Patent Application
20160313331
Fifty million Americans are infected with herpes simplex virus type 2 (HSV-2), but 80-90% of those infected are unaware that they carry HSV-2 [CDC, 2010; Paz-Bailey et al., 2007; Xu et al., 2006]. Regardless of whether patients have visible symptoms or not, they may shed infectious virus and transmit HSV-2 to sexual partners [Rattray et al., 1978; Tronstein et al:, 2011; Wald et al., 2000]. Antiviral drugs reduce, but do not eliminate, the risk of HSV-2 transmission [Sperling et al., 2008; Handsfield et al., 2007; Corey et al., 2004; DeJesus et al., 2003]. Patients who know they carry HSV-2 may take proactive steps to reduce the risk of transmission including antiviral drugs, condoms, disclosure to partners and awareness of subtle symptoms, all of which are effective tools in transmission reduction [Gupta et al., 2007; Rana et al., 2006; Warren, 2002; Wald et al., 2001].
The serological tests used to confirm a diagnosis of HSV-2 infection are imperfect. The most significant problems include (1) the HerpeSelect® HSV type-specific serological ELISA assay (Focus Diagnostics, a wholly-owned subsidiary of Quest Diagnostics, Inc.) may return false-positive results and (2) the confirmatory HSV Western blot test (i.e., the gold standard of HSV serology tests [Warren et al., 2011]) may return “indeterminate” results.
Patients with the potential for false-positives on the HSV-2 ELISA often score as “low-positives” with an index value of 1.1 to 3.5; 50% of these patients prove to be false-positive on the confirmatory HSV Western blot. However, confirmatory Western blot testing fails to resolve the serological status of about 50% of patients who obtained HSV-2 “low-positive” results from the HerpeSelect® ELISA test. Rather, Western blot testing typically returns indeterminate results for these patients. Thus, current HSV-2 serological testing leaves 2-4% of patients with ambiguous results [Ng'ayo et al., 2011; Golden et al., 2005], which for the purposes of this document are referred to as a “HSV-2 indeterminate” diagnosis.
Having an indeterminate diagnosis leaves patients wondering if they are infected with HSV-2, and causes needless anguish in patients who are not infected [Warren, 2002; Warren and Ebel, 2005]. Patients with an indeterminate diagnosis are forced to deal with the ramifications of a bona fide HSV-2 infection; specifically, they feel compelled to disclose their “HSV-2 status” to potential sex partners, risking possible rejection; they may take daily antiviral therapy to reduce the risk of infecting others; and they believe themselves to be 3 times more likely to acquire HIV infection than someone who does not have HSV-2 [Vergidis et al., 2009; Lingappa and Celum, 2007]. Repeat testing often fails to resolve their diagnosis, and thus patients may not know their HSV-2 infection status for months or years; this can have a profoundly negative impact on patients' self-perception and their quality of life.
There is thus an unmet need for an improved serological assay for diagnosis of HSV-2 infection that minimizes, or eliminates, HSV-2 indeterminate diagnoses. The present invention is a novel, flow cytometry-based serological assay that measures the affinity of serum antibody-binding to virus-infected cells (ABVIC) and is believed to be a more definitive HSV-2 serological test.
The present invention provides a serological assay for determining whether a subject is infected with one or the other or neither of herpes simplex -1 or -2 viruses. Broadly, the assay comprises the steps of providing an antibody-containing serum or plasma (collectively, “serum”) sample from the subject to be assayed. The serum sample contains antibodies that immunoreact with cell antigens present on HSV-1-infected (HSV-1+) cells, or HSV-2-infected (HSV-2+) cells, or cells infected with both HSV-1 and HSV-2, or cells infected with neither HSV-1 nor HSV-2.
The serum sample is divided into at least three subsample portions. A separate serum subsample portion is contacted and contact is maintained (incubated) of with each of: (a) antigens of cells uninfected with either HSV-1 or HSV-2 (HSV-1− or HSV-2−), (b) antigens of cells infected with HSV-1 and (c) antigens of cells infected with HSV-2, thereby binding antibodies present in each subsample to one or more of the recited cell antigens. Each such incubated subsample portion is separated from the antibody-bound antigens, to form at least three preadsorbed serum samples, wherein the preadsorbed serum subsample incubated with uninfected cell antigens (a) contains a reduced amount of antibodies that immunoreact with uninfected cells, the preadsorbed serum subsample incubated with antigens of cells infected with HSV-1 (b) contains a reduced amount of antibodies that immunoreact with HSV-1-infected cells when those antibodies were present in the provided serum sample, and the preadsorbed serum subsample incubated with antigens of cells infected with HSV-2 (c) contains a reduced amount of antibodies that immunoreact with HSV-2-infected cells when those antibodies were present in the provided serum sample. Each of the preadsorbed subsample portions is admixed and incubated with a mixture of antigens from cells uninfected by either HSV-1 or HSV-2, cells infected by HSV-1 and cells infected by HSV-2, and determining to which one or more antigens the antibodies present in each subsample portions bound, and thereby whether the subject was infected with HSV-1, HSV-2, both or neither.
