WO2013144722A2 - Methods for detection of anti-cytomegalovirus neutralizing antibodies - Google Patents
Methods for detection of anti-cytomegalovirus neutralizing antibodies Download PDFInfo
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- WO2013144722A2 WO2013144722A2 PCT/IB2013/001021 IB2013001021W WO2013144722A2 WO 2013144722 A2 WO2013144722 A2 WO 2013144722A2 IB 2013001021 W IB2013001021 W IB 2013001021W WO 2013144722 A2 WO2013144722 A2 WO 2013144722A2
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- hcmv
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Classifications
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
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Definitions
- HCMV Human cytomegalovirus
- HCMV infection is normally unnoticed, having at most mild and nonspecific symptoms.
- certain risk groups for example in immunosuppressed patients such as AIDS patients or transplant recipients, and after prenatal infection, HCMV infection has serious 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 therapies include the use of immunoglobulins and antiviral agents such as ganciclovir and its derivatives, which are most effective when used prophylactically or very early during infection in at risk populations.
- 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
- In vitro assays are important tools to evaluate candidate vaccines for their ability to interfere with HCMV infection.
- 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.
- antigen binding ELISAs can measure antibodies specific for HCMV antigens; however, only an assay in which neutralization of viral entry into cells is measured can establish and quantify the biological activity of HCMV antigen-specific antibodies (Abai et al., 2007 J Immunol Methods 332(1- 2):82-93).
- the degree to which neutralizing antibodies reduce HCMV infection of cells in the assay is determined by quantification of nuclei of infected cells based on expression of one or more viral proteins in the cell. Such analyses can be time consuming and difficult to employ in high throughput applications. There remains a need in the art for improved methods of screening potential HCMV vaccine candidates for neutralizing antibody induction.
- the present disclosure provides methods useful for determining levels of HCMV infection in host cells and, by extension, determining levels of neutralizing antibodies present in a sample.
- the present disclosure encompasses the recognition that HCMV viruses that have a fluorescent moiety permit detection of viral infection (e.g., by assessing fluorescence in cells after contacting the host cell with the virus).
- levels of HCMV infection are determined by fluorescence detection where the virus has been preincubated with a test sample (e.g., a serum sample) from a subject.
- a test sample e.g., a serum sample
- the subject has been administered a candidate HCMV vaccine.
- Figure 1 depicts exemplary ELISA anti-gB antibody titers after immunization with bivalent gB virus-like particles (VLPs) (gB/pp65 and gB/pp65 + gH-G/pp65). A potent and sustained immunity is induced by bivalent gB VLPs in rabbits after a single immunization.
- Figure 2 depicts exemplary FACS analysis of GFP expression in fibroblast cells indicative of neutralizing antibody response induced with gB/pp65 CMV VLPs in rabbits.
- Figure 3 depicts exemplary FACS analysis of GFP expression in fibroblast cells indicative of neutralizing antibody response induced with bivalent gB + gH CMV VLPs in rabbits.
- Sera were pooled and tested at indicated dilutions in comparison to CytogamTM at similar dilutions against GFP-expressing CMV virus (TB40) in HFF fibroblasts. 100,000 cells were collected during flow cytometric analysis of infected (GFP + ) cells.
- Figure 4 depicts exemplary percent neutralizations in HFF-1 cells. Depicted are neutralizations for 15RA09 group 7 (monovalent gB-G/monovalent gH-G adjuvanted with alum) pooled samples at PlVdl4, PlVd28, PlVd42, PlVd55, and P2Vdl4; and 15RA05 group 8 (empty MLV Gag) at P2Vdl4, in presence of 10% guinea pig complement against 1 :6 CMV- GFP-TB40-010512 in HFF-1 cells.
- 15RA09 group 7 monovalent gB-G/monovalent gH-G adjuvanted with alum
- 15RA05 group 8 empty MLV Gag
- Figure 5 depicts exemplary percent neutralizations in ARPE-19 cells. Depicted are neutralizations for 15RA09 group 7 (monovalent gB-G/monovalent gH-G adjuvanted with alum) pooled samples at PlVdl4, PlVd28, PI Vd42, PlVd55, and P2Vdl4; and 15RA05 group 8 (empty MLV Gag) at P2Vdl4, in presence of 2.5% rabbit complement against 1 :3 CMV-GFP- Towne-150612 in ARPE-19 cells.
- 15RA09 group 7 monovalent gB-G/monovalent gH-G adjuvanted with alum
- 15RA05 group 8 empty MLV Gag
- amino acid in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain.
- an amino acid has the general structure H 2 N-C(H)(R)-COOH.
- an amino acid is a naturally occurring amino acid.
- 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 1-amino acid.
- Standard amino acid refers to any of the twenty standard 1-amino acids commonly found in naturally occurring peptides.
- Nonstandard amino acid refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.
- synthetic amino acid encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions. Amino acids, including carboxy- and/or amino-terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, protecting groups, and/or substitution with other chemical groups that can change the peptide's circulating half-life without adversely affecting their activity.
- Amino acids may participate in a disulfide bond.
- Amino acids may comprise one or posttranslational modifications, such as association with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.).
- the term "amino acid” is used interchangeably with "amino acid residue,” and may refer to a free amino acid and/or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.
- Antigen refers to a substance containing one or more epitopes (either linear, conformational or both) that are recognized by antibodies.
- an antigen is or comprises a virus or a viral polypeptide.
- the term "antigen” refers to a subunit antigen (i.e., an antigen which is separate and discrete from a whole virus with which the antigen is associated in nature; e.g., an antigen which is associated with a virus-like particle).
- a subunit antigen i.e., an antigen which is separate and discrete from a whole virus with which the antigen is associated in nature; e.g., an antigen which is associated with a virus-like particle.
- the term "antigen” refers to killed, attenuated or inactivated viruses.
- an antigen is an "immunogen.”
- Approximately or about As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value.
- the term “approximately” or “about” refers to a range of values that fall 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 either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
- Amelioration is meant the prevention, reduction or palliation of a state, or improvement of 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).
- prevention refers to a delay of onset of a disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.
- Dosage form As used herein, the terms “dosage form” and “unit dosage form” refer to a physically discrete unit of a 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.
- Dosing regimen is a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
- a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
- a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses.
- expression refers to one or more of the following events: (1) production of an R A template from a DNA sequence ⁇ e.g., by transcription); (2) processing of an RNA transcript ⁇ e.g. , by splicing, editing, 5 ' cap formation, and/or 3' end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
- Fluorescence refers to a moiety that luminesces. Typically fluorescent moieties contain electrons which can absorb electromagnetic energy at one wavelength and emit electromagnetic energy at 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)).
