Method for detecting anti-cytomegalovirus neutralizing antibodies

Abstract

The present disclosure provides methods for detecting anti-cytomegalovirus neutralizing antibodies. In particular, the present disclosure provides methods for determining the level of HCMV infection in a host cell, and extends to determining the level of neutralizing antibodies present in a sample. The present disclosure encompasses this knowledge that an HCMV virus with a fluorescent moiety allows detection of viral infection (e.g., by assessing fluorescence in a cell after contacting the host cell with the virus). In some embodiments, the level of HCMV infection is determined by fluorescence detection where the virus has been pre-incubated with a test sample (e.g., a serum sample) from the subject. In some embodiments, the subject has been administered a candidate HCMV vaccine.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201380026015.9 and invention name “Method for detecting anti-cytomegalovirus neutralizing antibodies”. The original application is the PCT international application PCT / IB2013 / 001021 filed on March 27, 2013, which entered the Chinese national phase on November 18, 2014.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 61 / 616,204, filed March 27, 2012, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0004] The present application relates to the field of methods for detecting anti-cytomegalovirus neutralizing antibodies. Background Art

[0005] Human cytomegalovirus (HCMV) (a beta herpes virus) is a ubiquitous pathogen. In people with immune competence, HCMV infection is usually ignored, and it has the mildest and non-specific symptoms. In contrast, certain risk groups, such as in immunosuppressed patients (such as AIDS patients or transplant recipients), and after antenatal infection, HCMV infection has severe manifestations (Staras SA et al., 2006 Clin Infect Dis 43 (9): 1143-51; Hebart H et al., 2004 Hum Immunol 65 (5): 432-6; Rowshani AT et al., 2005 Transplantation 79 (4): 381-6). Existing treatment methods include the use of immunoglobulins and antiviral preparations (such as ganciclovir and its derivatives), which are most effective when used prophylactically in high-risk groups or when used in very early stages of infection. However, existing therapies are characterized by significant toxicity and limited efficacy, especially for late-onset disease (Boeckh M., 2004 Pediatr Transplant 8 (Suppl 5): 19-27; Limaye AP., 2004 Transplantation 78 (9): 1390-6), and these therapies have no effect on congenital HCMV disease. Developing an effective vaccine to prevent HCMV disease is considered an important public health priority (Arvin AM et al., 2004 Clin Infect Dis 39 (2): 233-9).

[0006] In vitro assays are important tools for assessing the ability of candidate vaccines to interfere with HCMV infection. For example, neutralization assays have been developed to study immune responses in infected individuals, as well as to assess vaccine immunogen candidates in both clinical and preclinical trials. In the case of HCMV, antigen binding ELISA can measure specific antibodies to HCMV antigens; however, only in the determination of neutralization reactions of viruses entering cells can the biological activity of HCMV antigen-specific antibodies be established and quantified (Abai et al., 2007 J Immunol Methods 332 (1-2): 82-93). Typically, in such neutralization assays of HCMV, the extent to which neutralizing antibodies reduce cell HCMV infection is determined in assays by quantitatively measuring the nuclei of infected cells based on the expression of one or more viral proteins in cells. Such analysis may be time-consuming and difficult to use in high-throughput applications. This area still needs an improved method for screening potential HCMV vaccine candidates for inducing neutralizing antibodies. Summary of the invention

[0007] Among other aspects, the present disclosure provides methods for determining the level of HCMV infection in a host cell, and by extension, the level of neutralizing antibodies present in a sample. The present disclosure encompasses the recognition that HCMV viruses with fluorescent moieties allow for the detection of viral infection (e.g., by assessing fluorescence in cells after contacting host cells with the virus). In some embodiments, the level of HCMV infection is determined by fluorescence detection, wherein the virus has been pre-incubated with a test sample (e.g., a serum sample) from a subject. In some embodiments, the subject has been administered a candidate HCMV vaccine.

[0008] Other features, purposes and advantages of the present disclosure will become apparent in the following detailed description. However, it should be understood that although the detailed description shows some embodiments of the present disclosure, it is provided only by way of example and not by way of limitation. From this detailed description, various changes and modifications within the scope of the present disclosure will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The drawings are for illustrative purposes only and are non-limiting.

[0010] Figure 1 Described are exemplary ELISA anti-gB antibody titers following immunization with bivalent gB virus-like particles (VLPs) (gB / pp65 and gB / pp65+gH-G / pp65). Strong and long-lasting immunity was induced in rabbits by bivalent gB VLPs after a single immunization.

[0011] Figure 2Describes an exemplary FACS analysis of GFP expression in fibroblasts indicating neutralizing antibody responses induced in rabbits with gB / pp65 CMV VLPs. Rabbits (n=6 / group) were immunized (IM) twice at weeks 0 and 8 and bled 2 weeks later. Sera were pooled and tested at the indicated dilutions and compared with Cytogam at similar dilutions. TM Comparison of HFF fibroblasts against CMV virus expressing GFP (TB40). + ) 100,000 cells were collected during flow cytometric analysis of the cells.

[0012] Figure 3 Described is an exemplary FACS analysis of GFP expression in fibroblasts indicating neutralizing antibody responses induced in rabbits with bivalent gB+gHCMV VLPs. Rabbits (n=6 / group) were immunized (IM) twice at weeks 0 and 8 and bled 2 weeks later. Sera were pooled and tested at the indicated dilutions and compared with Cytogam at similar dilutions. TM Comparison of HFF fibroblasts against CMV virus expressing GFP (TB40). + ) 100,000 cells were collected during flow cytometric analysis of the cells.

[0013] Figure 4 Exemplary neutralization percentages in HFF-1 cells are described. Neutralization of 15RA09 Group 7 (monovalent gB-G / monovalent gH-G with aluminum adjuvant) pooled samples at P1Vd14, P1Vd28, P1Vd42, P1Vd55, and P2Vd14, and 15RA05 Group 8 (empty MLV Gag) at P2Vd14 in HFF-1 cells in the presence of 10% guinea pig complement anti-1:6CMV-GFP-TB40-010512 is described.

[0014] Figure 5 Exemplary neutralization percentages in ARPE-19 cells are described. Neutralization of 15RA09 Group 7 (monovalent gB-G / monovalent gH-G with aluminum adjuvant) pooled samples at P1Vd14, P1Vd28, P1Vd42, P1Vd55, and P2Vd14, and 15RA05 Group 8 (empty MLVGag) at P2Vd14 in ARPE-19 cells in the presence of 2.5% rabbit complement anti-1:3CMV-GFP-Towne-150612 is described. DETAILED DESCRIPTION

[0015] definition

[0016] In order to make the present disclosure more easily understood, some terms are first defined below. The following terms and other terms are further defined throughout the specification.

[0017] Amino Acid: The term "amino acid" as used herein, in its broadest sense, refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some embodiments, an amino acid is an l-amino acid. "Standard amino acid" refers to any of the 20 standard l-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than a standard amino acid, whether it is synthetically prepared or obtained from a natural source. "Synthetic amino acid" as used herein encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (e.g., amides), and / or substitutions. Amino acids (including carboxyl-terminal and / or amino-terminal amino acids in peptides) can be modified by methylation, amidation, acetylation, protecting groups, and / or substitution with other chemical groups, which can change the circulating half-life of the peptide without negatively affecting its activity. Amino acids can participate in disulfide bonds. The amino acid may contain one or more post-translational modifications, such as binding to one or more chemical entities (e.g., a methyl group, an acetate group, an acetyl group, a phosphate group, a formyl moiety, an isoprenoid group, a sulfate group, a polyethylene glycol moiety, a lipid moiety, a carbohydrate moiety, a biotin moiety, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and may refer to a free amino acid and / or an amino acid residue of a peptide. Whether the term refers to a free amino acid or a residue of a peptide will be apparent from the context in which the term is used.

[0018] Antigen: As used herein, the term "antigen" refers to a substance containing one or more epitopes (linear epitopes, conformational epitopes, or both) recognized by antibodies. In certain embodiments, the antigen is a virus or a viral polypeptide or comprises a virus or a viral polypeptide. In some embodiments, the term "antigen" refers to a subunit antigen (i.e., an antigen separated from or separated from a complete virus, the antigen being essentially associated with the complete virus; e.g., an antigen associated with a virus-like particle). Alternatively or additionally, in some embodiments, the term "antigen" refers to a killed, attenuated, or inactivated virus. In certain embodiments, an antigen is an "immunogen."

[0019] About or approximately: The term "about" or "approximately" as used herein, applied to one or more target values, refers to a value similar to the reference value. In certain embodiments, unless otherwise mentioned or otherwise apparent from the context, the term "about" or "approximately" refers to a series of values ​​falling within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in any direction (greater than or less than) of the reference value (except where such a number would exceed 100% of the possible value).

[0020] Amelioration: As used herein, the term "amelioration" means to prevent, reduce or alleviate a state, or improve the state of a subject. Amelioration includes but does not require complete recovery or complete prevention of a disease, disorder or condition (e.g., HCMV infection). The term "prevention" refers to delaying the onset of a disease, disorder or condition. Prevention may be considered accomplished when the onset of a disease, disorder or condition has been delayed for a predetermined period of time.

[0021] Dosage form: The terms "dosage form" and "unit dosage form" as used herein refer to physically discrete units of therapeutic agent for the patient to be treated. Each unit contains a predetermined quantity of active material calculated to produce the desired therapeutic effect. It will be understood, however, that the total dosage of the composition will be decided by the attending physician within the scope of sound medical judgment.

[0022] Dosing regimen: As used herein, the term "dosing regimen" (or "treatment regimen") is a group of unit doses (usually more than one) that are administered individually to a subject, usually separated by a period of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more administrations. In some embodiments, the dosing regimen includes multiple administrations, each administration being separated by a period of time of the same length; in some embodiments, the dosing regimen includes multiple administrations and at least two different time periods separating the administrations.

[0023] Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' cap formation and / or 3' end formation); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0024] Fluorescence: The term fluorescence as used herein refers to a moiety that emits light. Typically the fluorescent moiety contains electrons that absorb electromagnetic energy of one wavelength and emit electromagnetic energy of a second wavelength. Some proteins or small molecules in cells are natural fluorescent substances (e.g., NADH, tryptophan, endogenous chlorophyll, phycoerythrin, or green fluorescent protein (GFP)). It should be understood that various mutants of fluorescent proteins have been engineered and can be used according to the present disclosure, such as EGFP, blue fluorescent protein (EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein (ECFP, Cerulean, CyPet), yellow fluorescent protein (YFP, Citrine, Venus, YPet), redox-sensitive GFP (roGFP), and monomeric GFP, etc. GFP and other fluorescent proteins can be expressed exogenously in cells alone or as fusion proteins. This approach allows fluorescent proteins to be used as reporter molecules for any number of biological events (e.g., subcellular localization and expression patterns).