The assay in one embodiment comprises the steps of providing a serum or plasma (collectively, “serum”) sample from the subject to be assayed, dividing the serum sample into at least three serum subsamples, preadsorbing the serum subsamples to at least three populations of antigens, preferably in the form of fixed cells, incubating the serum subsamples with at least three populations of free cells, incubating the serum subsamples with a detection antibody, and analyzing the serum subsamples with a cell sorting device or a flow cytometer.
A serological assay kit for determining whether a subject is infected with one, both or neither of herpes simplex -1 and -2 viruses is also contemplated. The kit comprises a) three separate vessels for serum preadsorption that separately contain i) antigens from uninfected cells in a physical matrix, (ii) antigens from HSV-1-infected cells in a same or different physical matrix, or (iii) antigens from HSV-2-infected cells in a same or different physical matrix from that of (i) or (ii). A fourth component of the kit are the test antigens, which may be provided in a variety of forms. In one embodiment, a fourth vessel is included in the kit that contains a mixture of three populations of uninfected cells, HSV-1-infected cells, and HSV-2 infected cells that have been (1) fixed and permeabilized and (2) differentially labeled with a fluorophore such that a cell sorting device or flow cytometer can differentiate each of the three populations. In this embodiment, preadsorbed serum separated from the antigen-containing matrices provided in kit vessels 1, 2, and 3, are separated from each matrix and combined with the test cells provided in kit vessel 4 to determine the relative abundance of HSV-1- and/or HSV-2-specific antibody in a cell sorting device or flow cytometer. Each of those four vessels contains a sufficient amount of the recited ingredient to carry out at least one assay. Instructions for carrying out an assay are preferably also be present in the kit.
The above-described serological assay kit further preferably includes a fifth vessel that contains labeled anti-human antibodies in an amount sufficient to carry out at least one assay. The label of the anti-human antibodies is preferably a fluorescent material whose fluorescence is distinguishable from the fluorescence of any other material present. It is also preferred that the mixture of fixed test cells of the fourth vessel-further include an exogenously-introduced fluorescent colorant by which cells containing uninfected, HSV-1, or HSV-2 antigens are distinguishable from each other by fluorescence, and are also distinguishable from any other fluorescent species utilized in the assay.
The present invention has several benefits and advantages.
One benefit is that many antibody assays are sufficient to distinguish HSV-seronegative from HSV-seropositive samples, but do not differentiate whether a person is infected with HSV-1, HSV-2, or both.
An advantage of the invention is that the HSV-1-specific antibody assay portion of the invention differentiates whether or not a person is infected with HSV-1, and corroborates the results of a Herpes Western Blot.
Another benefit of the invention is that the HSV-2-specific antibody assay portion differentiates whether or not a person is infected with HSV-2, and corroborates the results of a Herpes Western Blot.
Another advantage of the invention is that the preferred Type-Specific ABVIC assay combines (i) an uninfected control assay, (ii) a HSV-1-specific antibody assay, and (iii) a HSV-2-specific antibody assay.
A further benefit of the invention is that the preferred Type-Specific ABVIC assay is highly quantitative and permits for statistical interpretation of the probability that a person is HSV-1 and/or HSV-2 seropositive.
A further advantage is that the quantitative and statistical power of a preferred Type-Specific ABVIC assay permits the assay to resolve Indeterminate Test Results of Herpes Western Blot tests.
An additional benefit of the invention is that the increased sensitivity and quantitative power of the Type-Specific ABVIC assay relative to the Herpes Western Blot can permit a preferred Type-Specific ABVIC assay to be carried out more rapidly than the usual Western blot-formatted assay, while maintaining the ability to distinguish infection by HSV-1 from infection by HSV-2.
An additional advantage of the invention is that it can provide more sensitive results than the commercial HerpeSelect® test ELISA assay because a preferred type-specific ABVIC assay screens for the presence of antibodies against up to 75 HSV-1 or HSV-2 proteins that can be present in the fixed and permeabilized test cells described above. In contrast, the HerpeSelect® ELISA tests for antibodies against only 1 of 75 HSV-1 or HSV-2 proteins; namely, glycoprotein G.
