALS3BP in the biological sample in comparison to their levels in control is indicative of a symptomatic cCMV infection. According to some embodiments, the biological sample is amniotic fluid. According to one embodiment, the amniotic fluid is obtained between week 12 to week 36 or between week 14 to week 24 of pregnancy.
According to some embodiments, the method further comprises a step of obtaining the biological sample. According to some embodiments, the step of obtaining the biological sample is effected before determining the level of at least one biomarker.
100871 According to some embodiments, the medical intervention is selected from a treatment of cCMV, cessation of treatment of cCMV, adjusting treatment, watchful waiting, monitoring or recommendation to terminate the pregnancy. According to some embodiments, the treatment is pregnancy termination. According to some embodiments, the term "adjusting treatment" contemplates cessation of treatment or changing the administered dose or changing the treatment. According to some embodiments, the medical intervention comprises treatment and/or monitoring of a symptomatic cCMV. According to some embodiments, the medical intervention comprises treatment and/or monitoring of an asymptomatic cCMV.
100881 According to some embodiments, the present invention provides a method of providing a medical intervention in case of presence of symptomatic cCMV in fetus comprising detecting whether a fetus has a symptomatic cCMV by the methods of the present invention and treating the symptomatic cCMV, wherein medical intervention comprises treatment using an anti-CMV compound or pregnancy termination. According to some embodiments, the present invention provides providing a medical intervention in case of presence of symptomatic cCMV in fetus comprising: (I) detecting whether a fetus has a symptomatic cCMV by (i) determining a level of at least one biomarker in a biological sample; (ii) comparing the level of the at least one biomarker from the biological sample to its level in a control, wherein a difference in the level of the biomarker in the biological sample and in the control is indicative of a congenital CMV infection, and wherein the biomarker is selected from biomarkers of Group C and Group D; and (II) based on the result of (I) providing a medical intervention comprising treating cCMV or pregnancy termination.
[0089] According to some embodiments, the treatment of cCMV comprises treatment using an anti-CMV compound selected from an antiviral drug and antibodies against CMV.
According to one embodiment, the antiviral drug is valacyclovir or letermovir.
According to some embodiment, the treatment comprises administering to the mother the anti-CMV
compound. According to other embodiments, the treatment comprises administering the fetus the anti-CMV compound.
[0090] According to some embodiments, the present invention provides a method of providing a medical intervention in case of presence of asymptomatic cCMV in fetus comprising detecting whether a fetus has an asymptomatic cCMV by the methods of the present invention and treating the symptomatic cCMV, wherein medical intervention comprises treatment using an anti-CMV compound. According to some embodiments, the present invention provides providing a medical intervention in case of presence of asymptomatic cCMV in fetus comprising (I) determining whether the fetus in infected with CMV, (II) detecting whether a fetus has a symptomatic cCMV by (i) determining a level of at least one biomarker in a biological sample; (ii) comparing the level of the at least one biomarker from the biological sample to its level in a control, wherein a difference in the level of the biomarker in the biological sample and in the control is indicative of a congenital CMV infection, and wherein the biomarker is selected from biomarkers of Group C and Group D, and (111) based on the result of (I) and (II) providing a medical intervention comprising treating cCMV or cessation of treatment of the cCMV. Determining whether the fetus is infected by CMV may be performed by the methods of the present invention or by any other methods such as determining the viral load of CMV by e.g. PCR.
[0091] The terms "treating", "therapy" or "treatment" a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results.
Beneficial or desired clinical results include, but are not limited to, or ameliorating abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially ameliorating or alleviating clinical or esthetical symptoms of a condition, substantially preventing the appearance of clinical or esthetical symptoms of a disease, condition, or disorder, and protecting from harmful or annoying symptoms.
Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated;