[0025] Alternatively or additionally, specific or general proteins, nucleic acids, lipids or small molecules can be labeled with exogenous fluorophores, fluorescent dyes, which can be small molecules, proteins or quantum dots. Exemplary fluorophores include, but are not limited to, 1,5IAEDANS; 1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-carboxynaphtholfluorescein; 5-carboxytetramethylrhodamine (5-TAMRA); 5-hydroxytryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-amino-4-methylcoumarin; 7-aminoactinomycin D (7-AAD); 7-hydroxy-4-1-methylcoumarin; 9-amino-6-chloro-2-methoxyacridine (ACMA); ABQ; acid fuchsin; acridine orange; acridine red; acridine yellow; trypan yellow (Acriflavin); trypan yellow Feulgen SITSA (Acriflavin Feulgen SITSA); aequorin (luminescent protein); AFP (autofluorescent protein, Quantum Biotechnologies) (see sgGFP, sgBFP); Alexa Fluor 350.TM.; Alexa Fluor 430.TM.; Alexa Fluor 488.TM.; Alexa Fluor 532.TM.; Alexa Fluor 546.TM.; Alexa Fluor 568.TM.; Alexa Fluor 594.TM.; Alexa Fluor 633.TM.; Alexa Fluor 647.TM.; AlexaFluor 660.TM.; Alexa Fluor 680.TM.; Alizarin complex; Alizarin Red; Allophycocyanin (APC); AMC; AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; Aminactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7GLL; Atrazon; ATTO-TAG.TM.CBQCA; ATTO-TAG.TM.FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (bisaminophenyl) oxadiazole); BCECF (high pH); BCECF (low pH); berberine sulfate; β-lactamase; BFP blue-shifted GFP (Y66H); blue fluorescent protein; BFP / GFP FRET; Bimane; Bisbenzemide; Bisbenzemide (Hoechst); BisBTC; Blancophor FFG; Blancophor SV; BOBO.TM.-1; BOBO.TM.-3; Bodipy492 / 515; Bodipy493 / 503; Bodipy500 / 510; Bodipy505 / 515; Bodipy 530 / 550; Bodipy 542 / 563; Bodipy558 / 568; Bodipy 564 / 570; Bodipy576 / 589; Bodipy 581 / 591; Bodipy630 / 650-X; Bodipy 650 / 665-X; Bodipy 665 / 676; BodipyF1; Bodipy FL ATP; Bodipy F1-ceramide; Bodipy R6GSE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO.TM.-1; BO-PRO.TM.-3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcium; Calcium Blue; CalciumCrimson; Calcium Green; Calcium Green-1 Ca.sup.2+ dye; Calcium Green-2 Ca.sup.2+; Calcium Green-5N Ca.sup.2+; Calcium Green-C18 Ca.sup.2+; Calcium Orange; Calcofluor White; Carboxy-X-Rhodamine (5-ROX); Cascade Blue.TM.; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (cyan fluorescent protein); CFP / YF P FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hcp; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin phalloidin; C-phycocyanine; CPM I methylcoumarin; CTC; CTC formazan; Cy2.TM.;Cy3.1 8; Cy3.5.TM.; Cy3.TM.; Cy5.1 8; Cy5.5.TM.; Cy5.TM.; Cy7.TM.; Cyan GFP; cyclic AMP fluorescent sensor (FiCRhR); Dabcyl; Dansyl; Dansylamide; Dansylcadaverine; Dansyl chloride; DansylDHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3'DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-specific ratio); DiA (4-Di 16-ASP); dichlorodihydrofluorescein diacetate (DCFH); DiD-lipophilic tracer; DiD (DilC18 (5)); DIDS; dihydrorhodamine (Dihydorhodamine) 123 (DHR); Dil (DilC18 (3)); I-dinitrophenol; DiO (DiOC18 (3)); DiR; DiR (Di1C18 (7)); DM-NERF (high pH); DNP; dopamine; DsRed ; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF97; Eosin; Erythrophyll; Erythrophyll ITC; Ethidium bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (para-fuchsin); FIF (Formaldehyde-induced fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein diacetate; Fluorescent Emerald; Fluorescent Gold (Fluoro-Gold) (hydroxystilbene); Fluor-Ruby; FluorX; FM 1-43.TM.; FM 4-46; Fura Red.TM. (high pH); Fura Red.TM. / Fluo-3; Fura-2; Fura-2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red shift (rsGFP); non-UV excited wild-type GFP (wtGFP); UV excited wild-type GFP (wtGFP); GFPuv; Gloxalic Acid; Granular blue; Hematoporphyrin;Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; hydroxycoumarin; hydroxystilbene (fluorescent gold); serotonin; Indo-1 (high calcium); Indo-1 (low calcium); Indo dicarbocyanine (DiD); Indo tricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine rhodamine; Lissamine rhodamine B; Calcein / ethidin homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; Lyso Tracker Blue Probe Blue); Lysosomal blue and white probe; Lysosomal green probe; Lysosomal red probe; Lysosomal yellow probe; LysoSensor blue; LysoSensor green; LysoSensor yellow / blue; Mag green; Sudan red (Magdala Red) (Root Bark Red B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indigo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; I Maxilon Brilliant Flavin10GFF; Maxilon Brilliant Flavin 8GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Green Orange; Mitotracker Green Red; Plicamycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red (Nile Red); Nitrobenzoxedidole; Norepinephrine; Nuclear Fast Red; I Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green.TM.; Oregon Green.TM.488; Oregon Green.TM.500; Oregon Green.TM.514 Pacific Blue; Feulgen; PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP Cy5.5; PE-TexasRed (Red 613); Root Bark Red B (Sudan Red); Phorwite AR; Phorwite BKL; Phorwite Rev; PhorwiteRPA; Phosphine 3R;Photoresist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-1PRO-3; Primulin; Procion Yellow; Propidium lodid (P1); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B; Rhodamine B200; Rhodamine B extra); Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine: Phalloidin; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron I Brilliant Red B; Sevron; Sevron Yellow L; sgBFP.TM. (super luminescent BFP); sgGFP.TM. (super luminescent GFP); SITS (primulin; stilbene isothiosulfonic acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green (Sodium Green); SpectrumAqua; SpectrumGreen; SpectrumOrange; SpectrumRed; SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulforhodamine B and C; Supersulforhodamine; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO16; SYTO 17; SYTO 18; SYTO SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO63; SYTO 64; SYTO 80; SYTO 81;SYTO 82;SYTO 83;SYTO 84;SYTO 85;SYTOX Blue;SYTOX Green;SYTOX Orange;Tetracycline;Tetramethylrhodamine (TRITC); Texas Red.TM.; Texas Red-X.TM. conjugate; DiSC3; Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofiuor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC Tetramethylrhodamine isothiocyanate (TetramethylRodaminelsoThioCyanate); True Blue; Tru Red; Ultralite; Fluorescein Sodium B; Uvitex SFC; wt GFP; WW 781; X-rhodamine; XRITC; xylene orange; Y66F; Y66H; Y66W; yellow GFP; YFP; YO-PRO-1; YO-PRO3; YOYO-1; YOYO-3; Sybr green; thiazole orange (interchelating dye); semiconductor nanoparticles such as quantum dots; or caged fluorophores (which can be activated by light or other electromagnetic energy sources); or combinations thereof. ;

[0026] Fusion protein: The term "fusion protein" as used herein generally refers to a polypeptide comprising at least two fragments, each of which exhibits a high degree of amino acid identity with a peptide portion that (1) occurs naturally and / or (2) represents a functional domain of a polypeptide. Generally, a polypeptide containing at least two such fragments is considered a fusion protein if the two fragments are such portions that: (1) are not naturally included in the same peptide, and / or (2) have not been previously linked to each other in a single polypeptide, and / or (3) have been linked to each other by artificial action.

[0027] Gene: The term "gene" as used herein has the meaning as understood in the art. It will be understood by those of ordinary skill in the art that the term "gene" may include gene regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences. It will also be understood that the definition of a gene includes reference to nucleic acids that do not encode proteins but encode functional RNA molecules (e.g., tRNA, RNAi-inducing agents, etc.). For the purpose of clarification, we note that the term "gene" used in this application generally refers to the portion of a nucleic acid that encodes a protein; as will be apparent to those of ordinary skill in the art from the context, the term may optionally encompass regulatory sequences. This definition is not intended to exclude the application of the term "gene" to non-protein-coding expression units, but rather to clarify that in most cases, the term as used in this text refers to nucleic acids that encode proteins.

[0028] Gene product or expression product: As used herein, the term "gene product" or "expression product" generally refers to RNA transcribed from a gene (before and / or after processing) or a polypeptide encoded by RNA transcribed from the gene (before and / or after modification).

[0029] High throughput: The term "high throughput" as used herein broadly refers to research utilizing a large number of assays, which makes it important to format each individual sample, minimize preparation steps and complexity, and measure the assay results in parallel or in rapid succession. High throughput testing typically does not include manual, one-at-a-time assays, such as single independent assays, in which the preparation, execution, measurement, and data collection of one assay are all completed before the assay of the next reagent is completed. High throughput typically includes, for example, any assay in which a batch of samples (e.g., 24, 96, 384 or more test samples) are prepared and measured. Formatting the test in such a test sample means, for example, accelerating the assay process by making the measurements be performed in parallel or in rapid succession with the assistance of automation.

[0030] Immunogenicity: As used herein, the term "immunogenicity" means the ability to generate an immune response in a host animal against a non-host entity (e.g., HCMV antigens). In certain embodiments, this immune response forms the basis for protective immunity elicited by a vaccine against a specific infectious organism (e.g., HCMV).

[0031] Immune response: The term "immune response" as used herein refers to a response elicited in an animal. An immune response may refer to cellular immunity, humoral immunity, or may involve both. An immune response may also be limited to a portion of the immune system. For example, in certain embodiments, an immunogenic composition may induce an increased IFNγ response. In certain embodiments, an immunogenic composition may induce a mucosal IgA response (e.g., measured in nasal and / or rectal washes). In certain embodiments, an immunogenic composition may induce a systemic IgG response (e.g., measured in serum). In certain embodiments, an immunogenic composition may induce a virus neutralizing antibody or a neutralizing antibody response.

[0032] Improvement, increase or decrease: The terms "improvement," "increase" or "decrease," or grammatical equivalents, as used herein, indicate a value relative to a baseline measurement, such as a measurement in the same individual before the start of a treatment described herein or a measurement in a control individual (or multiple control individuals) in the absence of a treatment described herein.

[0033] Individual, subject, patient: The terms "subject", "individual" or "patient" as used herein refer to a human or non-human mammalian subject. The individual to be treated (also referred to as a "patient" or "subject") is an individual (fetus, infant, child, adolescent or adult) suffering from a disease (e.g., HCMV infection). In some embodiments, the subject is at risk for HCMV infection. In some embodiments, the subject is an immunosuppressed subject. For example, in some embodiments, the immunosuppressed subject is selected from HIV-infected subjects, AIDS patients, transplant recipients, child subjects, and pregnant subjects. In some embodiments, the subject has been exposed to HCMV infection. In some embodiments, the subject is a human.