Still further benefits and advantages will be apparent to the skilled worker from the description that follows.
HSV-2 shedding on the y-axis. The solid black line represents the best-fit linear regression model, y=3.77−0.95x, for these four matched averages (r2=0.98). Groups of immunized guinea pigs that exhibited a significant reduction in vaginal HSV-2 shedding relative to naïve guinea pigs are indicated by a single asterisk (*p<0.05) or double-asterisk (**p<0.001), as determined by one-way ANOVA and Tukey's post-hoc t-test.
The present invention contemplates an assay that can detect and differentiate between infection by one or both or neither of HSV-1 and HSV-2 for the purposes of disease diagnosis from a subject serum sample. One preferred illustrative embodiment contemplates a three cell population assay that is referred to herein as a flow cytometry-based serological assay that measures the virus type-specific affinity of serum antibody-binding to virus-infected cells (ABVIC).
The present invention contemplates use of an antibody-containing sample from a patient whose infection status with one, the other, both or none of HSV-1 and HSV-2 is to be determined. Usually, that sample is in the form of serum or plasma from a blood draw sample. The sample can also be an antibody-enriched sample such as an ammonium sulfate precipitate from a blood or other sample as are well known, or from a dried, e.g. lyophilized, serum or plasma sample. For convenience and because of their similarity, serum and plasma are collectively referred to herein as serum.
The patient (subject) sample is divided into at least three portions or subsamples. Each portion (subsample) is separately admixed and contacted with (a) antigens from uninfected cells, (b) with antigens from HSV-1-infected cells and (c) with antigens from HSV-2-infected cells.
That contact is maintained for a time period sufficient for antibodies within the subsample that immunoreact with the recited antigens to immunoreact (bind) therewith. That contact and maintenance is also referred to herein as incubation. Maintenance times can range from a few minutes to about 96 hours. Usually, the maintenance time is about 1 to about 8 hours, and more preferably about 2 to about 6 hours.
The above-mentioned cell antigens are themselves part of a physical matrix so that the reacted antibodies form physical matrix-bound antibodies (also referred to as matrix-bound antibodies). Illustrative physical matrices include, for example, 1) a protein-coated solid matrix (e.g., ELISA plate); 2) a cell-coated solid matrix (e.g., culture plate coated with fixed cells); 3) free-floating particles (e.g., live or fixed cells in liquid suspension); 4) a column of particles (e.g., live or fixed cells in a capillary tube); 5) protein-coated magnetic beads; 6) a slurry of protein-coated matrix (e.g., antigen-reacted CNBr-activated Sepharose® 4B) suspended in liquid, or packed into a flow-through column.
Slurries of fixed and permeabilized uninfected (UI) cells, HSV-1+ cells and HSV-2+ cells were used illustratively herein. As is well known in the biological arts, cell fixation can be achieved by a wide variety of chemicals including, but not limited to treatment with one or more of formaldehyde, paraformaldehyde, methanol, ethanol, and acetone. It is preferred that the cells used as each of the physical matrices be of the same type. Illustrative cell types include 1) human SK—N—SH neuroblastoma cells; 2) human U2OS osteosarcoma cells; 3) human 293 embryonic kidney cells; 4) monkey CV-1 kidney cells (Vero cells); 5) monkey COS cells; 6) mouse 3T3 cells; 7) hamster BHK-21 cells; 8) bovine BIEC cells; 9) bovine BUVEC cells; 10) human Caco-2 cells; 11) human HeLa cells; 12) monkey MA104 cells; 13) canine MDCK cells; 14) pig PK-15 cells; and 15) human WiDr cells.
It is noteworthy that HSV ICP0− viruses form plaques with an efficiency that is indistinguishable from Vero cells in 11 of 15 other cell lines tested to date. Specifically, work by the inventor and co-workers indicates that 0.5-2% of HSV ICP0− viruses form plaques in monolayers of human 293 cells, mouse 3T3 cells, hamster BHK-21 cells, bovine BIEC cells, bovine BUVEC cells, human Caco-2 cells, human HeLa cells, monkey MA104 cells, canine MDCK cells, pig PK-15 cells, and human WiDr cells. Thus, any cell line in this list, by definition, supports replication of wild-type HSV-1 and HSV-2 as well as the mutant HSV-1 virus specifically discussed in the text. Vero cells are one preferred cell type and are used hereinafter as illustrative.