- GFP green fluorescent protein
- mutants of fluorescent proteins have been engineered and may be used in accordance with 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, among others.
- GFP and other fluorescent proteins can be expressed exogenously in cells alone or as a fusion protein. This approach permits fluorescent proteins to be used as reporters for any number of biological events, such as subcellular localization and expression patterns.
- fluorophores include, but are not limited to, 1,5 IAEDANS; 1,8-ANS; 4- Methylumbelliferone; 5-carboxy-2,7-dichlorofiuorescein; 5- Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5- TAMRA); 5-Hydroxy Tryptamine (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
- 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.; Alexa Fluor 660.TM.; Alexa Fluor 680.TM.; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S;
- Aminomethylcoumarin (AMC A); AMCA-X; Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG.TM. CBQCA; ATTO-TAG.TM. FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9
- Bodipy 530/550 Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate;
- Bodipy TMR-X, SE Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO.TM.-l; BO- PRO.TM.-3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium
- Fluorescence Fluorescence
- FITC Fluorescein
- Flazo Orange Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43.TM.; FM 4-46; Fura Red.TM.
- LysoSensor Green LysoSensor Yellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-lndo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; I Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF;
- Noradrenaline 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; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26
- Rhodamine Rhodamine
- Rhodamine 110 Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine: Phalloidine; 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 Orange; Sevron Yellow L; sgBFP.TM.
- SYTO 40 SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84;
- Fusion protein generally refers to a polypeptide including at least two segments, each of which shows a high degree of amino acid identity to a peptide moiety that (1) occurs in nature, and/or (2) represents a functional domain of a polypeptide.
- a polypeptide containing at least two such segments is considered to be a fusion protein if the two segments are moieties that (1) are not included in nature in the same peptide, and/or (2) have not previously been linked to one another in a single polypeptide, and/or (3) have been linked to one another through action of the hand of man.
- the term “gene” may include gene regulatory sequences ⁇ e.g., promoters, enhancers, etc.) and/or intron sequences. It will further be appreciated that definitions of gene include references to nucleic acids that do not encode proteins but rather encode functional RNA molecules such as tRNAs, RNAi-inducing agents, etc.
- the term “gene” generally refers to a portion of a nucleic acid that encodes a protein; the term may optionally encompass regulatory sequences, as will be clear from context to those of ordinary skill in the art. This definition is not intended to exclude 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 document refers to a protein-coding nucleic acid.
- Gene product or expression product As used herein, the term “gene product” or
- expression product generally refers to an R A transcribed from the gene (pre-and/or postprocessing) or a polypeptide (pre- and/or post-modification) encoded by an RNA transcribed from the gene.
- High-throughput refers broadly to investigations with a large number of assays such that formatting of each individual sample, minimizing preparation steps and complications, and measuring of the assay results either in parallel or in rapid succession become important.
- High-throughput tests generally do not include manual, one-at-a-time assays, such as assays by a single individual in which the preparation, execution, measurement, and data collection for one assay are all completed before the assay on the next agent is done.
- High-throughput typically includes, for example, any assays in which a batch of samples (e.g., 24, 96, 384 or more test samples) are prepared and measured. Formatting the tests in such test samples is meant to accelerate the assay process by enabling measurement in parallel or in rapid succession, such as with the assistance of automation.
- Immunogenic means capable of producing an immune response in a host animal against a non-host entity (e.g., an HCMV antigen). In certain embodiments, this immune response forms the basis of the protective immunity elicited by a vaccine against a specific infectious organism (e.g., an HCMV).
- a non-host entity e.g., an HCMV antigen
- Immune response 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 part of the immune system.
- an immunogenic composition may induce an increased IFNy response.
- an immunogenic composition may induce a mucosal IgA response (e.g., as measured in nasal and/or rectal washes).
- an immunogenic composition may induce a systemic IgG response (e.g., as measured in serum).
- an immunogenic composition may induce virus- neutralizing antibodies or a neutralizing antibody response.
- “reduce,” or grammatical equivalents indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of the treatment described herein.
- patient refer to a human or a non-human mammalian subject.
- the individual (also referred to as “patient” or “subject") being treated is an individual (fetus, infant, child, adolescent, or adult) suffering from a disease, for example, HCMV infection.
- the subject is at risk for HCMV infection.
- the subject is an immunosuppressed subject.
- the immunosuppressed subject is selected from the group consisting of an HIV-infected subject, an AIDS patient, a transplant recipient, a pediatric subject, and a pregnant subject.
- the subject has been exposed to HCMV infection.
- the subject is a human.
- Isolated refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from 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%o, about 97%, about 98%>, about 99%, or more than about 99% of the other components with which they were initially associated.
- isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%o, about 98%>, about 99%, or more than about 99% pure.
- a substance is "pure” if it is substantially free of other components.
- calculation of percent purity of isolated substances and/or entities should not include excipients (e.g., buffer, solvent, water, etc.).
- Linker refers to, e.g., in a fusion protein, an amino acid sequence of an appropriate length other than that appearing at a particular position in the natural protein and is generally designed to be fiexible and/or to interpose a structure, such as an a-helix, between two protein moieties.
- a linker allows two or more domains of a fusion protein to retain 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the biological activity of each of the domains.
- a linker may also referred to as a spacer.
- nucleic acid refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
- a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage.
- nucleic acid refers to individual nucleic acid residues ⁇ e.g., nucleotides and/or nucleosides).
- nucleic acid refers to an oligonucleotide chain comprising individual nucleic acid residues.
- nucleic acid encompasses RNA as well as single and/or double-stranded DNA and/or cDNA.
- nucleic acid encompasses RNA as well as single and/or double-stranded DNA and/or cDNA.
- nucleic acid “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone.
- peptide nucleic acids which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present disclosure.
- nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and/or encode the same amino acid sequence. Nucleotide sequences that encode 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. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated.
- nucleic acid segment is used herein to refer to a nucleic acid sequence that is a portion of a longer nucleic acid sequence. In many embodiments, a nucleic acid segment comprises at least 3, 4, 5, 6, 7, 8, 9, 10, or more residues.
- a nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxy cytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 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-deaza
- nucleic acids e.g., polynucleotides and residues, including nucleotides and/or nucleosides
- nucleic acids e.g., polynucleotides and residues, including nucleotides and/or nucleosides
- compositions that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
- Polypeptide As used herein, a "polypeptide”, generally speaking, is a string of at least two amino acids attached to one another by a peptide bond. In some embodiments, a polypeptide may include at least 3-5 amino acids, each of which is attached to others by way of at least one peptide bond. Those of ordinary skill in the art will appreciate that polypeptides sometimes include "non-natural" amino acids or other entities that nonetheless are capable of integrating into a polypeptide chain, optionally.