[0034] Isolated: As used herein, the term "isolated" refers to a substance and / or entity that has been (1) separated from at least some of the components with which it was originally associated (whether in nature and / or in an experimental setting), and / or (2) artificially produced, prepared, and / or manufactured. An isolated substance and / or entity may be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% separated from the other components with which it was originally associated. In some embodiments, the isolated substance is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, calculations of the percent purity of isolated substances and / or entities should not include excipients (e.g., buffers, solvents, water, etc.).

[0035] Linker: The term "linker" as used herein refers to an amino acid sequence of appropriate length that does not occur at a specific position in a native protein (e.g., in a fusion protein) and is generally designed to be flexible and / or to insert a structure (e.g., an α-helix between two protein parts). In general, the linker allows two or more domains of the fusion protein to retain 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the biological activity of the respective domains. A linker may also be referred to as a spacer.

[0036] Nucleic acid: The term "nucleic acid" as used herein, in its broadest sense, refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain through a phosphodiester bond. In some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., nucleotide and / or nucleoside). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising a single nucleic acid residue. The terms "oligonucleotide" and "polynucleotide" used herein are interchangeable. In some embodiments, "nucleic acid" encompasses RNA and single-stranded and / or double-stranded DNA and / or cDNA. In addition, the terms "nucleic acid", "DNA", "RNA" and / or similar terms include nucleic acid analogs, i.e., analogs with non-phosphodiester backbones. For example, so-called "peptide nucleic acids" known in the art and having peptide bonds rather than phosphodiester bonds in the backbone are considered to be within the scope of the present disclosure. The term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences encoding the same amino acid sequence and / or a degenerate version of each. Nucleotide sequences encoding proteins and / or RNA may include introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Wherein, for example, in the case of chemically synthesized molecules, suitable nucleic acids may include nucleoside analogs, for example analogs with chemically modified bases or sugars, backbone modifications, etc. Unless otherwise indicated, nucleic acid sequences are given in a 5' to 3' direction. The term "nucleic acid fragment" as used herein refers to a nucleic acid sequence that is a portion of a longer nucleic acid sequence. In many embodiments, the nucleic acid fragment comprises at least 3, 4, 5, 6, 7, 8, 9, 10 or more residues. In some embodiments, the nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite bonds).In some embodiments, the present disclosure specifically relates to "unmodified nucleic acids," meaning that the nucleic acids (eg, polynucleotides and residues, including nucleotides and / or nucleosides) have not been chemically modified to facilitate or achieve delivery.

[0037] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" refers to substances that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0038] Polypeptide: As used herein, a "polypeptide" is generally a string of at least two amino acids connected to each other by peptide bonds. In some embodiments, a polypeptide may include at least 3 to 5 amino acids, each of which is connected to the others by at least one peptide bond. It will be understood by those of ordinary skill in the art that, optionally, a polypeptide sometimes includes "non-natural" amino acids or other entities that can still be incorporated into a polypeptide chain.

[0039] Substantial homology: As used herein, the phrase "substantial homology" refers to a comparison between amino acid or nucleic acid sequences. One of ordinary skill in the art will appreciate that two sequences are generally considered to be "substantially homologous" if they contain homologous residues at corresponding positions. Homologous residues can be identical residues. Alternatively, homologous residues can be non-identical residues with substantially similar structural and / or functional characteristics. For example, it is well known to one of ordinary skill in the art that certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids, and / or have "polar" or "non-polar" side chains. The substitution of one amino acid of the same type for another can often be considered a "homologous" substitution.

[0040] Comparison of amino acid or nucleic acid sequences can be performed using any of a variety of algorithms, as is known in the art, including those commercially available computer programs such as BLASTN for nucleotide sequences and BLASTP, Gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul, et al., Methods in Enzymology; Altschul, et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the above programs generally provide an indication of the degree of homology. In some embodiments, two sequences are considered to be substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are homologous over a stretch of related residues. In some embodiments, the related stretch is the entire sequence. In some embodiments, the related stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0041] Substantial identity: As used herein, the phrase "substantial identity" refers to a comparison between amino acid or nucleic acid sequences. One of ordinary skill in the art will appreciate that two sequences are generally considered to be "substantially identical" if they contain identical residues at corresponding positions. It is well known in the art that amino acid or nucleic acid sequence comparisons can be performed using any of a variety of algorithms, including those commercially available computer programs such as BLASTN for nucleotide sequences and BLASTP, Gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul, et al., Methods in Enzymology; Altschul, et al., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the above programs typically provide an indication of the degree of identity. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over a stretch of related residues. In some embodiments, the related stretch is the entire sequence. In some embodiments, the related stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0042] Suffering from: An individual "suffering from" a disease, disorder, or condition (eg, HCMV infection) has been diagnosed as having and / or exhibits one or more symptoms of the disease, disorder, or condition.

[0043] Susceptible: An individual who is "susceptible" to a disease, disorder, or condition (e.g., HCMV infection) is at risk for developing the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition does not exhibit any symptoms of the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition has been diagnosed as having the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition is an individual who has been exposed to conditions associated with the development of the disease, disorder, or condition (e.g., an individual has been exposed to HCMV).

[0044] Symptom Relief: According to the present disclosure, when the degree (e.g., intensity, severity, etc.) or frequency of one or more symptoms of a particular disease, disorder, or condition is reduced, then the symptoms are "relieved." For purposes of clarification, a delay in the onset of a particular symptom is considered a form of reducing the frequency of the symptom. It is not intended that the present disclosure be limited only to instances of symptom elimination. The present disclosure specifically contemplates treatments such that one or more symptoms are alleviated (and the subject's condition is thereby "improved"), although not completely eliminated.

[0045] Therapeutically effective amount: The term "therapeutically effective amount" as used herein refers to an amount sufficient to produce a therapeutic effect on the subject being treated at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by a test or marker) or subjective (i.e., the subject gives an indication of the effect or feels the effect). In particular, a "therapeutically effective amount" refers to an amount of a therapeutic protein or composition that effectively treats, improves or prevents a desired disease or condition or exhibits a detectable therapeutic or preventive effect (e.g., by improving symptoms associated with the disease, preventing or delaying the onset of the disease, and / or also alleviating the severity or frequency of disease symptoms). The therapeutically effective amount is typically administered in a dosage regimen that may include a plurality of unit doses. For any particular immunogenic composition, the therapeutically effective amount (and / or the appropriate unit dose within an effective dosage regimen) may vary, for example, according to the route of administration, in combination with other agents. In addition, the specific therapeutically effective amount (and / or unit dosage) for any particular patient may depend on a variety of factors, including: the condition being treated and the severity of the condition; the activity of the specific agent employed; the specific composition employed; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and / or rate of excretion or metabolism of the specific immunogenic composition employed; the duration of the treatment; and similar factors well known in the medical arts.

[0046] Treatment: As used herein, the term "treatment" refers to any administration of an immunogenic composition that partially or completely alleviates, ameliorates, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition (e.g., HCMV infection), or susceptibility to the disease. Such treatment may be performed on subjects who do not show signs of the relevant disease, disorder, and / or condition, and / or on subjects who only show early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be performed on subjects who show one or more established signs of the relevant disease, disorder, and / or condition. In certain embodiments, the term "treatment" refers to vaccinating a patient.

[0047] Tropism: The terms "tropism" or "host tropism" or "cell tropism" used herein generally refer to the ability of a virus or pathogen to infect a specific cell type in the context of a virus or other pathogen. Tropism can refer to a mode in which a virus or pathogen has evolved to preferentially target a specific host species or a specific cell type within these species. For example, HCMV can usually infect a very wide range of cells within its host, including parenchymal cells, connective tissue cells, and various hematopoietic cell types of almost any organ. Epithelial cells, endothelial cells, fibroblasts, and smooth muscle cells are the main targets of viral replication. However, the tropism of various cells varies greatly between different HCMV strains, such as changes from the UL128-131 locus. In some embodiments, the HCMV strain can infect fibroblasts, but cannot infect epithelial and / or endothelial cells. In some embodiments, the HCMV strain can infect fibroblasts, epithelial cells, and endothelial cells.

[0048] Vaccination: As used herein, the term "vaccination" refers to the administration of a composition intended to generate an immune response to, for example, a pathogenic agent (e.g., HCMV). For the purposes of the present disclosure, vaccinations can be administered before, during, and / or after exposure to a pathogenic agent, and in certain embodiments, shortly before, during, and / or after exposure to a pathogenic agent. In some embodiments, vaccinations include multiple administrations of a vaccine composition at appropriate intervals.

[0049] Vector: As used herein, "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is associated. In some embodiments, a vector is capable of extrachromosomal replication and / or expression of a nucleic acid to which it is linked in a host cell (e.g., a eukaryotic and / or prokaryotic cell). A vector capable of directing the expression of an operably linked gene is referred to herein as an "expression vector."

[0050] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0051] Among other aspects, the present disclosure provides methods for determining the level of HCMV infection in a host cell, and by extension, the level of neutralizing antibodies present in a sample. The present disclosure encompasses the recognition that HCMV viruses with fluorescent moieties allow for the detection of viral infection (e.g., by assessing fluorescence in cells after contacting host cells with the virus). In some embodiments, the level of HCMV infection is determined by fluorescence detection, wherein the virus has been pre-incubated with a test sample (e.g., a serum sample) from a subject. In some embodiments, a candidate HCMV vaccine has been administered to a subject.

[0052] I. HCMV infection and vaccines

[0053] Human cytomegalovirus (HCMV) (a kind of beta herpes virus) is a ubiquitous pathogen. Generally speaking, it is a complicated process for herpes virus to enter cells, which is initiated by absorption and receptor binding, and then fused with cell membrane by viral envelope. Fusion usually occurs at plasma membrane or endosomal membrane. HCMV infects multiple cell types in vivo, including epithelial cells, endothelial cells and fibroblasts (Plachter B etc., 1996Adv Virus Res 46:195-261). It fuses with the plasma membrane of fibroblasts (Compton T etc., 1992Virology 191:387-395), but enters retinal pigment epithelial cells and umbilical vein endothelial cells (Bodaghi B etc., 1999J Immunol 162:957-964; Ryckman BJ etc., 2006J Virol 80:710-722). The mechanism that herpes virus selects its entry pathway is still unclear. It is usually assumed that the entry pathway is mainly determined by the host cell, but there is evidence that it is determined by the tropism of the virion glycoprotein (Wang X et al., 1998 J Virol 72: 5552-5558). HCMV encodes two gH / gL complexes, gH / gL / gO and gH / gL / UL128 / UL130 / UL131. The complex containing gO is sufficient for fibroblast infection, while the complex containing pUL128 / UL130 / UL131 is important for HCMV infection of endothelial cells and epithelial cells. As used herein, the term "tropism" or "host tropism" or "cell tropism" generally refers to the ability of a virus or pathogen to infect a specific cell type in the context of viruses and other pathogens. Tropism can refer to a mode in which a virus or pathogen has evolved to preferentially target a specific host species or a specific cell type within these species. In some embodiments, the HCMV strain can infect fibroblasts, but cannot infect epithelial and / or endothelial cells. In some embodiments, the HCMV strain is capable of infecting fibroblasts, epithelial cells, and endothelial cells.