(d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s);
and/or (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s).
100921 The term "administering" or "administration of' a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered intrauterinally, intra umbilical cord,intravenously, arterially, intradermally, intramuscularly, intraperitonealy, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent.
Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. According to some embodiments, the composition is administered 1, 2, 3, 4, 5 or 6 times a day. According to other embodiments, the composition is administered 1, 2, 3, 4, 5 or 6 times a month. In some embodiments, the administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug, or to have the drug administered by another and/or who provides a patient with a prescription for a drug is administering the drug to the patient.
100931 According to another aspect, the present invention provide a method of treating a symptomatic or asymptomatic congenital cytomegalovirus in a fetus comprising diagnosing the symptomatic or asymptomatic cCMV in fetus according to the methods of the present invention and administering to the fetus or to the mother an anti-CMV active agent.
100941 According to another aspect, the present invention provides a method of determining the efficacy of the prevention or treatment of a symptomatic or asymptomatic congenital cytomegalovirus (cCMV) infection, the method comprises the steps of: (i) determining a level of at least one biomarker in a biological sample, wherein the at least one biomarker is selected from a biomarker of Group C and Group D; and (ii) comparing the level of the at least one biomarker from the biological sample to the level of the biomarker in a corresponding biological sample obtained from the same subject at an earlier sampling and/or to the level of the biomarker in the control, wherein a change in the level of the biomarkers in two consecutive measurements is indicative of efficacy of the prevention or therapy of the symptomatic or asymptomatic cCMV.
[0095] According some embodiments, the present invention provides a method of determining efficacy of prevention of a congenital cytomegalovirus (cCMV) infection or disease, the method comprising the steps of: (i) determining the level of at least one biomarker in a biological sample, wherein the at least one biomarker is selected from a biomarker of Group A, Group B, Group C or Group D; and (ii) comparing the level of the at least one biomarker from the biological sample to the level of the biomarker in the control, wherein the efficacy of the prevention is evaluated according to the change or lack of change in the level(s) of the biomarker(s). Any terms and definitions according to previous aspects and embodiment are valid and implemented in this aspect as well.
[0096] According to another embodiment, the present invention provides a method of determining the efficacy of therapy of a congenital cytomegalovirus (cCMV) infection or disease, the method comprising the steps of: (i) determining the level of at least one biomarker in a biological sample, wherein the at least one biomarker is selected from a biomarker of Group A, Group B, Group C or Group D; and (ii) comparing the level of the at least one biomarker from the biological sample to the level of the biomarker in a corresponding biological sample obtained from the same subject at an earlier sampling or to the level of the biomarker in the control, wherein the efficacy of the therapy is evaluated according to the change or lack of change in the level(s) of the biomarker(s).
Any terms and definition according to previous aspects and embodiment are valid and implemented in this aspect as well.
[0097] According to one embodiment, the present invention provides a method of determining efficacy of prevention of a symptomatic cCMV infection or disease, the method comprising the steps of: (i) determining the level of at least one biomarker in a biological sample, wherein the at least one biomarker is selected from a biomarker of Group C or Group D; and (ii) comparing the level of the at least one biomarker from the biological sample to the level of the biomarker in the control or in a corresponding biological sample obtained from the same subject at an earlier sampling, wherein the efficacy of the prevention is evaluated according to the change or lack of change in the level(s) of the biomarker(s).
[0098] According to another embodiment, the present invention provides a method of determining the efficacy of therapy of a symptomatic cCMV, the method comprising the steps of: (i) determining the level of at least one biomarker in a biological sample, wherein the at least one biomarker is selected from a biomarker of Group C or Group D;
and (ii) comparing the level of the at least one biomarker from the biological sample to the level of the biomarker in a corresponding biological sample obtained from the same subject at an earlier sampling or to the level of the biomarker in the control, wherein the efficacy of the therapy is evaluated according to the change or lack of change in the level(s) of the biomarker(s) between subsequently obtained biological samples.
100991 According to one embodiment, the present invention provides a method of determining efficacy of prevention of an asymptomatic cCMV infection or disease, the method comprising the steps of: (i) determining the level of at least one biomarker in a biological sample, wherein the at least one biomarker is selected from a biomarker of Group C or Group D; and (ii) comparing the level of the at least one biomarker from the biological sample to the level of the biomarker in a corresponding biological sample obtained from the same subject at an earlier sampling or to its level in the control, wherein the efficacy of the prevention is evaluated according to the change or lack of change in the level(s) of the biomarker(s).
101001 As used herein, the term "preventing" when used in relation to a condition, refers to administration of a composition which reduces the frequency of, or delays the onset of, symptoms of the medical condition in a subject relative to a subject which does not receive the composition.
101011 According to some embodiments, decreasing the difference between the level(s) of the biomarker(s) in the biological sample and its/their level in a control corresponds to effective treatment. According to some embodiments, cessation of the increase of the difference between the level(s) of the biomarker(s) in the biological sample and its/their level in a control corresponds to effective treatment.
101021 According to another aspect, the present invention provides a kit comprising means for determining a level of at least one biomarker in a biological sample, and instructions for use, wherein the biomarker is selected from CTSB, SIRPB1, LGALS3BP, AGRN, VCAM
I, COTL1, CD5L, ATP5B, KRT6A, SECTM1, FABP3, S100A6, SH3BGRL, ISG15, CRTAC I, RARRES2, BST2, B2M, EPYC, DKK1, HLA-C, CPQ, F5, GPNMB, LAMAS, CA3, DES, MEGF8, GALNT7, FAT4, LPHN2, AFM, MASPI, SOD3, GALNT2, LSR, HGF, PRG2, QS0X1, ERVMER34-1, PAPPA2, GOT1, RDX, IGFBP5, AHNAK, ADAM9, LAMP2, CPM, SBSN, CAST, SPINK5, 'TNMD, CDH15, KLKB1, PRSS8, COL3A1, C0L1A2, SYNEI, MYH14, GPNMB, LGALS3BP, CPA2, RARRES2, SCGB3A2, CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS1, PPBP, GP1BA and any combination thereof. According to another embodiment, the kit comprises means for determining a level of at least one biomarker in a biological sample, and instructions for use, wherein the biomarker is selected from SYNE1, MYH14, GPNMB, LGALS3BP, CPA2, RARRES2, SCGB3A2, CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS1, PPBP, GP1BA and any combination thereof According to some embodiments, the kit comprises means for comparing the level of the biomarker in the biological sample to its level in a control. According to some embodiments, the kit comprises means for comparing the level of the biomarker in the biological sample to its level in another biological sample.
According to some embodiments, the kit comprises means for comparing the level of the biomarker in the biological sample to a predefined threshold.
[0103] According to some embodiments, the kit is an assay kit. According to any one of the above embodiments, the kit is a diagnostic kit.
[0104] According to some embodiments, the instructions comprise instructions for diagnosing a congenital cytomegalovirus (cCMV) infection. According to some embodiments, the instructions comprise instructions for diagnosing a symptomatic congenital cytomegalovirus (cCMV) infection. According to some embodiments, the instructions comprise levels of the at least one biomarker in the control.
According to some embodiments, the instructions comprise instructions of comparing the levels in the biological sample to the levels in the control or in another biological sample. According to some embodiments, the instructions comprise instructions of comparing the levels of the biomarkers in the biological sample to a predefined threshold. According to some embodiments, the kit comprises means for determining the levels of the at least one biomarker in the control or in another biological sample. According to a further embodiment, the instructions comprise means to differentiate between symptomatic and asymptomatic infected subjects. According to some embodiments, the cCMV is a symptomatic cCMV.
According to some embodiments, the means for determining the level of at least one biomarker comprises an agent specific for determining the level of the at least one biomarker.
According to certain embodiments, the agent is an antibody capable of binding to the at least one biomarker or a combination of antibodies. According to some embodiments, the antibodies are selected from monoclonal antibodies, secondary antibody capable of binding an antibody specific to the at least one biomarker, tagged antibody such as fluorescently tagged, and any combination thereof.
[0105] According to some embodiments, the means for determining the level of at least one biomarker in a biological sample is an immunoassay kit. According to some embodiments, the means is ELISA kit. According to some embodiments, the means are means for performing ELISA. According to some embodiments, the means are means for performing immunoassay test. Thus, according to some embodiments, the present invention provides a kit comprising at least one ELISA kit for determining the level of at least one biomarker in a biological sample, and instructions for use, wherein the biomarker is selected from a biomarker of Group A, Group B, Group C and Group D. Thus, according to some embodiments, the present invention provides a kit comprising at least one ELISA kit for determining the level of at least one biomarker in a biological sample, and instructions for use, wherein the biomarker is selected from a biomarker of Group C and Group D. According to other embodiments, the means is any method capable of determining levels of a plurality of biomarkers, such as luminex.
[0106] According to some embodiments, the present invention provides a kit comprising means for determining a level of at least one biomarker from biomarkers of Group C and D
in a biological sample, and instructions for use in diagnosis symptomatic congenital CMV
infection. According to one embodiment, the present invention provides a kit comprising means for determining the levels of 2, 3, 4, 5, 6, 7 or 8 said biomarkers.
According to some embodiments, the present invention provides a kit comprising means for determining a level of at least one biomarker from biomarkers of Group C and D in a biological sample, and instructions for use in diagnosis asymptomatic congenital CMV infection wherein the instructions to diagnose asymptomatic CMV comprise instructions to detect whether the subject is infected with CMV and negating that the subject has a symptomatic CMV.
[0107] According to some embodiments, the kit of the present invention comprises means for determining the level of RARRES2 in the biological sample. According to other embodiments, the kit of the present invention comprises means for determining the level of LGALS3BP. According to one embodiment, the kit of the present invention comprises means for determining the levels of RARRES2 and LGALS3BP. According to some embodiments, the kit comprises means for determining the level of GPNMB. According to some embodiments, the kit comprises means for determining the levels of RARRES2 and GPNMB
or LGALS3BP and GPNMB. According to one embodiment, the kit of the present invention comprises means for determining the levels of RARRES2, LGALS3BP and GPNMB.
According to some embodiments, the kit comprises means for determining the level of SYNE1. According to some embodiments, the kit comprises means for determining the level of ANGPTL6. According to some embodiments, the kit comprises means for determining the level of GAS1. According to some embodiments, the kit comprises means for determining the level of CHGB. According to some embodiments, the kit comprises means for determining the level of ADAM9. According to some embodiments, the kit of the present invention further comprises means for determining a level of at least one biomarker selected from SYNE1, MYH14, GPNMB, CPA2, SCGB3A2, CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS1, PPBP, and GP IBA. Thus, according to some embodiments, the kit of the present invention comprises means for determining levels of RARRES2 and LGALS3BP and further comprises means for determining a level of at least one biomarker selected from SYNE], MYH14, GPNMB, CPA2, SCGB3A2, CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS!, PPBP, and GP IBA. According to some embodiments, the means is ELISA kit. Thus, according to some embodiments, the present invention provides a kit comprising at least one ELISA kit for determining the level of at least one biomarker in a biological sample, and instructions for use, wherein the biomarker is selected from RARRES2, LGALS3BP and a combination thereof. According to other embodiments, the kit of the present invention further comprises ELISA kit for determining the level at least one biomarker selected from SYNE1, MYH14, GPNMB, CPA2, SCGB3A2 in the biological sample. According to any one of the above embodiments, the kit comprises any other means to determine the levels of a plurality of biomarkers, instead of ELISA kit.
101081 According to any one of the above embodiments, the kit further comprises reference levels of the biomarkers as in the control or means to determine the reference levels of the biomarkers or numerical values of the levels of the biomarker in the control or the threshold value of the biomarker above or below which the subject is diagnosed as having a cCMV
and/or having a symptomatic cCMV. The terms "reference level" and "threshold"
are used herein interchangeably and refer to a level of the biomarker in the control above which or below which or in comparison to which a subject is diagnosed according to the teaching of the present invention, i.e. having cCMV infection and/or having a symptomatic cCMV
infection.
101091 According to some embodiments, the kit further comprises means for processing the biological sample. According to some embodiments, the processing the biological sample comprises any procedure to allow measurement or improve measurement of the levels of the biomarkers, such as purification, dilution, concentration, separation of biomarkers etc.
101101 According to some embodiment, the kit is for use in diagnosing a congenital CMV
infection. According to some embodiments, the cCMV is a symptomatic CMV, thus the kit is for use in diagnosing or detecting a symptomatic congenital CMV infection.
According to other embodiments, the cCMV is an asymptomatic CMV, thus the kit is for use in diagnosing or detecting an asymptomatic congenital CMV infection.