- Substantial homology is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered to be “substantially homologous” if they contain homologous residues in corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues will appropriately similar structural and/or functional characteristics.
- amino acids are typically classified as “hydrophobic” or “hydrophilic” amino acids., and/or as having "polar” or “non-polar” side chains Substitution of one amino acid for another of the same type may often be considered a “homologous" substitution.
- amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial 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,
- 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 relevant stretch of residues.
- the relevant stretch is a complete sequence.
- the relevant 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.
- Substantial identity is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered to be “substantially identical” if they contain identical residues in corresponding positions. As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial 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.
- 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%o, 97%), 98%), 99%> or more of their corresponding residues are identical over a relevant stretch of residues.
- the relevant stretch is a complete sequence.
- the relevant 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.
- an individual who is "susceptible to" a disease, disorder, or condition is at risk for developing the disease, disorder, or condition.
- an individual who is susceptible to a disease, disorder, or condition does not display any symptoms of the disease, disorder, or condition.
- an individual who is susceptible to a disease, disorder, or condition has not been diagnosed with the disease, disorder, and/or condition.
- an individual who is susceptible to a disease, disorder, or condition is an individual who has been exposed to conditions associated with development of the disease, disorder, or condition (e.g., the individual has been exposed to HCMV).
- Symptoms are reduced: According to the present disclosure, "symptoms are reduced” when one or more symptoms of a particular disease, disorder or condition is reduced in magnitude (e.g., intensity, severity, etc.) or frequency. For purposes of clarity, a delay in the onset of a particular symptom is considered one form of reducing the frequency of that symptom. It is not intended that the present disclosure be limited only to cases where the symptoms are eliminated. The present disclosure specifically contemplates treatment such that one or more symptoms is/are reduced (and the condition of the subject is thereby "improved"), albeit not completely eliminated.
- therapeutically effective amount refers to an amount sufficient to confer a therapeutic effect on the treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment.
- the therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect).
- therapeutically effective amount refers to an amount of a therapeutic protein or composition effective to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity or frequency of symptoms of the disease.
- a therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses.
- a therapeutically effective amount (and/or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration, on combination with other pharmaceutical agents.
- the specific therapeutically effective amount (and/or unit dose) for any particular patient may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; 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 like factors as is well known in the medical arts.
- treatment refers to any administration of an immunogenic composition that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of a particular disease, disorder, and/or condition (e.g., HCMV infection) or the predisposition toward the disease.
- a particular disease, disorder, and/or condition e.g., HCMV infection
- Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition.
- such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.
- the term “treating” refers to the vaccination of a patient.
- Tropism As used herein, the terms “tropism” or “host tropism” or “cell tropism” in the context of viruses and other pathogens generally refer to the ability of the virus or pathogen to infect a particular cell type. Tropism may refer to a way in which the virus or pathogen has evolved to preferentially target specific host species or specific cell types within those species. For example, HCMV can typically infect a remarkably broad cell range within its host, including parenchymal cells, connective tissue cells of virtually any organ and various hematopoietic cell types. Epithelial cells, endothelial cells, fibroblasts and smooth muscle cells are predominant targets for virus replication.
- an HCMV strain is able to infect fibroblasts, but not epithelial and/or endothelial cells. In some embodiments, an HCMV strain is able to infect fibroblasts, epithelial cells and endothelial cells.
- Vaccination refers to the administration of a composition intended to generate an immune response, for example to a disease-causing agent (e.g., HCMV).
- a disease-causing agent e.g., HCMV
- vaccination can be administered before, during, and/or after exposure to a disease-causing agent, and in certain embodiments, before, during, and/or shortly after exposure to the agent.
- vaccination includes multiple administrations, appropriately spaced in time, of a vaccinating composition.
- Vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is associated.
- vectors are capable of extra-chromosomal replication and/or expression of nucleic acids to which they are linked in a host cell such as a eukaryotic and/or prokaryotic cell.
- vectors capable of directing the expression of operatively linked genes are referred to herein as "expression vectors.”
- the present disclosure provides methods useful for determining levels of HCMV infection in host cells and, by extension, determining levels of neutralizing antibodies present in a sample.
- the present disclosure encompasses the recognition that HCMV viruses that have a fluorescent moiety permit detection of viral infection (e.g., by assessing fluorescence in cells after contacting the host cell with the virus).
- levels of HCMV infection are determined by fluorescence detection where the virus has been preincubated with a test sample (e.g., a serum sample) from a subject.
- a test sample e.g., a serum sample
- the subject has been administered a candidate HCMV vaccine.
- HCMV Human cytomegalovirus
- HCMV Human cytomegalovirus
- a ⁇ -herpesvirus is a ubiquitously occurring pathogen.
- entry of herpesviruses into cells is a complex process initiated by adsorption and receptor binding and followed by fusion of the virus envelope with a cell membrane. Fusion generally occurs at either the plasma membrane or an endosomal membrane.
- HCMV infects multiple cell types in vivo, including epithelial cells, endothelial cells and fibroblasts (Plachter B et al., 1996 Adv Virus Res 46: 195-261).
- HCMV encodes two gH/gL complexes: gH/gL/gO and gH/gL/UL128/UL130/UL131.
- the gO-containing complex is sufficient for fibroblast infection, whereas the pUL128/UL130/UL131-containing complex is important for HCMV infection of endothelial and epithelial cells.
- the terms "tropism” or "host tropism” or “cell tropism” in the context of viruses and other pathogens generally refer to the ability of the virus or pathogen to infect a particular cell type.
- Tropism may refer to a way in which the virus or pathogen has evolved to preferentially target specific host species or specific cell types within those species.
- an HCMV strain is able to infect fibroblasts, but not epithelial and/or endothelial cells. In some embodiments, an HCMV strain is able to infect fibroblasts, epithelial cells and endothelial cells.
- HCMV infects 50-85% of adults by 40 years of age (Gershon AA et al, 1997 in
- HCMV disease is the cause of significant morbidity and mortality in immunocompromised individuals, such as recipients of hematopoietic cell transplants (HCT) and solid-organ transplants (SOT) (Pass RF 2001 Cytomegalovirus. In Fields Virology. 4 th edition, Philadelphia; Lippincott Williams & Wilkens:2675-2705).
- HCT hematopoietic cell transplants
- SOT solid-organ transplants
- HCMV disease can occur either from new infection transmitted from the donor organ or HCT, or can recur as a result of reactivation of latent virus in the recipient.