[0054] HCMV infects 50% to 85% of adults under 40 years of age (Gershon AA et al., 1997 in Viral Infections of Humans, 4th edition, New York; Plenum Press: 229-251). Most healthy individuals who acquire HCMV after birth rarely develop symptoms (if any). However, HCMV disease causes significant morbidity and mortality in immunocompromised individuals (e.g., recipients of hematopoietic cell transplants (HCT) and solid organ transplants (SOT)) (PassRF 2001 Cytomegalovirus. In Fields Virology. 4th edition, Philadelphia; Lippincott Williams & Wilkens: 2675-2705). In SOT or HCT populations, HCMV disease may occur due to new infections transmitted by donor organs or HCT, or may recur due to reactivation of latent viruses in recipients. In HIV-infected individuals, despite the availability of antiretroviral therapy, HCMV infection accelerates the progression of AIDS and death (Deayton JR et al., 2004 Lancet 363:2116-2121). In addition, HCMV is the most common intrauterine infection in the United States and causes congenital malformations that cause death or severe birth defects, including deafness and mental retardation, in approximately 8,000 infants each year (Stagon S et al., 1986 JAMA 256:1904-1908).

[0055] The immune response that controls HCMV is not fully understood. By analogy with other human herpes viruses, it can be inferred that both cellular and humoral immune responses play an important role (Kohl S1992Current topics in Microbiology and Immunology 179:75-88). For mouse CMV, it is shown that both cytotoxic T cell responses and passive transport of neutralizing antibodies are sufficient to prevent fatal attacks (challenge) (Rapp M et al., 1993Multidisciplinary Approach to Understanding Cytomegalovirus Disease:327-332; Reddehase MJ et al., 198J Virology61:3102-3108).

[0056] In immunocompromised individuals, control of HCMV is primarily associated with a cell-mediated immune response; CD8 + and CD4 +Both T lymphocytes appear to be important for protection against CMV disease (Gamadia LE et al., 2003 Blood 101:2686-2692; Cobbold M et al., 2005 J Exp Med 202:379-386). The cellular immune response to CMV includes CD4 T cells directed against a variety of antigens found in the viral envelope (the region of the viral particle between the envelope and the capsid). + Helper T lymphocytes and CD8 + Cytotoxic T lymphocyte responses. Recent studies on CMV-specific CD4 + and CD8 + T cell studies have used overlapping peptides from a series of CMV open reading frames to identify antigens recognized after CMV infection (Sylwester AW et al., 2005 J Exp Med 202:673-685). CMV envelope phosphoprotein 65 (pp65) and surface glycoprotein gB are the most frequently recognized antigens by CD4 + pp65 is also the most frequently recognized antigen by CD8 + One of the antigens recognized by T cells.

[0057] In contrast to transplantation, the humoral immune response of the mother to the virus seems to be important for preventing HCMV disease in newborns. Antibodies to surface glycoproteins (particularly gB) seem to be crucial for preventing maternal-fetal transfer of HCMV (Fowler KB et al., 2003 JAMA 289: 1008-1011). In addition, in earlier vaccination studies, it was shown that the prevention of reinfection was related to neutralizing antibodies (Adler SP et al., 1995 J Infectious Diseases 171: 26-32). The humoral immune response to HCMV is based on the response to the viral envelope glycoproteins (e.g. gB and gH) present in the outer envelope of the virion.

[0058] In the case of HCMV, it is difficult to directly assess immunological effector functions because the virus is strictly species-specific and no animal model systems are available. However, murine CMV and guinea pig CMV have been used to evaluate vaccine strategies in these host species.

[0059] A CMV vaccine that induces both protective T cell and neutralizing antibody responses has the potential to prevent infection or ameliorate CMV disease resulting from congenital infection or transplantation.

[0060] The first live attenuated HCMV vaccine candidate tested in humans is based on the AD169 strain modified in the laboratory. Subsequent experiments conducted with the Towne strain of clinical isolates (isolate) modified in another laboratory confirmed that the live attenuated vaccine can induce neutralizing antibodies and CD4+ and CD8+ T lymphocyte responses. The efficacy of the Towne vaccine was evaluated in a series of studies in kidney transplant recipients. Although the Towne vaccine does provide a protective effect on HCMV disease, it cannot prevent HCMV infection after transplantation (Plotkin SA et al., 1984Lancet 1:528-530). The Towne vaccine was also evaluated in a placebo-controlled study of seronegative mothers whose children attended kindergartens (group daycare), and in this case it was impossible to prevent these women from acquiring infection from their children infected with HCMV (Adler SP et al., 1995J Infectious Diseases 171:26-32). The explanation for these studies is that the Towne vaccine is over-attenuated. To explore this possibility, a series of genetic recombinants (wherein the corresponding region of the Towne genome was replaced with a region of the "Toledo" strain of CMV that was not attenuated) produced the construction of Towne / Toledo "chimeras" containing some but not all mutations that make the Towne vaccine attenuated (Heineman TC et al. 2006 J Infect Disease 193: 1350-1360). The safety and tolerability of the four Towne / Toledo "chimeras" are being tested in Phase I trials. Long-term safety concerns about the potential risk of establishing latent HCMV infection have hindered further development of live, attenuated vaccines.

[0061] The main subunit CMV vaccine candidate is based on the envelope glycoprotein gB (the recombinant gB vaccine of purification is manufactured by Sanofi-Pasteur Vaccines), because this protein can cause high titer virus neutralizing antibody response during natural infection. The recombinant gB vaccine causes neutralizing antibody response and has excellent safety properties, but it excludes other glycoprotein targets of neutralizing antibody response and more importantly excludes T lymphocyte target. The vaccine needs MF59 adjuvant to optimize immunogenicity. In recent experiments, this vaccine provides a total of 50% effectiveness (Pass RF etc., 2009N Engl J Med 360:1191-1199) to preventing CMV infection in 2 phase clinical trials in young women. Other viral proteins being evaluated as subunit vaccine candidates include pp65 and IE1, both of which cause T cell response.

[0062] DNA vaccines induce powerful (robust) cellular and humoral immune responses in animals and match the specificity and accuracy (precision) in vaccine design well. DNA vaccines for CMV have been developed, and gB, IE1 and pp65 proteins have been focused on as candidate target immunogens. Bivalent CMV DNA vaccine candidates (Wloch MK, 2008 J Infectious Diseases 297: 1634-1642) using the plasmid DNA encoding pp65 and gB, and trivalent vaccine candidates (Jacobson MA, 2009 Vaccine 27: 1540-1548) also comprising the third plasmid encoding IE1 gene product, have been developed by Vical Vaccines (U.S. Patent No. US 7,410,795). Regardless of the route of administration, the immunogenicity of the independent trivalent DNA vaccine is minimal. However, after applying live attenuated CMV (Towne), the CMV DNA vaccine does appear to safely induce the observed memory response for CMV antigens.

[0063] In the method of vector vaccine, the target gene product is expressed in the environment of non-replicating (usually viral) vector. An example of this is the canarypox vector called ALVAC developed by Virogenetics and Sanofi-Pasteur Vaccines, which is an attenuated poxvirus that cannot be replicated in mammalian cells. ALVAC expressing CMV gB and ALVAC expressing pp65 have been tested in clinical trials (U.S. Patent No. 6,267,965). ALVAC-CMV (gB) does not induce neutralizing antibodies, but does induce higher neutralizing antibody titers (Adler SP et al. 1999 J Infectious Diseases 180: 843-846) after subsequent infection with Towne strain CMV, although it does not seem to strengthen neutralizing antibody titers (Bernstein DI et al., 2002 J Infectious Diseases 185: 686-690) after subsequent immunization with gB subunit / MF59 vaccine. Canarypox vectors expressing pp65, ALVAC-CMV (pp64) induced a durable CTL response in all initially seronegative volunteers, with frequencies comparable to naturally seropositive individuals (Berencsi K et al. 2001 J Infectious Diseases 183: 1171-1179). Another approach for expressing gB as a vector vaccine is to use the alphavirus replicon system of AlphaVax Inc (U.S. Pat. No. 7,419,674). This approach involves a single-cycle RNA replicon vector system derived from an attenuated strain of alphavirus (Venezuelan Equine Encephalitis (VEE) virus) to produce virus-like replicon particles (VRPs) expressing pp65, IE1 or gB proteins (Berstein et al., 2010 Vaccine 28: 484-493). A two-component alphavirus replicon vaccine was used to express three CMV proteins in a soluble form of CMV gB (Towne strain), and the pp65 / IE1 fusion protein (Reap EA et al., 2007 Vaccine 25:7441-7449) was found to be safe and induce high levels of neutralizing antibodies and multifunctional CD4+ and CD8+ antigen-specific T cell responses. The geometric mean titer (GMT) of the high-dose group was about half of the GMT of the 12 naturally infected CMV seropositive individuals tested in the assay.

[0064] The candidate for HCMV vaccination in preclinical development is currently a "dense body" vaccine. Dense bodies (densebody, DB) are enveloped, replication-defective particles formed during the replication of CMV in cell culture. They contain envelope glycoproteins and a large amount of pp65 proteins. DB is non-infectious and immunogenic, but potential HCMV infection cannot occur in vaccine recipients. DB has been shown to be able to induce virus neutralizing antibodies and T cell responses in mice without viral gene expression (Pepperl S et al., 2000 J Virol 74: 6132-6146 PCT Publication No. WO 00 / 53729 and U.S. Patent No. 6,713,070).

[0065] The other candidate for vaccination against HCMV is virus-like particle (VLP). Retrovirus is an enveloped RNA virus, which belongs to Retroviridae. After the host cell is infected by the retrovirus, RNA is transcribed into DNA through the enzyme reverse transcriptase. Then DNA is incorporated into the genome of the host cell by integrase and replicated as a part of the host cell DNA afterwards. Retroviridae includes the following genus: Alpharetrovirus, Betaretrovirus, Gammearetrovirus, Deltaretrovirus, Epsilonretrovirus, Lentivirus and Spumavirus. The host of this Retroviridae is generally a vertebrate. Retrovirus produces infectious virions, which contain a spherical nucleocapsid (viral genome compounded with viral structural proteins) surrounded by a lipid bilayer from the host cell membrane.

[0066] Retroviral vectors can be used to produce enveloped virions that are infectious and either replication competent or replication defective. Replication competent infectious retroviral vectors contain all the necessary genes for virion synthesis and continue to propagate themselves once infection of the host cell occurs. Replication defective infectious retroviral vectors do not spread after the initial infection. This is achieved by replacing most of the coding regions of the retrovirus with the gene or nucleotide sequence to be transferred; rendering the vector incapable of making the proteins required for additional replication cycles.