101 1 1] According to some embodiment, the present invention provides a use of a kit comprising means for determining the level of at least one biomarker in a biological sample, in the diagnosis of a congenital CMV, wherein the at least one biomarker is selected from a biomarker of Group A, Group B, Group C and Group D. According to some embodiments, the cCMV is a symptomatic cCMV. According to some embodiments, the at least one biomarker is selected from a biomarker of Group C and Group D. According to a further embodiment, the instructions comprise means to differentiate between symptomatic and asymptomatic infected subjects.
101121 According to some embodiments, the present invention provides a use of a kit comprising means for determining the level of at least one biomarker in a biological sample, in diagnosis of a symptomatic congenital CMV, wherein the at least one biomarker is selected from a biomarker of Group C and Group D.
101131 According to some embodiments, the present invention provides a use of a kit for diagnosing symptomatic cCMV, wherein the kit comprises means for determining levels of RARRES2 and/or LGALS3BP in a biological sample. According to yet another embodiment, the kit further comprises means for determining the levels of at least one biomarker selected from SYNE I, MYH14, GPNMB, CPA2, SCGB3A2, CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS], PPBP, and GP1BA. According to yet another embodiment, the means for determining the level of at least one biomarker in a biological sample is ELISA.
101141 The terms "comprising", "comprise(s)", "include(s)", "having", "has"
and "contain(s)," are used herein interchangeably and have the meaning of "consisting at least in part of'. When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present.
Related terms such as "comprise" and "comprises" are to be interpreted in the same manner.
The terms "have", "has", having" and "comprising" may also encompass the meaning of "consisting of' and "consisting essentially of', and may be substituted by these terms. The term "consisting of' excludes any component, step or procedure not specifically delineated or listed. The term "consisting essentially of' means that the composition or component may include additional ingredients, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed compositions or methods.
101151 Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
[0116] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
[0117] Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.
EXAMPLES
[0118] Example 1.
[0119] Methods [0120] Amniotic fluid samples were obtained from women with primary CMV
infection at .. 21-23 weeks' gestation as part of the routine diagnosis of fetal infection.
[0121] We analyzed the proteome of 24 amniotic fluid samples: 14 CMV-positive samples and 10 CMV-negative samples. Of the CMV-positive samples, 8 were samples of asymptomatic fetuses (who were born as asymptomatic neonates and remained asymptomatic for at least one year after birth), and 6 were of CMV-positive symptomatic fetuses who were terminated in view of severe cerebral lesions. At a later stage, the validation cohort was extended to include 21 symptomatic and 28 asymptomatic samples and 15 CMV-negative samples. All samples were analyzed in parallel to avoid assay-to-assay variations.
[0122] Sample preparation [0123] The samples (150 microliters) were loaded onto a specific column for serum depletion, followed by an in-solution tryptic digestion and a desalting step.
101241 Liquid chromatography mass spectrometry 101251 The resulting peptides were analyzed using nanoflow liquid chromatography (nanoAcquity) coupled to high resolution, high mass accuracy mass spectrometry (Q