- HCMV infection accelerates progression to AIDS and death, despite availability of antiretroviral therapy (Deayton JR et al, 2004 Lancet 363:2116-2121).
- HCMV is the most common intrauterine infection and causes congenital abnormalities resulting in 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).
- Control of HCMV in immunocompromised persons is primarily associated with cellular immune responses; both CD8 + and CD4 + 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 + helper T-lymphocyte and CD8 + Cytotoxic T-lymphocyte responses to a number of antigens, found in the viral tegument, the region of the viral particle between the envelope and capsid.
- the maternal humoral immune response against the virus seems to be important in preventing HCMV disease in the newborn.
- protection from re -infection is correlated with neutralizing antibodies (Adler SP et al., 1995 J Infectious Diseases 171 :26-32).
- the humoral immune response to HCMV is dominated by responses to viral envelope glycoproteins present in the outer envelope of the virus particle (e.g., gB and gH).
- a CMV vaccine that induces both protective T cell and neutralizing antibody responses has the potential to prevent infection or ameliorate CMV disease due to congenital infection or transplantation.
- the first live, attenuated HCMV vaccine candidate tested in humans was based on the laboratory-adapted AD 169 strain. Subsequent trials with another laboratory-adapted clinical isolate, the Towne strain, confirmed that live attenuated vaccines could elicit neutralizing antibodies, as well as CD4+ and CD8+ T lymphocyte responses. The efficacy of the Towne vaccine was assessed in a series of studies in renal transplant recipients. Although the Towne vaccine did provide a protective impact on HCMV disease it failed to prevent HCMV infection after transplantation (Plotkin SA et al., 1984 Lancet 1 :528-530).
- Towne vaccine was also evaluated in a placebo-controlled study of seronegative mothers who had children attending group daycare where it failed to prevent these women from acquiring infection from their HCMV-infected children (Adler SP et al, 1995 J Infectious Diseases 171 :26-32). An interpretation of these studies was that the Towne vaccine was overattenuated. To explore this possibility a series of genetic recombinants in which regions of the unattenuated "Toledo" strain of CMV were substituted for the corresponding regions of the Towne genome, resulting in the construction of Towne/Toledo "chimeras" that contain some, but not all, of the mutations that contribute to the Towne vaccine attenuation (Heineman TC et al. 2006 J Infect Disease
- the leading subunit CMV vaccine candidate is based on the envelope
- glycoprotein, gB (purified recombinant gB vaccine is manufactured by Sanofi-Pasteur Vaccines) due to this protein's ability to elicit high-titer, virus-neutralizing antibody responses during natural infection.
- the recombinant gB vaccine elicits neutralizing antibody responses and has an excellent safety profile, however, it excludes other glycoprotein targets of neutralizing antibody response and more importantly T-lymphocyte targets.
- the vaccine requires MF59 adjuvant to optimize immunogenicity. In the most recent trial, this vaccine provided an overall 50% efficacy for prevention of CMV infection in a Phase 2 clinical trial in young women (Pass RF et al, 2009 N Engl J Med 360: 1191-1199).
- Other viral proteins being evaluated as subunit vaccine candidates include pp65 and IE1, both of which elicit T-cell responses.
- DNA vaccines elicit robust cellular and humoral immune responses in animals and are well suited to specificity and precision in vaccine design.
- DNA vaccines have been developed for CMV and have focused on gB, IE1 and pp65 proteins as the candidate target immunogens.
- a bivalent CMV DNA vaccine candidate (Wloch MK, 2008 J Infectious Diseases 297: 1634-1642), using plasmid DNA encoding pp65 and gB and a trivalent vaccine candidate (Jacobson MA, 2009 Vaccine 27: 1540-1548) that also includes a third plasmid encoding the IE1 gene product have been developed by Vical Vaccines (US Patent No. 7,410,795).
- the trivalent DNA vaccine alone had minimal immunogenicity irrespective of route of administration.
- the CMV DNA vaccine did appear to safely prime for a memory response to CMV antigens observed after administration of a live, attenuated CMV (Towne).
- the gene product of interest is expressed in the context of a non-replicating (usually viral) carrier.
- a non-replicating usually viral
- ALVAC canarypox vector
- Virogenetics and Sanofi-Pasteur Vaccines which is an attenuated poxvirus that replicates abortively in mammalian cells.
- ALVAC expressing CMV gB and ALVAC expressing pp65 US Patent No. 6,267,965 have been tested in clinical trials.
- ALVAC- CMV(gB) did not induce neutralizing antibodies but did prime for higher neutralizing antibody titers after subsequent infection with the Towne strain CMV (Adler SP et al., 1999 J Infectious Diseases 180:843-846), although it did not appear to boost neutralizing antibody titers after subsequent immunization with gB subunit/MF59 vaccine (Bernstein DI et al., 2002 J Infectious Diseases 185:686-690).
- Another approach used to express gB as a vectored vaccine is the use of an alphavirus replicon system by AlphaVax Inc (US Patent No. 7,419,674).
- This approach involves a propagation-defective single-cycle RNA replicon vector system derived from an attenuated strain of an alphavirus, Venezuelan Equine Encephalitis (VEE) virus, to produce virus-like replicon particles (VRPs) expressing pp65, IE1 or gB protein (Berstein et al, 2010 Vaccine 28:484-493).
- VEE Venezuelan Equine Encephalitis
- a two component alphavirus replicon vaccine was used to express the three CMV proteins as a soluble form of CMV gB (Towne strain) and a pp65/IEl fusion protein (Reap EA et al., 2007 Vaccine 25:7441-7449) was found to be safe and induced high levels of neutralizing antibody and polyfunctional CD4+ and CD8+ antigen-specific T cell responses.
- the Geometric Mean Titre (GMT) for the high dose group was about half the GMT in 12 naturally infected, CMV seropositive individuals tested in the assay.
- DBs Dense bodies
- DBs are non-infectious and immunogenic but incapable of establishing latent HCMV infection in the vaccine recipient. DBs have been shown to be capable of inducing virus neutralizing antibodies and T-cell responses in mice in the absence of viral gene expression (Pepperl S et al, 2000 J Virol 74:6132-6146, PCT Publication No. WO 00/53729 and US Patent No. 6,713,070).
- Additional candidates contemplated for vaccination against HCMV are virus like particles (VLPs).
- Retroviruses are enveloped RNA viruses that belong to the family
- Retroviridae After infection of a host cell by a retrovirus, RNA is transcribed into DNA via the enzyme reverse transcriptase. DNA is then incorporated into the host cell's genome by an integrase enzyme and thereafter replicates as part of the host cell's DNA.