[0067] Alternatively or additionally, retroviral vectors can be used to produce virus-like particles (VLPs), which lack the genome from a retrovirus and are both non-infectious and non-replicative. Due to the beneficial properties of VLPs, VLPs can be used as antigen delivery systems. In addition, because VLPs are non-infectious, they can be used as immunogenic compositions (e.g., vaccines) and are safely administered. The general structure of VLPs is similar to the virion of the above-mentioned envelope, but lacks the genome from a retrovirus, so that viral replication will be unlikely to occur. The expression of the capsid protein (e.g., Gag) of some viruses (e.g., murine leukemia virus, e.g., Moloney murine leukemia virus (MMLV)) causes self-assembly into a particle similar to a corresponding natural virus, which does not have the genetic material of the virus.

[0068] A wide variety of VLPs have been prepared. For example, VLPs comprising a single or multiple capsid proteins (with or without envelope proteins and / or surface glycoproteins) have been prepared. In some cases, VLPs are non-enveloped and assembled by expressing only one major capsid protein, such as from hepadnaviruses (e.g., Engerix spp.) as shown. TM ,GSK and Recombivax HB TM , Merck), papillomavirus (eg Cervarix TM ,GSK and Gardasil TM , Merck), parvovirus (paroviruse) or polyomavirus preparation VLP.In some embodiments, VLP is enveloped and may include multiple antigenic proteins present in the corresponding natural virus.VLP is generally similar to its corresponding natural virus and may be a multivalent particle structure.In some embodiments, antigenic proteins may be present in VLP as components of VLP structure and / or on the surface of VLP.In some embodiments, antigen presentation in the case of VLP is conducive to inducing neutralizing antibodies against the antigen compared with other forms of antigen presentation (such as soluble antigens not associated with VLP).Most of neutralizing antibodies often recognize tertiary or quaternary structures, which often require antigenic proteins (such as envelope glycoproteins) to be presented in their natural viral conformation.Alternatively or additionally, VLP can be used to present antigens in the case of inducing cellular immunity (such as T cell response).In some embodiments, using antigen combinations in VLP systems can produce improved immune responses.

[0069] II. Detectable HCMV

[0070] As described above, among other aspects, the present disclosure provides methods for determining the level of HCMV infection in a host cell, and by extension, the level of neutralizing antibodies present in a sample. The present disclosure encompasses the recognition that HCMV viruses with fluorescent moieties allow for the detection of viral infection (e.g., by assessing fluorescence in cells after contacting host cells with the virus). In some embodiments, the level of HCMV infection is determined by fluorescence detection, wherein the virus has been pre-incubated with a test sample (e.g., a serum sample) from a subject. In some embodiments, a candidate HCMV vaccine has been administered to a subject.

[0071] The method provided utilizes HCMV virus comprising a fluorescent moiety. Any HCMV virus capable of infecting host cells as described herein can be modified to include a fluorescent moiety. In some embodiments, for infecting fibroblasts, an HCMV virus comprising all or part of a gH / gL / gO complex can be modified to include a fluorescent moiety. In some embodiments, for infecting endothelial cells and / or epithelial cells, an HCMV virus comprising all or part of a gH / gL / UL128 / UL130 / UL131 complex can be modified to include a fluorescent moiety. The modified HCMV strain responsible for fluorescence detection is known in the art and can be used according to the present disclosure. For example, UL32-EGFP-HCMV-TB40 is an in vitro recombinant (ATCC; VR-1578) of a plasmid of HCMV strain TB40 and the TB40UL32 gene fused to GFP. The UL32-EGFP-HCMV-TB40 recombinant strain produced a recombinant HCMV virus with GFP fused to the C-terminus of the tegument phosphoprotein pp150 (product of the UL32 gene) (Sampaio et al., 2005 Journal of Virology 79(5):2754). Since GFP is associated with viral structural proteins, the viral particles emit green fluorescence under appropriate illumination. The UL32-EGFP-HCMV-TB40 strain has been shown to have tropism for fibroblasts (Sampaio et al., 2005 Journal of Virology 79(5):2754). Another HCMV strain responsible for fluorescence detection is HB15-t178b, which contains the CMV strain AD 169 genome and a GFP reporter gene cassette (Saccoccio et al., 2011 Vaccine 29(15):2705).

[0072] It should be understood that the term fluorescence used herein refers to a luminescent portion. Typically the fluorescent portion comprises an electron that absorbs electromagnetic energy of one wavelength and emits electromagnetic energy of a second wavelength. Some proteins or small molecules in cells are naturally fluorescent (e.g., NADH, tryptophan, endogenous chlorophyll, phycoerythrin, or green fluorescent protein (GFP)). It will be understood that various mutants of fluorescent proteins have been engineered and can be used according to the present disclosure, such as EGFP, blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, CyPet), yellow fluorescent protein (e.g., YFP, Citrine, Venus, YPet), redox-sensitive GFP (e.g., roGFP), and monomeric GFP, etc. GFP and other fluorescent proteins can be expressed exogenously in cells alone or as fusion proteins. This approach allows fluorescent proteins to be used as reporter molecules for any number of biological events (e.g., subcellular localization and expression patterns).

[0073] Alternatively or additionally, specific or general proteins, nucleic acids, lipids or small molecules can be labeled with exogenous fluorophores, fluorescent dyes, which can be small molecules, proteins or quantum dots. Exemplary fluorophores include, but are not limited to, 1,5IAEDANS; 1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-carboxynaphtholfluorescein; 5-carboxytetramethylrhodamine (5-TAMRA); 5-hydroxytryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-amino-4-methylcoumarin; 7-aminoactinomycin D (7-AAD); 7-hydroxy-4-1-methylcoumarin; 9-amino-6-chloro-2-methoxyacridine (ACMA); ABQ; acid fuchsin; acridine orange; acridine red; acridine yellow; trypan yellow (Acriflavin); trypan yellow Feulgen SITSA (Acriflavin Feulgen SITSA); aequorin (luminescent protein); AFP (AutoFluorescent Protein, QuantumBiotechnologies) (see sgGFP, sgBFP); Alexa 350; Alexa 430; Alexa 488; Alexa 532; Alexa 546; Alexa 568; Alexa 594; Alexa 633; Alexa 647; Alexa 660; Alexa 680; Alizarin complex; Alizarin red; Allophycocyanin (APC); AMC; AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; Aminactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7GLL; Atrazon; ATTO-TAG TM CBQCA; ATTO-TAG TM FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (bisaminobenzene oxadiazole); BCECF (high pH); BCECF (low pH); berberine sulfate; β-lactamase; BFP blue-shifted GFP (Y66H); blue fluorescent protein; BFP / GFP FRET; Bimane; Bisbenzemide; Bisbenzemide (Hoechst); Bis-BTC; Blancophor FFG; Blancophor SV; BOBO TM -1;BOBO TM -3; Bodipy492 / 515; Bodipy493 / 503; Bodipy500 / 510; Bodipy 505 / 515; Bodipy 530 / 550; 581 / 591; Bodipy 630 / 650-X; Bodipy 650 / 665-X; Bodipy 665 / 676; Bodipy F1; Bodipy FL ATP; Bodipy F1-ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; BodipyTR ATP; Bodipy TR-X SE; BO-PRO TM -1; BO-PRO TM-3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcium Crimson; Calcium Crimson Blue TM ;Calcium Green TM ;Calcium Green TM -1Ca 2+ Dye; Calcium Green TM -2Ca 2+ ;Calcium Green TM -5N Ca 2+ ;Calcium Green TM -C18 Ca 2+ ;CalciumOrange TM ;Calcofluor White;Carboxy-X-rhodamine (5-ROX);Cascade CascadeYellow TM ; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (cyan fluorescent protein); CFP / YF P FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hcp; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin phalloidin; C-phycocyanine; CPM I methylcoumarin; CTC; CTC formazan; Cy2 TM ;Cy3.1 8;Cy3.5 TM ;Cy3 TM ;Cy5.1 8;Cy5.5 TM ;Cy5 TM ;Cy7 TM; Cyan GFP; cyclicAMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansamide; Dansylcadaverine; Dansyl chloride; DansylDHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3'DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-specific ratio); DiA (4-Di 16-ASP); dichlorodihydrofluorescein diacetate (DCFH); DiD-lipophilic tracer; DiD (DilC18 (5)); DIDS; dihydrorhodamine (Dihydorhodamine) 123 (DHR); Dil (DilC18 (3)); I-dinitrophenol; DiO (DiOC18 (3)); DiR; DiR (Di1C18 (7)); DM-NERF (high pH); DNP; dopamine; DsRed ;DTAF;DY-630-NHS;DY-635-NHS;EBFP;ECFP;EGFP;ELF97;Eosin;Erythrophyll;Erythrophyll ITC;Ethidium bromide;Ethidium homodimer-1 (EthD-1);Euchrysin;EukoLight;Europium (111) chloride;EYFP;Fast Blue;FDA;Feulgen (para-purpurin);FIF (Formaldehyd-induced fluorescence);FITC;Flazo Orange;Fluo-3;Fluo-4;Fluorescein (FITC);Fluorescein diacetate;Fluoro Emerald;Fluoro-Gold (Fluoro-Ruby);FluorX; 1-43; 4-64; Fura Red TM(high pH); Fura RedTM / Fluo-3; Fura-2; Fura-2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP(S65T); GFP red shift (rsGFP); non-UV excited wild-type GFP (wtGFP); UV excited wild-type GFP (wtGFP); GFPuv; Gloxalic Acid; Granular blue; Hematoporphyrin; Hoechst 33258; Hoechst33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbene (fluorescent gold); Serotonin; Indo-1 (high calcium); Indo-1 (low calcium); Indo dicarbocyanine (DiD); Indo tricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine rhodamine; Lissamine rhodamine B; Calcein / ethidin homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow / Blue; Mag Green; Sudan Red (Magdala Red); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indigo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; I Maxilon Brilliant Flavin 10GFF; Maxilon Brilliant Flavin 8GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Green Orange; Mitotracker Green Red; Plucamicin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyrronine Diphenyl Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxedidole; Norepinephrine; Nuclear Fast Red; Nuclear Yellow;NylosanBrilliant lavin E8G; Oregon; Oregon 488; Oregon 500; Oregon 514 Pacific Blue; Feulgen; PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP Cy5.5; PE-TexasRed (Red 613); Radix Red B (Sudan Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; Photoresist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-1PRO-3; Primrose; Procion Yellow; Propidium lodid (P1); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY7; Quinacrine Mustard); Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine green; Rhodamine phallic acid; Rhodamine: phalloidin; Rhodamine red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron I Brilliant Red B; Sevron; Sevron Yellow L; sgBFPTM (super luminescent BFP); sgGFP TM(super luminescent GFP); SITS (primulin; stilbene isothiosulfonic acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; Spectrum Aqua; Spectrum Green; Spectrum Orange; Spectrum Red; SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium); stilbene; Sulforhodamine B and C; Supersulforhodamine; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Texas Red- Conjugates; DiSC3; Thiazine Red R; Thiazolyl Orange; Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofiuor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC Tetramethyl RodaminelsoThioCyanate; True Blue; Tru Red; Ultralite; Fluorescein Sodium B; Uvitex SFC; wt GFP; WW781; X Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO3; YOYO-1; YOYO-3; Sybr Green; Thiazole Orange (interchelating dye) dye); semiconductor nanoparticles such as quantum dots; or caged fluorophores (which can be activated with light or other electromagnetic energy sources); or combinations thereof.