Exactive HFX). Each sample was analyzed on the instrument separately in a random order in discovery mode.
[0126] Data processing 101271 Raw data was processed with MaxQuant v1.6Ø16. The data was searched with the Andromeda search engine against the human and HCMV proteome databases appended with common lab protein contaminants and the following modifications:
Carbamidomethylation of C as a fixed modification and oxidation of Ni, deamidation of N and Q and protein N-terminal acetylation as variable ones.
[0128] The LFQ (Label-Free Quantification) intensities were extracted and used for further calculations using Perseus v1.6Ø7. Decoy hits were filtered out, as well as proteins that were identified on the basis of a modified peptide only and GO annotations added. The LFQ
intensities were log transformed and only proteins that had at least 7 valid values in at least one experimental group were kept. The remaining missing values were imputed.
[0129] Validation assays and panels:
[0130] We have validated the proteome findings using specific protein ELISA
assays.
[0131] We have validated the RARRES2 (chemerin) and LGALS3BP proteome analysis in amniotic fluid samples using a RARRES2 and LGALS3BP protein ELISA assays. The assays used were: Quantikine ELISA assays for Human Galectin-3BP/M AC-2BP
Immunoassay and Quantikine ELISA Human Chemerin Immunoassay (R&D systems).
[0132] Results [0133] In total, we identified and quantified 1066 proteins in the fluid samples. All proteins were of human origin. We obtained qualitative and quantitative data for each identified protein, along with the relevant relative intensity comparisons between samples. The fold change for each comparison was calculated based on the protein LFQ intensity for each experiment. We grouped the samples into: Uninfected, sick (symptomatic) and not sick (infected asymptomatic) ¨ to compare between all 3 (ANOVA) and the pairs, and p-values for the Infected ¨ sick (symptomatic) to infected ¨ not sick (asymptomatic) comparison were calculated.
[0134] Comparison of the amniotic fluid proteins content of the discovery cohort led to the identification of proteins that were differentially excreted between CMV-positive and CMV-negative cases.
[0135] Comparison of the amniotic fluid proteins content of the discovery cohort led to the identification of proteins that were differentially excreted between CMV-positive (infected) and CMV-negative (not infected) cases (Fig. 1) and Table 1.