- the Retroviridae family includes the following genus Alpharetrovirus, Betaretrovirus, Gammearetrovirus, Deltaretrovirus, Epsilonretrovirus, Lentivirus and Spumavirus.
- the hosts for this family of retroviruses generally are vertebrates. Retroviruses produce an infectious virion containing a spherical nucleocapsid (the viral genome in complex with viral structural proteins) surrounded by a lipid bilayer derived from the host cell membrane.
- Retroviral vectors can be used to generate enveloped virions that are infectious and either replication-competent or replication-defective.
- Replication-competent infectious retroviral vectors contain all of 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 accomplished by replacement of most of the coding regions of the retrovirus with genes or nucleotide sequences to be transferred; so that the vector is incapable of making proteins required for additional rounds of replication.
- retroviral vectors can be used to generate virus-like particles (VLPs) that lack a retrovirus-derived genome and are both non-infectious and non- replicating. Because of VLPs advantageous properties, VLPs may be utilized as an antigen delivery system. Furthermore, because VLPs are non-infectious, they can be administered safely as an immunogenic composition (e.g., a vaccine). VLPs are generally structurally similar to enveloped virions described above, but lack a retrovirus-derived genome, making it unlikely that viral replication will occur.
- VLPs include single or multiple capsid proteins either with or without envelope proteins and/or surface glycoproteins.
- VLPs are non-enveloped and assemble by expression of just one major capsid protein, as shown for VLPs prepared from hepadnaviruses (e.g., EngerixTM, GSK and Recombivax HBTM, Merck), papillomaviruses (e.g., CervarixTM , GSK and GardasilTM, Merck), paroviruses, or polyomaviruses.
- VLPs are enveloped and can comprise multiple antigenic proteins found in the corresponding native virus. VLPs typically resemble their corresponding native virus and can be multivalent particulate structures.
- antigenic proteins may be presented internally within the VLP, as a component of the VLP structure, and/or on the surface of the VLP.
- presentation of an antigen in the context of a VLP is advantageous for induction of neutralizing antibodies against the antigen as compared to other forms of antigen presentation, e.g., soluble antigens not associated with a VLP.
- Neutralizing antibodies most often recognize tertiary or quaternary structures; this often requires presenting antigenic proteins, like envelope glycoproteins, in their native viral conformation.
- VLPs may be useful for presenting antigens in a context which induces cellular immunity (e.g., T cell response).
- use of antigen combinations in VLP systems can generate improved immune response.
- the present disclosure provides methods for determining levels of HCMV infection in host cells and, by extension, determining levels of neutralizing antibodies present in a sample.
- the present disclosure encompasses the recognition that HCMV viruses that have a fluorescent moiety permit detection of viral infection (e.g., by assessing fluorescence in cells after contacting the host cell with the virus).
- levels of HCMV infection are determined by fluorescence detection where the virus has been preincubated with a test sample (e.g., a serum sample) from a subject.
- a test sample e.g., a serum sample
- the subject has been administered a candidate HCMV vaccine.
- Provided methods utilize an HCMV virus that includes a fluorescent moiety. Any
- HCMV virus capable of infecting a host cell described herein can be engineered to include a fluorescent moiety.
- an HCMV virus that includes all or a portion of a gH/gL/gO complex can be engineered to include a fluorescent moiety.
- an HCMV virus that includes all or a portion of a gH/gL/UL128/UL130/UL131 complex can be engineered to include a fluorescent moiety.
- Modified HCMV strains that are amenable to fluorescent detection are known in the art and may be used in accordance with the present disclosure.
- UL32-EGFP-HCMV-TB40 is an in vitro recombination of HCMV strain TB40 with a plasmid carrying the TB40 UL32 gene fused to GFP (ATCC; VR-1578).
- the UL32-EGFP-HCMV- TB40 recombinant strain gives rise to a recombinant HCMV virus with GFP fused to the C terminus of the tegument phosphoprotein ppl50, the product of the UL32 gene (Sampaio et al, 2005 Journal of Virology 79(5):2754). Because GFP is associated with a viral structural protein, virus particles fluoresce green under appropriate illumination.
- the UL32-EGFP-HCMV-TB40 strain has been demonstrated to have tropism for fibroblast cells (Sampaio et al., 2005 Journal of Virology 79(5):2754).
- An additional HCMV strain that is amenable to fluorescent detection is HB15-tl78b, which contains the CMV strain AD 169 genome and a GFP reporter cassette (Saccoccio et al, 2011 Vaccine 29(15):2705).
- fluorescence refers to a moiety that luminesces. Typically fluorescent moieties contain electrons which can absorb
- Some proteins or small molecules in cells are naturally fluorescent (e.g., NADH, tryptophan, endogenous chlorophyll, phycoerythrin, or green fluorescent protein (GFP)).
- GFP green fluorescent protein
- mutants of fluorescent proteins have been engineered and may be used in accordance with 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, among others.
- GFP and other fluorescent proteins can be expressed exogenously in cells alone or as a fusion protein. This approach permits fluorescent proteins to be used as reporters for any number of biological events, such as subcellular localization and expression patterns.
- proteins, nucleic acids, lipids or small molecules can be labeled with an extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot.
- extrinsic fluorophore a fluorescent dye which can be a small molecule, protein or quantum dot.
- exemplary f uorophores include, but are not limited to, 1,5 IAEDANS; 1,8-ANS; 4- Methylumbelliferone; 5-carboxy-2,7-dichlorofiuorescein; 5- Carboxyfiuorescein (5-FAM); 5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5- TAMRA); 5-Hydroxy Tryptamine (5 -HAT); 5-ROX (carboxy-X-rhodamine); 6- Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7-Amin
- Biotechnologies see sgGFP, sgBFP; Alexa Fluor® 350; Alexa Fluor® 430; Alexa Fluor® 488; Alexa Fluor® 532; Alexa Fluor® 546; Alexa Fluor® 568; Alexa Fluor® 594; Alexa Fluor® 633; Alexa Fluor® 647; Alexa Fluor® 660; Alexa Fluor® 680; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X;
- Chromomycin A Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPM I
- Dihydorhodamine 123 Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di 16-ASP);
- DIDS Dihydorhodamine 123 (DHR); Dil (DilC18(3)); I Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY- 635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro- Gold (Hy
- Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO- 1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein/Ethidium 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 Green; LysoSensor
- Rhodamine 5 GLD Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine: Phalloidine; 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 Orange; Sevron Yellow L;
- sgBFPTM (super glow BFP); sgGFPTM (super glow GFP); SITS (Primuline; Stilbene
- SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6- methoxy-N-(3 sulfopropyl)quinolinium); Stilbene; Sulphorhodamine B and C; Sulphorhodamine Extra; 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; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82
- TIER (Calcofiuor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; Tricolor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H;
- Y66W Yellow GFP; YFP; YO-PRO-1; YO- PRO 3; YOYO-l;YOYO-3; Sybr Green; Thiazole orange (interchelating dyes); semiconductor nanoparticles such as quantum dots; or caged fluorophore (which can be activated with light or other electromagnetic energy source), or a combination thereof.