[0074] III. Infection Assay

[0075] The determination of HCMV infection titer generally comprises contacting host cells susceptible to HCMV infection with serially diluted viruses (e.g., HCMV comprising a fluorescent moiety) in the absence of any test substance allowing infection of the cells. The infection titer of the virus preparation can be calculated (e.g., by flow cytometry) by determining the number of target cells expressing a reporter gene construct (e.g., a fluorescent moiety, such as GFP).

[0076] To assess the presence and / or activity of neutralizing antibodies in the serum of a subject to whom a candidate vaccine has been administered, the serum can be pre-incubated with HCMV (e.g., HCMV comprising a fluorescent moiety such as GFP) for a period of time (e.g., at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, or longer) sufficient to reduce the infectivity of HCMV with neutralizing antibodies. The pre-incubated mixture of the serum and HCMV (e.g., HCMV comprising a fluorescent moiety) can then be used to contact host cells susceptible to HCMV infection in a permissive infection. One of ordinary skill in the art will be able to determine the appropriate dilution of serum for infection determination. For example, in some embodiments, the dilution tested is 1:6, 1:12, 1:24, 1:48, 1:96, 1:192, or a combination thereof.

[0077] It should be appreciated that the conditions allowing infection can vary according to multiple factors, including viral strains, host cell types, temperature, cell confluence, viral concentration, etc. Those of ordinary skill in the art can appropriately modify infection conditions. In some embodiments, infection conditions include host cells and virus incubation for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours or longer. It should also be appreciated that monitoring for cell infection can include visual cell morphology assessment (e.g., for cell swelling and rounding) and / or visual fluorescence assessment (e.g., detecting fluorescence on equipment such as a fluorescent microscope). After infection, host cells can be collected (e.g., by washing and trypsinization and / or scraping) and analyzed by fluorescence detection methods available in the art and as described herein.

[0078] Any host cell susceptible to HCMV infection can be used for the methods described herein. Exemplary host cells include, but are not limited to, human fibroblasts such as human foreskin fibroblasts (HFF), and human epithelial cells such as retinal pigment epithelial cells (ARPE-19). The culture medium in which the host cells are grown during the infection assay can vary depending on the cell type. For example, in some embodiments, the HFF infection culture medium comprises MEM+5% FBS+1% PenStrep. In some embodiments, the APRE infection culture medium comprises DMEM:F-12+1% FBS.

[0079] IV. Fluorescence Detection and Neutralizing Antibody Assessment

[0080] Fluorescence can be detected using any appropriate method, including, for example, flow cytometry analysis, fluorescence activated cell sorting, or flow microfluorometry. It will be appreciated that the sensitivity of fluorescence detection generally depends on the number of copies of the fluorescent entity in the detection system, the efficiency of the detection instrument, and the fluorescence brightness of the fluorescent entity relative to background fluorescence arising from endogenous bioluminescent entities in the sample and from fluorescent entities non-specifically associated with the sample. The brightness of the fluorescent entity, in turn, depends on the quantum efficiency of the fluorescent entity producing the fluorescent signal and the light absorption capacity of the fluorescent entity (quantified by the extinction coefficient).

[0081] It will be appreciated that reporter molecules such as green fluorescent protein (GFP) can be used to identify and / or separate cells based on the level of surface and / or intracellular fluorescence. In general, the correlation between fluorescence intensity and protein product (e.g., viral infection and production) will be observed. For example, in some embodiments, a high level of fluorescence intensity in a host cell is associated with a high level of viral infection in the host cell.

[0082] In some embodiments, the overall fluorescence level of the cell is assessed (e.g., regardless of the subcellular localization of the fluorescence). In some embodiments, the fluorescence level of the cell is assessed at a specific subcellular location of the cell (e.g., the nucleus). In general, flow cytometry allows the phenotype of a large number of cells to be determined quantitatively, however many flow cytometry applications do not include the ability to image cells because they are quantitative. In some embodiments, the detection and quantification of cell fluorescence is sufficient to assess infection. It will be appreciated that in some cases, it is desirable to determine the location of fluorescence within the cell. In some embodiments, the flow cytometric analysis of cells can be combined with a visual assessment of fluorescence. Dual analysis of fluorescence levels and location can be performed using multiple devices (e.g., flow cytometry and fluorescence microscopy) or a single device. Such devices that allow fluorescence analysis and / or quantification, combined with location analysis, are available in the art. For example, ImageStream x Both the intensity and location of fluorescence are quantified and allow imaging of more than 50,000 cells per minute.

[0083] The cellular fluorescence level can be quantitatively measured by any appropriate method known in the art. The cellular fluorescence level can be compared with a reference level. In some embodiments, the reference level is a predetermined reference level or a historical reference level. In some embodiments, the reference level is obtained by comparing in parallel with a reference sample (e.g., a positive and / or negative control).

[0084] The advent of screening and selection methods using flow cytometry and cell sorting has dramatically increased the number of cells that can be screened. For example, millions of cells can be screened in a short period of time, and subpopulations and single cells can be isolated from mixed cell populations even when their frequency in the population is as low as 10. -6 When. It will be appreciated that one of the advantages of the provided method is that sample evaluation can be achieved in a high-throughput manner. In some embodiments, the provided method is compared with known infection and / or neutralizing antibody detection methods (such as staining of viral proteins, fluorescence microscopy, ELISPOT, etc.), and flux has increased by 10%, 20%, 30%, 40%, 50%, 60%, 70% or more. The provided method can be carried out in a multi-well plate manner, and is therefore particularly suitable for medium to high-throughput screening. In some embodiments, the multi-well plate has 96 holes. In some embodiments, the multi-well plate has another number of holes, including but not limited to plates with 6, 12, 24, 384, 864 or 1536 holes. The terms "multi-well plate" and "microtiter plate" are used interchangeably.

[0085] In some embodiments, living cells are analyzed. In some embodiments, cells are fixed prior to analysis.

[0086] In some embodiments, the present disclosure provides methods for measuring anti-HCMV neutralizing antibodies by relevant fluorescence detection and / or viral infection of host cells. For example, HCMV comprising a fluorescent moiety (e.g., GFP) can be pre-incubated with serum from an object immunized with a candidate HCMV vaccine. Neutralizing antibodies present in serum will reduce the infection of host cells that are usually susceptible to HCMV infection to HCMV (e.g., HCMV comprising a fluorescent moiety). A pre-incubated mixture comprising HCMV (e.g., HCMV comprising a fluorescent moiety) and serum can be subsequently used to contact such host cells, and the fluorescence level of the host cells can be assessed. Based on the fluorescence level in the host cells, the infection level of the host cells can be determined. The infection level of the host cells is generally negatively correlated with the presence and amount of neutralizing antibodies in the serum, and conversely, is related to the efficacy of the candidate vaccine in inducing a therapeutic response (e.g., a protective immune response). For example, the more effective a candidate vaccine is at inducing neutralizing antibodies in a subject, the more neutralizing antibodies are present in the serum, which in turn corresponds to a reduction in the ability of HCMV comprising a fluorescent moiety to infect host cells after pre-incubation with the serum, and further corresponds to a reduction in fluorescence detection in the host cells after contacting them with HCMV comprising a fluorescent moiety. In some embodiments, the methods of the present disclosure also include selecting and / or identifying a candidate vaccine (e.g., a HCMV candidate vaccine) as a vaccine that induces neutralizing antibodies based on the correlations described herein.

[0087] Example

[0088] The following examples describe some exemplary modes of making and practicing certain compositions described herein. It should be understood that these examples are for illustrative purposes only and are not meant to limit the scope of the compositions and methods described herein.

[0089] Example 1: Immunization of rabbits with virus-like particles

[0090] This example describes exemplary immunization of rabbits with virus-like particles containing various recombinant HCMV antigens.

[0091] HEK 293T cells (ATCC, CRL-11268) were transiently transfected with expression plasmids encoding various recombinant HCMV antigens using the calcium phosphate method. The expression of various HCMV antigens by HEK 293 cells was confirmed by flow cytometry. 48 to 72 hours after transfection, the supernatant containing VLPs was harvested and filtered through a membrane with a pore size of 0.45 μm, and then concentrated and purified by ultracentrifugation of a 20% sucrose cushion in a SW32 Beckman rotor (25,000rpm, 2 hours, 4°C). The precipitate was resuspended in sterile endotoxin-free PBS (GIBCO) to obtain a 500-fold concentrated VLP stock solution. The total protein of the aliquot was determined by a Bradford assay quantitative kit (BioRad). The purified VLPs were stored at -80°C until use.

[0092] Rabbits were immunized intramuscularly with VLPs at t = 0 and t = 8 weeks as shown in Table 1 below. Sera were collected at t = 4, 6, 8, 10, 13 and 16 weeks.

[0093] Table 1.

[0094] Tested products # (n = 6 / group) dose Description of inspection product 1 100 μg gB / pp65 bivalent VLP 6 100 μg / each gB / pp65 bivalent VLP + gH-G / pp65 bivalent VLP (1:1 ratio)

[0095] Enzyme-linked immunosorbent assay (ELISA) was performed to determine the HCMV IgG content in rabbit serum. Figure 1 Shows strong and long-lasting immunity in rabbits immunized with gB / pp65 bivalent VLPs (top) and gB / pp65 bivalent VLPs + gH-G / pp65 bivalent VLPs (bottom).

[0096] Example 2: Neutralization activity based on flow cytometry

[0097] This embodiment describes the evaluation of neutralizing antibody responses to HCMV from sera of immunized animals. The purpose of this embodiment includes, but is not limited to: 1) confirming the immunogenicity of CMV clinical candidate components from other animal studies; 2) evaluating dosage and potential synergy and / or antagonism with administration routes (e.g., IM versus IP administration); and 3) evaluating immune enhancement and immune persistence.

[0098] Serum samples were collected at t=0 weeks (POV) and t=2 weeks (P2Vdl4) time points from rabbits immunized with Test Article #6 from Example 1. Samples from this group were pooled, heat inactivated (HI), and then tested for neutralizing antibody activity as described below.

[0099] Serum samples were diluted 1 / 6 to 1 / 96. HFF cells (P=9, 24 hours after inoculation) and freshly harvested TB40-230212 viruses were used. TB40-230212 viruses are descendants of TB40-010212. TB40-010212 was obtained from ATCC (ATCC VR-1578; human herpes virus 5; UL32-EGFP-HCMV-TB40). Viral infection continued for 15 days in T150 infection bottles (Table 2). Microscopic observation indicated that the virus was infectious. Infection medium was TB40-HFF-1 infection medium (MEM+5% FBS+1% Pen / Strep) or VR1814-APRE-19 infection medium (DMEM:F-12+1% FBS).

[0100] Table 2.