101361 Table 1. Proteins that were differentially excreted between CMV-positive (infected) and CMV-negative (not infected) subjects.
Increased Decreased Gene names Protein names Gene names Protein names th CTSB Cathepsin B MEGF8 Multiple epidermal grow factor-like domains protein 8 N-Signal-regulatory SIRPB1 GALNT7 acetylgalactosaminyltransferase protein beta-1 Galectin-3-binding LGALS3BP FAT4 Protocadherin Fat 4 protein AGRN Agiin LPHN2 Latrophilin-2 Vascular cell VCAM1 AFM Afamin adhesion protein I
Coactosin-like Mannan-binding lectin serine COTL I MASP I
protein protease 1 Extracellular superoxide CD5L CD5 antigen-like SOD3 dismutase [Cu-Zn]
ATP synthase Polypeptide N-ATP5B subunit beta, GALNT2 acetylgalactosaminyltransferase mitochondria' 2 Keratin, type 11 Lipolysis-stimulated lipoprotein cytoskeletal 6A receptor Secreted and SECTM I transmembrane HO' Hepatocyte growth factor protein I
Fatty acid-binding FABP3 PRG2 Bone marrow proteoglycan protein, heart S100A6 Calcyclin QS0X1 Sulfhydry,1 oxidase 1 SH3 domain-binding Endogenous retrovirus group SH3BGRL glutamic acid-rich- MER.34 member 1 Env like protein polyprotein Interferon-stimulated TSG15 PAPPA2 Pappalysin-2 gene 15 Cartilage acidic A spartate ant inotransfera se, protein I cytoplasmic Retinoic acid RARRES2 receptor responder RDX Radixin protein 2 Bone marrow Insulin-like growth factor-stromal antigen 2 binding protein 5 Beta-2-B2M AHNAK Desmoyokin microglobulin Disintegrin and EPYC Epiphycan ADAM9 metalloproteinase domain-containing protein 9 Dickkopf-related Lysosome-associated protein 1 membrane glycoprotein 2 Major HLA-C Histocompatibility CPM Carboxypeptidase M
Complex, Class 1, C
CPQ Carboxypeptidase Q SBSN Suprabasin F5 Coagulation factor V CAST Calpastatin GPNMB
Transmembrane SP1NK5 Serine protease inhibitor Kazal-glycoprotein NMB type 5 Laminin subunit LAMAS TNMD Tenomodulin alpha-5 Carbonic anhydrase CA.3 CDH15 Cadherin- 15 DES Desmin KLKB I Plasma kallikrein PRSS8 Prostasin _______________________________________________ COL3 Al Collagen alpha-1(11D chain COL1A2 Collagen alpha-2(I) chain 101371 Importantly, we identified a set of proteins which demonstrated a clearly distinct abundance between severely symptomatic and asymptomatic cases in the amniotic fluid samples of 14 fetuses infected with CMV (Fig. 2A). In fact, the secretion pattern of these identified proteins in infected asymptomatic fetuses appeared to resemble the pattern observed CMV-negative samples (Fig. 2B and Table 2), further supporting their potential role in disease discrimination.
101381 Table 2. Proteins that were differentially excreted between symptomatic CMV
(infected) and asymptomatic (infected) CMV
Increased Decreased Protein names Gene names Protein names Gene names SYNE1 Nesprin-1 CPM Carboxypeptidase M
Platelet-derived MYI114 Myosin-14 PDGERB growth factor receptor beta GPNMB brane Transmem Angiopoietin-glycoprotein NMB related protein 6 Galectin-3-binding LGALS3BP DMKN Dermokine protein CPA2 Carboxypeptidase A2 CHGB Secretogranin-1 Disintegrin and Retinoic acid receptor ADAM9 metalloproteinase .
responder protein 2 domain-containing protein 9 =