- Determination of infectious titer of HCMV typically includes contacting a host cell that is susceptible to infection by HCMV with serial dilutions of the virus (e.g., HCMV that includes a fluorescent moiety), under conditions that allow cell infection in the absence of any test substance.
- the number of target cells expressing a reporter gene construct e.g., a fluorescent moiety, e.g., GFP
- GFP fluorescent moiety
- the serum may be pre -incubated with HCMV (e.g., HCMV that includes a fluorescent moiety, e.g., GFP) for a period of time sufficient for neutralizing antibodies to reduce infectivity of the HCMV (e.g., at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, or more).
- HCMV e.g., HCMV that includes a fluorescent moiety
- Serial dilutions of the pre- incubated mixture of serum and HCMV may then be used to contact a host cell that is susceptible to infection by HCMV under conditions that allow infection.
- dilutions tested are 1 :6, 1 : 12, 1 :24, 1 :48, 1 :96, 1 :192, or combinations thereof.
- infection conditions include incubation of the host cell with virus 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 more.
- monitoring for infection of cells may include visual cellular morphology assessment (e.g., for cellular swelling and rounding) and/or visual fluorescence assessment (e.g., detecting fluorescence on a device such as a fluorescence microscope).
- host cells may be collected (e.g., by washing and trypsinizing and/or scraping) and analyzed by fluorescent detection methods available in the art and described herein.
- Any host cell susceptible to infection by HCMV can be used in the methods described herein.
- exemplary host cells include, but are not limited to human fibroblast cells, such as human foreskin fibroblasts (HFF), and human epithelial cells such, as retinal pigmented epithelial cells (ARPE-19).
- HFF infection medium includes MEM + 5% FBS + 1% PenStrep.
- APRE infection medium includes DMEM:F-12 + 1% FBS.
- Fluorescence may be detected using any appropriate method, including, for example, flow cytometry analysis, fluorescent 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 that arises from endogenous biological fluorescent entities in the sample and from non-specific association of the fluorescent entity with the sample. The brightness of the fluorescent entity, in turn, depends on the quantum efficiency of the fluorescent entity that produces the fluorescence signal and the light absorbing capability (quantified by the extinction coefficient) of the fluorescent entity.
- cells can be identified and/or isolated based on levels of surface and/or intracellular fluorescence using reporter molecules such as green fluorescent protein (GFP).
- GFP green fluorescent protein
- a correlation between fluorescence intensity and protein production e.g., viral infection and production
- high levels of fluorescence intensity in a host cell correlates with high level of viral infection of the host cell.
- cells are assessed for overall fluorescence level (e.g., irrespective of subcellular localization of the fluorescence).
- cells are assessed for fluorescence level in a particular subcellular location (e.g., the nucleus) of the cell.
- flow cytometry permits quantitative phenotyping of large numbers of cells, however, many flow cytometry applications do not include an ability to image cells as they are quantitated. In some embodiments, detection and quantitation of fluorescence of cells is sufficient for assessing infection. It will be appreciated that in some cases it is desirable to determine the localization of fluorescence within the cell. In some embodiments, flow cytometry analysis of cells may be combined with visual assessment of fluorescence. Dual analysis of fluorescence level and localization may be performed using multiple devices (e.g., flow cytometry and fluorescence microscopy) or on a single device. Such devices that permit fluorescence analysis and/or quantitation in conjunction with localization analysis are available in the art. For example, ImageStream x (Amnis ® ) quantifies both intensity and localization of fluorescence and permits imaging of more than 50,000 cells per minute.
- ImageStream x Amnis ®
- Cell fluorescence levels may be quantitated by any appropriate method known in the art. Cell fluorescence levels may be compared to a reference level. In some embodiments, the reference level is a predetermined or historical reference level. In some embodiments, the reference level is obtained by side-by-side comparison with a reference sample (e.g., a positive and/or negative control).
- a reference sample e.g., a positive and/or negative control
- sample assessment can be achieved in a high-throughput manner.
- provided methods increase throughput by 10%, 20%, 30%, 40%, 50%, 60%, 70%, or more as compared to known infection and/or neutralizing antibody detection methods (e.g., staining of viral proteins, fluorescence microscopy, ELISPOT, etc.).
- multi-well plates have 96 wells.
- the multi-well plates have another number of wells, which include but is not limited to plates with 6, 12, 24, 384, 864 or 1536 wells.
- the terms "multi-well plate” and "microtiter plate” are used interchangeably.
- live cells are analyzed. In some embodiments, cells are fixed prior to analysis.
- the present disclosure provides methods for measuring anti-HCMV neutralizing antibodies by correlating fluorescence detection and/or viral infection of host cells.
- an HCMV that includes a fluorescent moiety e.g., GFP
- an HCMV candidate vaccine may be pre- incubated with serum from a subject immunized with an HCMV candidate vaccine.
- Neutralizing antibodies present in the serum will reduce HCMV (e.g., HCMV that includes a fluorescent moiety) from infecting a host cell that is normally susceptible to infection by HCMV.
- the pre- incubated mixture including HCMV (e.g., HCMV that includes a fluorescent moiety) and serum can then be used to contact such a host cell and fluorescence levels of the host cell may be assessed.
- a level of infection of the host cell can be determined.
- Level of infection of the host cell generally is inversely related to the presence and amount neutralizing antibody in the serum, which in turn, correlates with efficacy of the candidate vaccine to elicit a therapeutic response (e.g., a protective immune response).
- a therapeutic response e.g., a protective immune response.
- the more efficacious a candidate vaccine at inducing neutralizing antibodies in a subject the more neutralizing antibodies will be present in the serum, in turn corresponding to a decrease in ability of an HCMV that includes a fluorescent moiety to infect a host cell after preincubation with the serum, further corresponding to a decrease in fluorescence detection in the host cell after contacting it with an HCMV that includes a fluorescent moiety.
- methods of the disclosure further include selecting and/or identifying, based on a correlation described herein, a candidate vaccine (e.g., an HCMV candidate vaccine) as a vaccine that induces neutralizing antibodies.
- This Example describes exemplary immunization of rabbits with virus-like particles containing various recombinant HCMV antigens.