[0101]

[0102] T150 bottles inoculated with HFF were infected with CMV TB40-010212 (5 vials) + 20 ml of infectious medium (MEM-Minimum Essential Medium Eagle Cat. No. M4655 + 5% FBS + 1% P / S from Sigma). After 15 days in a CO2 incubator / 37°C, the bottles were completely infected. The virus was harvested by removing almost all of the supernatant from the bottle and kept sterile in a 50 mL Falcon tube. The remaining supernatant, about 5 to 7 mL, was used to help scrape the cells with a cell scraper (NUNC, Cat#179707). After scraping, strong up and down pipetting (up to 10X) promotes the release of the virus from the cells. Add half of the original supernatant volume from the Falcon tube (cca.8 mL) to obtain more potent virus.

[0103] Five vials of TB40-010212 (ancestor) from liquid nitrogen were infected in one T150 bottle and incubated for 1 hour at 37°C, 5% CO2. After incubation, 12 ml of infection medium was added. The virus was concentrated in 15 mL of supernatant.

[0104] Cytogam TM(CMV-IGIV-cytomegalovirus immune globulin intravenous injection-human; CSL Behring; commercially available concentration 2.5g / 50ml or 50mg / ml) was used as a positive control. A 1 / 20 dilution was made as the base concentration (1 to 20 dilution: 100μl of Cytogam TM 50mg / mL + 1900μL infection medium) and subsequently used for dilutions, using the same dilutions for rabbit serum. Cytogam TM Heat inactivated under the same conditions as rabbit serum (described in Tables 3-5 below).

[0105] Table 3. Infection assay

[0106]

[0107] Table 4. POV, P2Vd14 and Cytogam TM Dilution of pooled serum

[0108]

[0109]

[0110] Table 5. HFF-1 cell growth and infection medium

[0111]

[0112] Each specific concentration of serum or Cytogam TM The final virus dilution was combined with 1 / 2 and rotated at 37°C for 1 hour. Six well plates of HFF cells with 95% to 100% confluence were carefully rinsed twice with warm PBS, and then 100 μL mixture / well of virus serum mixture + 200 μL infection medium were applied in duplicate to avoid drying during 4 hours. Incubate at 37°C / 5% CO2 for 4 hours (vibration every 60 minutes). After 4 hours of incubation, carefully aspirate the contents from each well with a pipette and add 3mL fresh infection medium. The plate was kept in an incubator at 37°C / 5% CO2 for 10 days. Usually, daily checks for viral infection in cells were performed after 5 days after infection. The swelling and rounding of cells were observed by optical microscopy or by detecting green fluorescence with a fluorescence microscope.

[0113] For sample collection, aspirate the medium from each well. Rinse each well twice with PBS (HyClone DPBS / modified without calcium and magnesium; Cat#SH30028.02) and add 100 μL of 1X trypsin-EDTA (Sigma; Cat#T4174-100ml) and 100 μL of PBS. Keep the sample in a CO2 incubator for 2 to 3 minutes until the cells are thoroughly trypsinized. Add 1 mL of PBS+5% FBS to each well to stop trypsin. Collect the sample into a clear flow-intended tube (two wells per tube for the same sample) (BD Falcon, 5ml polystyrene round bottom, REF 352054). Check the plate under an optical microscope to confirm that all cells have been collected (if not, add an additional 500 μL PBS+5% FBS to collect the remainder). Centrifuge the sample at 900 rpm for 10 minutes. Discard the supernatant and save the pellet. Vortex the tube to disperse the pellet in the remaining buffer. Perform the remaining steps with minimal light exposure. Add 200 μL of fixative (BD Biosciences, BD Cytofix, Fixation Buffer Cat#554655, 100 ml) and incubate on ice for 15 minutes. Add 1 mL of PBS + 5% FBS to each tube of mixture to stop Cytofix. Centrifuge the sample at 900 rpm for 10 minutes. Discard the supernatant and keep the pellet. Reconstitute the pellet in 500 μL PBS + 5% FBS and vortex vigorously before placing it on the flow cytometer.

[0114] The samples were subjected to flow cytometry and analyzed using Cellquest software. The FSC and SSC parameters were set to "linear", while all other parameters were set to "logarithmic". 100,000 cells were collected during flow cytometry analysis of infected cells. The general conditions for flow using HFF cells and TB40 CMV were FSC El and 4.83; SSC 325; FL1 427, but the parameters could be slightly changed around the existing conditions to get a more appropriate dot plot.

[0115] Figure 2 Described is an exemplary FACS analysis of GFP expression in fibroblasts indicating neutralizing antibody responses induced in rabbits with gB / pp65 CMV VLPs. Rabbits (n=6 / group) were immunized (IM) twice at weeks 0 and 8 and bled 2 weeks later. Sera were pooled and tested at the indicated dilutions against Cytogam at similar dilutions. TM Comparison of HFF fibroblasts against CMV virus expressing GFP (TB40). + ) 100,000 cells were collected during flow cytometric analysis of the cells.

[0116] Figure 3 Described is an exemplary FACS analysis of GFP expression in fibroblasts indicating neutralizing antibody responses induced in rabbits with bivalent gB+gHCMV VLPs. Rabbits (n=6 / group) were immunized (IM) twice at weeks 0 and 8 and bled 2 weeks later. Sera were pooled and tested at the indicated dilutions with Cytogam at similar dilutions. TM Comparison of HFF fibroblasts against CMV virus expressing GFP (TB40). + ) 100,000 cells were collected during flow cytometric analysis of the cells.

[0117] Example 3. Exemplary microneutralization assay for detection of neutralizing antibodies by flow cytometry

[0118] This example describes the detection of functional anti-CMV neutralizing antibodies in serum samples from vaccinated animals by flow cytometry.

[0119] Materials / Equipment

[0120] The following materials and equipment were used in this assay: Human foreskin fibroblast (HFF-1) cells-ATCC#SCRC-1041; Human Arising Retinal Pigment Epithelia-(ARPE-19)-ATCC#CRL-2302; Human Herpesvirus 5 HCMV (UL32-EGFP-HCMV-TB40)-ATCC#VR-1578; Human CMV-GFP-Towne TS15-rR (obtained from Dr. M. McVoy, VCU-Virginia); Goat antiserum to rabbit IgG (GAR)-MPCappel, Cat#: 55620; Complement serum from rabbit-Sigma-Aldrich Cat#S7764-5ml; Standard guinea pig complement-Cedarlane Cat#CL-5000; Sterile distilled water - Gibco, Cat#15230; Dulbecco's modified Eagle's medium (DMEM) - HyClone; Cat#SH30243.01 (growth medium); Minimal essential medium Eagle (MEM) - Sigma; Cat#M4655-500ml (infection medium); Fetal bovine serum (FBS) - HyClone; Cat#SH30396.03; Penicillin-streptomycin solution (Pen / Strep) - Sigma; Cat#P0781-100ml; 1X trypsin-EDTA - Sigma; Cat#T4174-100ml; Modified phosphate buffered saline (DPBS - without calcium and magnesium) - HyClone; Cat#SH30028.02; Fixation buffer - BD Biosciences; BD Cytofix Cat#554655-100ml; Cytogam (CMV-IGIV- Cytomegalovirus Immune Globulin Intravenous / Human - CSL Behring, commercially available at 2.5g / 50ml; Dimethyl Sulfoxide (DMSO) - Sigma-Aldrich Cat#D1435-500ml; Biosafety Cabinet; Incubator 5% CO2, 37°C; Centrifuge; Vortex; Sample collection tubes for flow cytometry (BD Falcon, 5ml polystyrene round bottom, REF 352054); 6-well plates; Multichannel micropipette reservoirs, 5ml and 10ml graduated pipettes; Single channel micropipette adjustable from 10μl to 1000μl with disposable tips; Cell scraper - NUNC; Cat#179707; 50ml Falcon tubes - BD358206; Rotor - for eppendorf tubes; Fluorescence microscope; FACSCAN machine.

[0121] Obtaining and harvesting viruses

[0122] HFF-1 cells (95% confluent monolayers seeded in T150 flasks) were infected with 1.5 ml of UL32-EGFP-HCMV-TB40 ("TB40"). ARPE-19 cells were infected with HCMV-GFP-Towne-TS15-rR ("Towne") in the same manner. Since these viruses are light sensitive, cells were infected in the absence of light (light off). The flask was incubated in a CO2 incubator / 37°C for 30 minutes to allow contact between cells and virus.

[0123] Then add 25 ml of infection medium to the cells. (See Tables 6 and 7). Keep the bottle in a CO2 incubator / 37°C until the cells are fully infected (in the case of TB40, observe the swelling and rounding of the cells by optical microscopy or green fluorescence using a fluorescent microscope; for Towne, infection is detected by green fluorescence). The time required for a good infection depends on the infectivity of the ancestral virus.

[0124] Harvest starts with removing almost all of the supernatant from the bottle and keeping it sterile in a 50ml Falcon tube. About 5ml of residual supernatant is convenient for scraping cells with a cell scraper. Scrape completely and blow up and down vigorously (up to 10X) to release the virus from the cells. Add 10ml volume of the original supernatant from the Falcon tube and centrifuge at 900rpm for 10 minutes. Remove the precipitation and retain about 15ml of concentrated virus. Add 5% of the total amount of virus in a cryoprotectant (DMSO). Prepare 1ml aliquots, label and keep at -80°C for a short time or keep in liquid nitrogen for a long time.

[0125] Table 6. HFF-1 cell growth and infection medium for CMV-GFP-TB40

[0126]

[0127] Table 7. ARPE-19 cell growth and infection medium for CMV-GFP-Towne

[0128]

[0129] Experimental setup and methods

[0130] Before use, the serum samples before and after immunization were kept at 56°C for 30 minutes for heat inactivation. The commercially available serum Cytogam, which serves as a positive control, was also heat inactivated. Serum dilution was performed from 1 / 6 to the desired dilution, in duplicate. Cytogam was tested at comparable dilutions, and the stock reagent was diluted 1:20 in advance to adjust the Ig content of human / rabbit serum (see Table 8).

[0131] Table 8. Serum and virus dilutions (1 in 2 virus dilution shown as an example)

[0132]

[0133] Specific concentrations of serum (Cytogam) and virus were combined and rotated at 37°C for 1 hour. In some assays, complement was included. For assays using complement and HFF cells, 10% standard guinea pig complement was added to each specific serum (Cytogam) / virus mixture. Virus controls were treated in the same manner. For assays using complement and ARPE-19 cells, 2.5% rabbit complement was added to each mix, regardless of the species used (rabbit or serum). The serum and virus were rotated at 37°C for 1 hour.

[0134] Before applying the virus-serum mixture, 95% confluent cells (seeded in 6-well plates) were carefully rinsed twice with warm PBS in duplicate. 100 μl of the mixture / well + 200 μl infection medium was added to avoid drying during the 4-hour incubation. The cells were incubated at 37°C / 5% CO2 for 4 hours (shaking every 60 minutes). After the 4-hour incubation, the contents of each well were carefully pipetted or poured out, and 3 ml of appropriate fresh warm infection medium was added. The plate was incubated at 37°C / 5% CO2 until fully infected. After 5 days of incubation, the cells were analyzed for viral infection. The infection of HFF-1 cells was determined by observing the swelling and rounding of the cells using an optical microscope or the green fluorescence of a fluorescence microscope. ARPE-19 cell infection was detected as green fluorescence using a fluorescence microscope.