Secretoglobin family 3A GAS1 Growth arrest-member 2 specific protein 1 PPBP Platelet basic protein Platelet GP1BA glycoprotein lb alpha chain 101391 One of the promising markers is RARRES2 protein. First, we have measured the RARRES2 concentrations in the 31 available amniotic fluid samples (14 symptomatic and 17 asymptomatic cases). This assay confirmed the significant differences between the concentrations of RARRES2 in amniotic fluid samples of symptomatic versus asymptomatic cCMV cases. We have further expanded the validation cohort to include additional amniotic fluid samples from the two categories (symptomatic/asymptomatic) to yield a total of 21 symptomatic and 28 asymptomatic cases. This validation cohort of 49 samples from CMV-infected fetuses included 22 external blinded-samples (obtained from Italy) from the 2 categories (10 symptomatic and 12 asymptomatic). The results are presented in Fig. 3.
101401 The combined assay results (performed for a total of 20 symptomatic and asymptomatic cases) confirmed the significant differences between the concentrations of RARRES2 in amniotic fluid samples of symptomatic versus asymptomatic cCMV
cases (p<0.001; Mann-Whitney test and student T-test), and allowed for the definition of cutoff concentration values for reliable prenatal disease prediction: (for example, sensitivity of 95.2% or 90.5%, specificity of 92.9% or 96.4%, positive predictive value of 90.9% or 95.0%, and negative predictive value of 96.3% or 93.1% for cutoff concentrations of 40 ng/ml or 51 ng/ml, respectively).
101411 We have also validated the LGALS3BP (Galectin-3-binding protein) proteome analysis in amniotic fluid samples using a LGALS3BP protein ELISA assay.
101421 To this end, first we have measured the LGALS3BP concentrations in the available amniotic fluid samples from the discovery panel (the same samples that had been subjected to proteome analysis: 4 amniotic fluid samples of symptomatic cCMV, 8 amniotic fluid samples of asymptomatic cCMV, and 8 amniotic fluid samples of uninfected fetuses).
This assay confirmed the significant differences between the concentrations of in amniotic fluid samples of symptomatic versus asymptomatic cCMV cases. We have further expanded the validation cohort to include additional amniotic fluid samples from the two categories (symptomatic/asymptomatic) to yield a total of 21 symptomatic and 28 asymptomatic cases. This validation cohort of 49 samples from CMV-infected fetuses included 22 external blinded-samples (obtained from Italy) from the 2 categories (10 symptomatic and 12 asymptomatic). The results are presented in Fig. 4.

[0143] The combined assay results (performed for a total of 20 symptomatic and asymptomatic cases) confirmed the significant differences between the concentrations of LGALS3BP in amniotic fluid samples of symptomatic versus asymptomatic cCMV
cases (p<0.001; Mann-Whitney test and student T-test), and allowed for the definition of cutoff concentration values for reliable prenatal disease prediction: (for example, sensitivity of 90.5%, specificity of 96.4%, positive predictive value of 95.0%, and negative predictive value of 93.1% for a cutoff concentration of 2475 ng/ml).
[0144] Conclusions [0145] Our findings identify novel protein-based biomarkers that allow reliable prenatal discrimination between CMV-infected and uninfected and between moderate-to-severely symptomatic and asymptomatic fetuses and neonates with cCMV infection, and can therefore serve to identify fetuses at risk for cCMV disease. Specifically it was shown that detecting the concentration of RARRES2 above 40 ng/ml in amniotic fluid allows diagnosing a symptomatic cCMV with sensitivity of 95.2%, specificity of 92.9%, positive predictive value of 90.9%, and negative predictive value of 96.3% %. In additional it was shown that detection of LGALS3BP in amniotic fluid above 2475 ng/ml and allowed for reliable prenatal disease prediction with sensitivity of 90.5%, specificity of 96.4%, positive predictive value of 95.0%, and negative predictive value of 93.1%. It is clear that use of both these markers allows even higher levels of specificity and sensitivity. These biomarkers assays would be valuable in prenatal counseling and clinical studies in the setting of intrauterine CMV infection. The findings could also provide new insights into the pathogenesis of cCMV disease, and lead to the development of new therapeutic interventions.
[0146] Although the present invention has been described herein above by way of preferred .. embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