- HEK 293T cells (ATCC, CRL-11268) were transiently transfected using calcium phosphate methods with expression plasmids encoding various recombinant HCMV antigens. Expression of various HCMV antigens by the HEK 293 cells was confirmed by flow cytometry. After 48 to 72 hours of transfection, supernatants containing the VLPs were harvested and filtered through 0.45 ⁇ pore size membranes and further concentrated and purified by ultracentrifugation through a 20% sucrose cushion in a SW32 Beckman rotor (25,000 rpm, 2 hours, 4°C).
- Enzyme-linked Immunosorbent Assay was performed to determine rabbit serum HCMV IgG content.
- Figure 1 shows potent and sustained immunity in rabbits immunized with gB/pp65 bivalent VLPs (upper) and gB/pp65 bivalent VLPs + gH-G/pp65 bivalent VLPs (lower).
- Example 2 Flow Cytometry-Based Neutralization Activity
- This Example describes assessment of neutralizing antibody responses to HCMV in serum from immunized animals.
- the objectives of this Example included, but were not limited to: 1) confirming the immunogenicity of CMV clinical candidate components selected from other animal studies; 2) assessing dose and potential synergy and/or antagonism with administration routes (e.g., IM vs. IP administration); and 3) assessing immunological boosting and durability of immunity.
- Serum samples were diluted 1/6 to 1/96.
- TB40-230212 virus is descendant of TB40-010212.
- TB40-010212 was obtained from ATCC (ATCC VR-1578; Human herpesvirus 5; UL32-EGFP-HCMV-TB40).
- Virus infection duration was 15 days in a T150 infection flask (Table 2). Microscopic observations indicated the virus was infective.
- Infection medium was TB40-HFF-1 infection medium (MEM+5%FBS+l%Pen/Strep) or VR1814-APRE- 19 infection medium (DMEM:F-12+1%FBS).
- T150 flask incubated at 37°C, 5%C0 2 for 1 hour. Twelve mL of infection media was added after incubation. Virus was concentrated in 15 mL of supernatant.
- CytogamTM (CMV-IGIV - Cytomegalovirus Immune Globulin Intravenous -
- Incubation was at 37°C/5% C0 2 /4 hrs (rocking was every 60 minutes). After a 4-hour incubation, contents were carefully aspirated from each well with a pipette and 3 mL of fresh infectious media were added. Plates were kept in the incubator at 37°C/5% C0 2 for 10 days. Daily checking of virus infection in cells was performed usually after 5 days post-infection. Swelling and rounding cells were visualized by light microscope or green fluorescence detection with a fluorescent microscope.
- FIG. 1 depicts exemplary FACS analysis of GFP expression in fibroblast cells indicative of neutralizing antibody response induced with gB/pp65 CMV VLPs in rabbits.
- Figure 3 depicts exemplary FACS analysis of GFP expression in fibroblast cells indicative of neutralizing antibody response induced with bivalent gB + gH CMV VLPs in rabbits.
- Sera were pooled and tested at indicated dilutions in comparison to CytogamTM at similar dilutions against GFP-expressing CMV virus (TB40) in HFF fibroblasts. 100,000 cells were collected during flow cytometric analysis of infected (GFP + ) cells.
- Example 3 Exemplary Micro-Neutralization Assay for Detection of Neutralizing Antibodies by Flow Cytometry
- This Example describes detection of functional anti-CMV neutralizing antibodies by flow cytometry in sera samples from vaccinated animals.
- Fibroblasts 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.
- HFF-1 cells (95% confluent monolayer seeded in T 150 flask) are infected with
- TB40 1.5 ml of UL32-EGFP-HCMV-TB40
- ARPE-19 cells are infected with HCMV-GFP- Towne-TS15-rR ("Towne") in the same manner. Since these viruses are light sensitive, cells are infected with the light off. The flasks are incubated in a C02 incubator/37°C for 30 minutes to allow attachment between cells and virus.
- FBS Fetal bovine
- FBS Fetal bovine
- Pre -bleed and Post-immunized serum samples are heat inactivated at 56°C for 30 minutes prior to use.
- Commercial sera, Cytogam that serves as positive control is heat inactivated, as well.
- Serum dilutions are made starting from 1/6 up to desired dilutions in duplicates. Cytogam is tested at comparable dilutions, a prior diluting the stock reagent 1 :20 to adjust to the Ig content of human/rabbit sera (see Table 8).
- Table 8 Sera and virus dilutions (as an example lin2 virus dilution is shown)
- complement is included.
- 10% standard guinea pig complement is added to each particular sera (Cytogam)/virus mixture.
- Virus control is treated the same way.
- 2.5% rabbit complement is added to each mix regardless which species is used (rabbit or sera). The sera and virus are rotated at 37°C for 1 hr.
- CHO cells were transfected at a cell density between 1.5E06 to 2.0E06 cells/mL with plasmids of monovalent gB-G and monovalent gH-G (prepared as described in
- Stuffer DNA was added to make total DNA concentration up to 1 ⁇ g/mL cell culture.
- the plasmids used for transfection were first purified by MaxiPrep or GigaPrep plasmid purification kits (Qiagen).
- the PEIMAX used for transfection to deliver DNA to the cells was provided at a ratio of 6: 1 (PEL DNA wt/wt).
- the cell culture was harvested 72 hours post transfection by centrifuging at 4000 rpm for 20 minutes, using rotor JS-4.2A by Beckman Coulter, in 1 Litre bottles. The supernatant was filtered through 0.8/0.45 ⁇ filter (AcroPak 500 Capsule, Pall).
- TFF Tangential Flow Filtration
- the diafiltered benzonase treated material was then loaded onto an anion exchange chromatography column (AEX for reduction of DNA and host proteins) where the flowthrough was collected.
- AEX anion exchange chromatography column
- the flowthrough was then sterile filtered through 0.45 ⁇ and aliquoted in different volumes.
- Rabbits were treated with 50 ⁇ g of both monovalent gB-G and monovalent gH-G (mixed together at a 1 : 1 ratio) VLP composition.
- blood was collected from all rabbits in the study pre- 1st immunization and then post- 1st immunization days 28, 42 and 55 and post-2nd immunization at day 14.
- microneutralization assay in fibroblast cells based on a GFP-expressing CMV virus (TB40) and flow cytometric analysis of infected (GFP+) HFF-1 cells as previously described.
- Rabbit sera collected pre- and post-immunizations as described were pooled and tested for neutralizing activity in the presence of guinea pig complement against HCMV expressing GFP in HFF fibroblasts relative to a positive control CMV hyperglobulin, CytogamTM and a negative control consisting of empty Gag VLP (lacking antigenic proteins gB-G and/or gH-G).