[0135] Sample collection and preparation for flow cytometry

[0136] Before sample collection, the presence of a good infection is confirmed under an optical microscope and, if possible, by analyzing the GFP incorporated into the cells using a fluorescence microscope. Aspirate or pour out the culture medium from each well. Rinse the cells twice with PBS (HyClone DPBS / modified, without calcium and magnesium; Cat#SH30028.02). Add 100 μl of 1X trypsin-EDTA (Sigma; Cat#T4174-100ml) and 200 μl of PBS to HFF-1 cells. Add 200 μl of 1X Trypsin-EDTA and 100 μl of PBS to ARPE-19 cells. Keep the cells in a CO2 incubator for 2 to 3 minutes until the cells are completely digested with trypsin. Add 1 ml of PBS+5% FBS to each well to stop trypsin. Collect the cells into a transparent flow-purpose tube (two wells per tube for the same sample) (BD Falcon, 5 ml polystyrene round bottom, REF 352054). The wells were observed under a light microscope and if cells remained, an additional 500 μl of PBS + 5% FBS was added to collect additional cells.

[0137] Centrifuge the Falcon tube at 900rpm for 10 minutes. Discard the supernatant and retain the pellet. Vortex the tube to disperse the pellet into the residual buffer. Perform the remaining steps in the dark. Add 200μl Cytofix (BD Biosciences, BD Cytofix, Fixation Buffer Cat#554655 100ml) to each tube and incubate the samples on ice for 15 minutes. Add 1ml of the mixture PBS+5% FBS to each tube to terminate Cytofix. Centrifuge the tube at 900rpm for 10 minutes. Pour off the supernatant and retain the pellet. Reconstitute the pellet in 500μl of PBS+5% FBS, shake vigorously and perform flow cytometry.

[0138] Example 4: Neutralizing activity of exemplary VLPs in rabbits

[0139] Plasmid transfection CHO cells (prepared as described in PCT / US2012 / 64556) with monovalent gB-G and monovalent gH-G at a cell density of 1.5E06 to 2.0E06 cells / mL. Add stuffer DNA to make total DNA concentration up to 1 μg per mL cell culture. Plasmid for transfection is first purified by MaxiPrep or GigaPrep plasmid purification kit (Qiagen). PEIMAX for transfection to deliver DNA to cells is provided with a (PEI:DNAwt / wt) ratio of 6:1. 72 hours after transfection, in a 1 liter bottle, cell culture was harvested by centrifugation at 4000rpm for 20 minutes using the rotor JS-4.2A of Beckman Coulter. Supernatant is filtered through a 0.8 / 0.45 μm filter (AcroPak500Capsule, Pall). The supernatant of filtration is then concentrated through tangential flow filtration (TFF for concentrating VLP), and for the buffer diafiltration containing histidine.The Benzonase (Novogen 99% purity (D00127703), every μ L contains 250 units) of 70 μ L is suspended in 400 ml PBS supplemented with MgCl2 to have a final concentration of 2 mM.The TFF retentate part is mixed with the Benzonase diluted in PBS solution and kept at room temperature for 1 hour in a rotary oscillator (head to tail rotation), and then the pipe is placed on 4 ℃ and spend the night.Then the material processed by the nuclease (benzonase) of diafiltration is loaded into anion exchange chromatography column (for reducing the AEX of DNA and host protein), wherein collects flowthrough.Then the flowthrough is aseptically filtered by 0.45 μ m and divided into different volumes.

[0140] The monovalent gB-G and monovalent gH-G VLP compositions prepared as described were mixed together, with aluminum adjuvant, and then tested for neutralization activity using the microneutralization assay described in Example 3 in female New Zealand white rabbits aged 6 to 8 weeks (a minimum of 5 animals per test group). Rabbits were intramuscularly immunized 3 times with 0.5 ml (250 μl at two sites proximal to the thigh muscle behind the coccyx), once on day 0 (priming) and once on day 57 (boosted at week 8), once on day 162 (boosted at week 24). Rabbits were treated with 50 μg of the VLP compositions of both monovalent gB-G and monovalent gH-G (mixed together in a 1:1 ratio). To evaluate the humoral immune response in rabbits, blood was collected from all rabbits in the study before the first immunization and then on days 28, 42, and 55 after the first immunization and on day 14 after the second immunization.

[0141] Neutralizing antibody responses to HCMV were determined using a microneutralization assay based on a GFP-expressing CMV virus (TB40) in fibroblasts and flow cytometric analysis of infected (GFP+) HFF-1 cells as described previously. Rabbit sera collected before and after immunization as described were pooled and tested in the presence of guinea pig complement relative to positive controls CMV hyperglobulin, Cytogam TM and negative controls consisting of empty Gag VLPs (lacking antigenic proteins gB-G and / or gH-G) for neutralizing activity against GFP-expressing HCMV in HFF fibroblasts.

[0142] Figure 4 The percentage neutralization of HFF-1 cells incubated with CMV-GFP-TB40-010512 virus in the presence of 10% guinea pig complement and rabbit serum is shown (group 7 pooled sera from 15RA09 were used as positive data representatives, where animals were treated three times with monovalent gB-G and monovalent gH-GVLPs, and group 8 from the 15RA05 study (empty Gag) was used as a negative control representative). As in Figure 4 As shown in , the combination of monovalent gB-G and monovalent gH-G VLP compositions elicited a rapid, synergistic, and sustained neutralizing antibody response against fibroblast infection in rabbits, whereas empty Gag VLPs showed no neutralizing antibody response. ("GFP-TB40-010512" stands for Human Herpesvirus 5 HCMV (UL32-EGFP-HCMV-TB40)-ATCC# VR-1578 (described in Example 3) grown on May 1, 2012.)

[0143] Pooled rabbit sera were also tested for neutralizing antibody responses to HCMV in epithelial cells based on HCMV virus expressing GFP (Towne TS15-rR) using a microneutralization assay and flow cytometric analysis of infected (GFP+) ARPE-19 cells as described previously. Rabbit sera collected before and after immunization as described were pooled and tested in the presence of 2.5% rabbit complement relative to positive controls CMV superglobulin, Cytogam TM Neutralization activity of HCMV against GFP expressing in ARPE-19 epithelial cells and negative control consisting of empty Gag VLPs (lacking antigenic proteins gB-G and / or gH-G).

[0144] Figure 5The percentage neutralization of ARPE-19 cells incubated with CMV-GFP-Towne-150612 virus in the presence of 2.5% rabbit complement and rabbit serum is shown (pooled sera from Group 7 of 15RA09 were used as positive data representatives, where animals were treated three times with monovalent gB-G and monovalent gH-G VLP compositions, and Group 8 from the 15RA05 study (empty Gag) was used as a negative control representative). As in Figure 5 As shown in , the combination of monovalent gB-G and monovalent gH-G VLP compositions elicited a rapid, synergistic, sustained neutralizing antibody response against epithelial cell infection in rabbits, whereas empty Gag VLPs showed no neutralizing antibody response. ("GFP-Towne-150612" stands for human CMV-GFP-Towne TS15-rR (obtained from Dr. M. McVoy, VCU-Virginia, and described in Example 3) grown on June 15, 2012.)

[0145] Other Implementations

[0146] Other embodiments of the present disclosure will be apparent to those skilled in the art in view of the specification or practice of the disclosure disclosed herein. It is intended that the description and examples be considered exemplary only, with the true scope of the present disclosure being indicated by the following claims. The contents of any reference referenced herein are incorporated herein by reference in their entirety.

Claims

1. A method comprising the following steps: (i) mixing serum from a subject that has been immunized with the HCMV candidate vaccine with HCMV comprising a fluorescent moiety to form a mixture; (ii) contacting a host cell susceptible to HCMV infection with the mixture of (i) under conditions permissive for infection; (iii) evaluating the fluorescence level of the host cells that have been contacted with the mixture by flow cytometry; and (iv) determining the level of infection of the host cell based on the assessed fluorescence level.

2. The method of claim 1, wherein the immunized subject is a human.

3. The method of claim 1, wherein the serum comprises anti-HCMV neutralizing antibodies.

4. The method of claim 1, wherein the HCMV candidate vaccine comprises VLPs.

5. The method of claim 4, wherein the VLP comprises one or more of gB, gH, gH-G, and pp65 from HCMV.

6. The method of claim 1, wherein the fluorescent moiety is a fluorescent protein.

7. The method of claim 6, wherein the fluorescent protein is selected from the group consisting of green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, and combinations thereof.

8. The method of claim 6, wherein the fluorescent protein is green fluorescent protein.

9. The method of claim 1, wherein the HCMV comprising a fluorescent moiety is a TB40-GFP strain.

10. The method of claim 1, wherein the HCMV comprising a fluorescent moiety is an AD 169-GFP strain.

11. The method of claim 1, wherein the mixture of (i) is incubated for at least 15 minutes, at least 30 minutes, at least 1 hour, or at least 2 hours prior to step (ii).

12. The method of claim 1, wherein the host cell susceptible to HCMV infection is a fibroblast.

13. The method of claim 12, wherein the fibroblasts are human foreskin fibroblast (HFF) cells.

14. The method of claim 1, wherein the host cell susceptible to HCMV infection is an epithelial cell.

15. The method of claim 14, wherein the epithelial cells are retinal pigment epithelial cells (ARPE-19).

16. The method of claim 1, wherein the evaluating step (iii) is performed in a high-throughput manner.

17. The method of claim 1, wherein the evaluating step (iii) comprises comparing the fluorescence level to a reference.

18. The method of claim 17, wherein the reference is a historical reference.

19. The method of claim 17, wherein the reference is a parallel reference.

20. The method of any one of claims 1 to 19, further comprising determining an anti-HCMV neutralizing antibody titer in serum based on the determining step.

21. The method of claim 20, further comprising evaluating the efficacy of the candidate HCMV vaccine based on the determining step.

22. The method of claim 21, further comprising selecting the candidate HCMV vaccine as a vaccine that induces neutralizing antibodies if the fluorescence level of the host cell that has been contacted with the mixture is lower than a reference fluorescence level.

23. The method of claim 1, wherein the contacting step (ii) comprises incubating for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, or at least 8 hours.

24. The method of claim 1, wherein infection of the host cells is also monitored by visual cell morphology assessment (e.g., for cell swelling and rounding) or visual fluorescence assessment (e.g., detecting fluorescence on an instrument such as a fluorescence microscope).

25. The method of claim 24, wherein the visual cell morphology assessment comprises monitoring swelling and rounding of cells.

26. The method of claim 24, wherein the visual fluorescence assessment comprises detecting fluorescence.

27. The method of claim 26, wherein the fluorescence is detected on a fluorescence microscope.

28. The method of claim 1, wherein the level of fluorescence of the nucleus of the host cell is assessed.

29. The method of claim 1, wherein the level of fluorescence is assessed throughout the host cell.

30. The method of claim 1, further comprising the step of quantifying the fluorescence level of the host cell.

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