Claims

PCT/11,2021/050125

1. A method of diagnosing a symptomatic congenital cytomegalovirus (symptomatic cCMV) in a fetus, wherein the method comprises (i) determining a level of at least one biomarker in a biological sample; and (ii) comparing the level of the at least one biomarker from the biolocal sample to its level in a control, wherein a difference in the level of the biomarker in the biological sample and in the control is indicative of presence of the symptomatic cCMV infection, wherein the at least one biomarker is selected from the biomarkers of Group C and Group D, wherein:
Group C comprises biomarkers RARRES2, LGALS3BP, GPNMB, Nesprin-1 (SYNE] ), Myosin-14 (MYH14), Carboxypeptidase A2 (CPA2), and Secretoglobin family 3A member 2 (SCGB3A2); and Group D comprises biomarkers CPM, Platelet-derived growth factor receptor beta (PDGFRB), Angiopoietin-related protein 6 (ANGPTL6), Dermokine (DMKN), Secretogranin-1 (CHGB), ADAM9, Growth arrest-specific protein 1 (GAS1), Platelet basic protein (PPBP), and Platelet glycoprotein rb alpha chain (GPI BA).

2. The method according to claim 1, wherein the biological sample is obtained from a subject selected from a fetus and pregnant woman.

3. The method according to claim 1 or 2, wherein the biolocal sample is selected from an amniotic fluid, fetal blood, plasma or serum, fetal blood, plasma or serum from the umbilical cord, placental biopsy, and maternal blood or serum.

4. The method according to claim 3, wherein the biolocal sample is amniotic fluid.

5. The method according to any one of claims 1 to 4, wherein: (i) an increase in the level of at least one biomarker of Group C in the biological sample in comparison to its level in the control; (ii) a decrease in the level of at least one biomarker of Group D
in the biological sample in comparison to its level in the control; or (iii) both (i) and (ii) is indicative of a symptomatic cCMV infection.

6. The method according to any one of claims 1 to 5, wherein the diagnosis of the symptomatic cCMV comprises determining the level of RARRES2.

PCT/11,2021/050125

7. The method according to claim 6, wherein an increase in the level of RARRES2 in the biological sample in comparison to its level in the control is indicative of symptomatic cCMV infection.

8. The method according to any one of claims 1 to 7, wherein the diagnosis of the symptomatic cCMV comprises determining the level of LGALS3BP.

9. The method according to claim 8, wherein an increase in the level of LGALS3BP in the biological sample in comparison to its level in the control is indicative of a symptomatic cCMV infection.

10. The method according to any one of claims 1 to 9, wherein the diagnosis of the symptomatic cCMV comprises determining the level of GPNMB.

11. The method according to claim 10, wherein an increase in the level of GPNMB in the biological sample in comparison to its level in the control is indicative of a symptomatic cCMV infection.

12. The method according to claim any one of claims 1 to 11, wherein the method comprises determining the level of at least two biomarkers in the biological sample.

13. The method according to claim 12, wherein the at least two of the biomarkers are RARRES2 and LGALS3BP.

14. The method according to claim 13, wherein an increase in the levels of and/or of LGALS3BP in the biological sample in comparison to their levels in the control is indicative of a symptomatic cCMV infection.

15. The method according to claim 12, wherein increase in the levels of RARRES2 and of GPNMB or of LGALS3BP and GPNMB in the biological sample in comparison to their levels in the control is indicative of a symptomatic cCMV infection.

16. The method according to any one of claims 1 to 15, wherein the method comprises determining the level of at least 3 biomarkers in the biological sample.

17. The method according to claim 16, comprising determining the level of RARRES2, LGALS3BP and at least one biomarker selected from SYNE1, MYH14, GPNMB, CPA2, PCT/11,2021/050125 SCGB3A2, CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS1, PPBP, and GP1BA.

18. The method according to claim 17, wherein increase in the levels of RARRES2 and of LGALS3BP, and (i) an increase in the level of at least one biomarker selected from SYNE1, MYH14, GPNMB, CPA2, and SCGB3A2 in comparison to their levels in the control, (ii) a decrease in the level of at least one biomarker selected from CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS1, PPBP, and GP1BA in comparison to their levels in the control, or (iii) both (i) and (ii) is indicative of a symptomatic cCMV infection.