- Figure 4 shows the percent neutralization in HFF-1 cells incubated with CMV-
- GFP-TB40-010512 virus in presence of 10% Guinea Pig complement and rabbit serum group 7 pooled sera from 15RA09 was used as a positive data representative where animals were treated with monovalent gB-G and monovalent gH-G VLPs three times and also group 8 from 15RA05 study (empty Gag) was used as a negative control representative).
- group 7 pooled sera from 15RA09 was used as a positive data representative where animals were treated with monovalent gB-G and monovalent gH-G VLPs three times and also group 8 from 15RA05 study (empty Gag) was used as a negative control representative).
- the combination of monovalent gB-G and monovalent gH-G VLP composition elicited a rapid, synergistic sustained neutralizing antibody response in rabbits against fibroblast cell infection, while the empty Gag VLP showed no neutralizing antibody response.
- GFP-TB40-010512 denotes Human herpesvirus 5 HCMV (UL32-EGFP-HCMV-TB40
- Figure 5 shows the percent neutralization in ARPE-19 cells incubated with CMV-
- GFP-Towne-150612 virus in presence of 2.5% rabbit complement and rabbit serum group 7 pooled sera from 15RA09 was used as a positive data representative where animals were treated with monovalent gB-G and monovalent gH-G VLP compositions three times and also group 8 from 15RA05 study (empty Gag) was used as negative control representative).
- group 7 pooled sera from 15RA09 was used as a positive data representative where animals were treated with monovalent gB-G and monovalent gH-G VLP compositions three times and also group 8 from 15RA05 study (empty Gag) was used as negative control representative).
- group 7 pooled sera from 15RA09 was used as a positive data representative where animals were treated with monovalent gB-G and monovalent gH-G VLP compositions three times and also group 8 from 15RA05 study (empty Gag) was used as negative control representative).
- the combination of monovalent gB-G and monovalent gH-G VLP composition elicited a rapid,
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CA2867789A CA2867789C (en) | 2012-03-27 | 2013-03-27 | Methods for detection of anti-cytomegalovirus neutralizing antibodies |
AU2013239383A AU2013239383B2 (en) | 2012-03-27 | 2013-03-27 | Methods for detection of anti-cytomegalovirus neutralizing antibodies |
JP2015502478A JP6265344B2 (en) | 2012-03-27 | 2013-03-27 | Method for detecting anti-cytomegalovirus neutralizing antibody |
BR112014023913-4A BR112014023913B1 (en) | 2012-03-27 | 2013-03-27 | METHODS FOR DETECTION OF ANTICYTOMEGALOVIRUS NEUTRALIZING ANTIBODIES |
CN201380026015.9A CN104321444A (en) | 2012-03-27 | 2013-03-27 | Methods for detection of anti-cytomegalovirus neutralizing antibodies |
KR1020147030138A KR102146340B1 (en) | 2012-03-27 | 2013-03-27 | Methods for detection of anti-cytomegalovirus neutralizing antibodies |
KR1020207021568A KR102314245B1 (en) | 2012-03-27 | 2013-03-27 | Methods for detection of anti-cytomegalovirus neutralizing antibodies |
MX2014011504A MX354822B (en) | 2012-03-27 | 2013-03-27 | Methods for detection of anti-cytomegalovirus neutralizing antibodies. |
EP19199107.4A EP3613431B1 (en) | 2012-03-27 | 2013-03-27 | Methods for detection of anti-cytomegalovirus neutralizing antibodies |
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US14/387,870 US10006096B2 (en) | 2012-03-27 | 2013-03-27 | Methods for detection of anti-cytomegalovirus neutralizing antibodies |
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US15/991,044 US10161010B2 (en) | 2012-03-27 | 2018-05-29 | Methods for detection of anti-cytomegalovirus neutralizing antibodies |
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US10611800B2 (en) | 2016-03-11 | 2020-04-07 | Pfizer Inc. | Human cytomegalovirus gB polypeptide |
EP3836962A4 (en) * | 2018-08-14 | 2022-04-27 | Merck Sharp & Dohme Corp. | A virus neutralization assay utilizing imaging cytometry |
US11629172B2 (en) | 2018-12-21 | 2023-04-18 | Pfizer Inc. | Human cytomegalovirus gB polypeptide |
US11857622B2 (en) | 2020-06-21 | 2024-01-02 | Pfizer Inc. | Human cytomegalovirus GB polypeptide |
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US10006096B2 (en) * | 2012-03-27 | 2018-06-26 | Variation Biotechnologies Inc. | Methods for detection of anti-cytomegalovirus neutralizing antibodies |
JP6850483B2 (en) * | 2018-03-06 | 2021-03-31 | 株式会社CeSPIA | High-throughput antibody detection method based on the Cell-based assay method |
WO2019232453A1 (en) * | 2018-06-01 | 2019-12-05 | Gravid Therapies, Inc. | Methods, kits, and products for detection of cytomegalovirus infection |
CN111474339A (en) * | 2020-04-17 | 2020-07-31 | 浙江康佰裕生物科技有限公司 | Method for labeling poxvirus particles by fluorescence and application thereof |
CN112362649B (en) * | 2020-11-09 | 2024-07-12 | 长春金赛药业有限责任公司 | Detection method of rhGH neutralizing antibody |
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WO2023141485A2 (en) * | 2022-01-19 | 2023-07-27 | Trustees Of Dartmouth College | Systems and methods for deterring viral transmission using neural networks |
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US10611800B2 (en) | 2016-03-11 | 2020-04-07 | Pfizer Inc. | Human cytomegalovirus gB polypeptide |
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RU2660712C2 (en) | 2018-07-09 |
RU2014142815A (en) | 2016-05-20 |
CN104321444A (en) | 2015-01-28 |
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BR112014023913A2 (en) | 2017-06-20 |
JP6265344B2 (en) | 2018-01-24 |
BR112014023913B1 (en) | 2022-08-09 |
AU2018271276B2 (en) | 2021-06-03 |
AU2018271276A1 (en) | 2018-12-20 |
WO2013144722A3 (en) | 2013-11-28 |
US10161010B2 (en) | 2018-12-25 |
AU2013239383A1 (en) | 2014-10-16 |
CA2867789A1 (en) | 2013-10-03 |
CA2867789C (en) | 2022-06-14 |
KR20200095575A (en) | 2020-08-10 |
EP2831285B1 (en) | 2019-10-30 |
KR102146340B1 (en) | 2020-08-24 |
EP3613431A1 (en) | 2020-02-26 |
EP2831285A2 (en) | 2015-02-04 |
MX2014011504A (en) | 2015-03-13 |
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