19. The method according to claim 18, wherein an increase in the levels of RARRES2, LGALS3BP and GPNMB in the biological sample in comparison to their levels in the control is indicative of a symptomatic cCMV infection.

20. The method according to any one of claims 5 to 19, wherein the biological sample is an amniotic fluid.

21. The method according to any one of claims 1 to 20, wherein the method compfises a step of obtaining the biological sample.

22. The method according to claim 21, wherein the sample is obtained at or before week 36 of pregnancy.

23. The method according to any one of claims 1 to 22, wherein the control comprises samples selected from (i) samples from infected asymptomatic subjects; (ii) samples from non-infected subjects, or (iii) both (i) and (ii).

24. The method according to any one of claims 1 to 23, wherein the biomarker is a protein biomarker.

25. The method according to any one of claims 1 to 24, wherein determining the level of the at least one biomarker comprises use of an enzyme-linked immunosorbent assay (ELISA) or any other immune-detection assay or mass spectroscopy methods.

26. The method according to any one of claims 1 to 25, wherein the method further comprises diagnosing whether the fetus is infected with CMV.

PCT/11,2021/050125

27. The method according to claim 26, comprising differentiating between symptomatic and asymptomatic CMV in the fetus.

28. A method of diagnosing an asymptomatic congenital cytomega1ovirus in a fetus, wherein the method comprises (i) detecting whether the fetus is infected with CMV and (ii) diagnosing whether the fetus has a symptomatic CMV by the method according to any one of the claims 1 to 26, wherein lack of symptomatic CMV in the infected fetus is indicative of presence of the asymptomatic cCMV.

29. A method for recommending a medical intervention comprising diagnosing a symptomatic congenital cytomegalovirus according to any one of claims 1 to 24 or an asymptomatic CMV according to claim 28, and recommending the medical intervention based on the obtained results.

30. The method according to claim 29, wherein the medical intervention is selected from a treatment of the symptomatic cCMV, treatment of the asymptomatic cCMV, adjusting treatment, watchful waiting, monitoring, and recommendation to terminate the pregnancy.

31. The method according to claim 30, wherein the treatment comprises treatment with an anti-CMV active agent.

32. A method of treating a congenital cytomegalovirus in a fetus comprising diagnosing the symptomatic cCMV in fetus according to the methods of any one of claims 1 to 28 and administering to the fetus or the mother an anti-CMV active agent.

33. A kit comprising means for determining levels of at least two biomarker in a biological sample, wherein the at least two biomarkers are selected from biomarkers of Group C and Group D, and instructions for use.

34. The kit according to claim 33, wherein the instructions comprise instructions for diagnosing a symptomatic or asymptomatic congenital cytomegalovirus (cCMV) infection.

35. The kit according to any one of claims 33 to 34, wherein the means for determining the level of the at least two biomarkers comprises agents specific for determining the levels of the biomarkers.

PCT/11,2021/050125

36. The kit according to claim 35, wherein the agents are antibodies capable of binding to the at least two biomarker or a combination of the antibodies.

37. The kit according to claim 36, wherein the means is ELISA kit or means for performing ELISA.

38. The kit according to any one of claims 33 to 37, wherein the kit comprises means for determining the levels of RARRES2 and LGALS3BP.

39. The kit according to any one of claims 33 to 38, wherein the kit comprises means for determining the levels of RARRES2 and GPNMB or of LGALS3BP and GPNMB.

40. The kit according to any one of claims 33 to 39, wherein the kit comprises means for determining the levels of RARRES2, LGALS3BP and GPNMB.

41. The kit according to claims to any one of claims 33 to 40, wherein the kit further comprises means for determining the level of at least one biomarker selected from SYNE1, MYH14, GPNMB, CPA2, SCGB3A2, CPM, PDGFRB, ANGPTL6, DMKN, CHGB, ADAM9, GAS I, PPBP, GP IBA.

42. The kit according to any one of claims 33 to 41, wherein the means for detecting the levels of the biomarkers is ELISA or immune detection assay, or any other method for determining the levels of a plurality of biomarkers.

43. The kit according to any one of claims 33 to 42, further comprising means for comparing the level of the biomarker in the biological sample to the control.

44. Use of a kit according to any one of claims 33 to 39 in diagnosis of a symptomatic or asymptomatic congenital CMV.

45. A method of determining the efficacy of the prevention or treatment of a congenital cytomegalovirus (cCMV) infection, the method comprises the steps of: (i) determining a level of at least one biomarker in a biological sample, wherein the at least one biomarker is selected from a biomarker of Group C and Group D; and (ii) comparing the level of the at least one biomarker from the biological sample to the level of the biomarker in a corresponding biological sample obtained from the same subject at an earlier sampling and/or to the level of the biomarker in the control, wherein a change in the level of the biomarkers in two consecutive measurements is indicative of efficacy of the prevention or therapy of the cCMV.