The cells used can themselves be attached to a solid, physical matrix (e.g., plastic dish, magnetic beads, agarose, etc.) or can be suspended in a liquid solution such as an aqueous medium like a buffer solution such as PBS. The physical matrix-bound (immunoreacted) antibodies are thereafter separated from any unreacted antibodies present in the reacted subsample. This separation can be carried out by centrifugation and decantation or pipetting out the supernatant liquid, pipette removal as where the antigen-bound antibody is on the walls of a culture plate, elution and the like.
The above-discussed admixing and incubation of each of three serum samples with a different one of the cell antigen-matrices is also referred to herein as preadsorption. That preadsorption is preferably carried out with fixed cells. It is preferred that the same cell type be used for each of the preadsorptions to minimize possible differing cross-reactivities.
The purpose of the uninfected cell antigen matrix, in whichever specific form it is used, is to serve as a “PreAdsorption Treatment Control” that has little to no effect on the population of human serum antibodies in a test sample taken from a patient seeking to determine if they are infected with HSV-1 and/or HSV-2 (leftmost panels of raw data in
Similarly, the purpose of the HSV-1+ cell antigen matrix, in whichever specific form it is used, is to remove (1) HSV-type-common antibodies and (2) HSV-1-specific antibodies from a patient's serum sample. Hence, the effluent that is removed after incubation with the HSV-1+ cell antigen matrix yields a highly enriched population of HSV-2-specific antibodies, which may be used to determine if a patient has been infected with the HSV-2 virus (center panels of raw data in
Likewise, the purpose of the HSV-2+ cell antigen matrix, in whichever specific form it is used, is to remove (1) HSV-type-common antibodies and (2) HSV-2-specific antibodies from a patient's serum sample. Hence, the effluent that is that is removed after incubation with the HSV-2+ antigen matrix provides a highly enriched population of HSV-1-specific antibodies, which may be used to determine if a patient has been infected with the HSV-1 virus (rightmost panels of raw data in
As a result of the preadsorptions, at least three preadsorbed serum subsamples are formed. Thus, the preadsorbed serum subsample incubated with uninfected cell antigens (a) contains a reduced amount of antibodies that immunoreact with uninfected cells. The preadsorbed serum subsample incubated with antigens of cells infected with HSV-1 (b) contains a reduced amount of antibodies that immunoreact with HSV-1-infected cells when those antibodies were present in the provided serum sample, and thereby a relatively enhanced amount of antibodies that immunoreact with HSV-2, when those antibodies were present in the provided serum sample. Similarly, the preadsorbed serum subsample incubated with antigens of cells infected with HSV-2 (c) contains a reduced amount of antibodies that immunoreact with HSV-2-infected cells when those antibodies were present in the provided serum sample, and a relatively enhanced amount of antibodies that immunoreact with HSV-1, when those antibodies were present in the provided serum sample.
Each of the preadsorbed subsample portions is incubated (as discussed above) with a mixture of second matrix-linked test antigens from cells uninfected by either HSV-1 or HSV-2, test antigens from cells infected by HSV-1 and test antigens from cells infected by HSV-2 to permit antibodies present within each subsample to immunoreact with test antigens present. The amount of immunoreaction, including little or no immunoreaction, is then determined for each of the subsamples with the test antigen mixture to determine with which test antigens, if any, the antibodies from the preadsorbed subsamples immunoreacted.
The second matrix-linked test antigens utilized in this portion of the assay can be the same test antigen-physical matrix constructs discussed before, or different constructs. In one preferred embodiment, the mixture of the three test antigen-physical matrices is comprised of fixed and permeabilized cells that are (a) unstained, (b) weakly stained with a cellular dye, fluorophore, or colorant and (c) strongly stained with the same exogenously-provided cellular dye, fluorophore, or colorant, so that each population of test cells can be distinguished from each other on the basis of their relative amounts of dye color or fluorescence.
A particularly preferred exogenously-provided (not normally present as part of the cells) cellular colorant is fluorescent upon irradiation, typically with defined wavelengths of light in the ultraviolet, visible, or infrared range (200-800 nm), and its fluorescence can be detected by a flow cytometer. Illustrative useful exogenously-provided chemically reactive (covalently-linkable) fluorescent colorants include but are not limited to 5- (and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), (CellTrace™ Violet), and 2,5-dioxopyrrolidin-1-yl-7-(2-(((1E,3E,4E)-1,5-dichloro-6-oxohexa-1,4-dien-3-ylidene)amino)-5-hydroxyphenyl)octanoate (CellTrace™ Far Red DDAO-SE) that couple to amino groups such as epsilon-amino groups of lysine residues via N-hydroxysuccinimide ester exchange, and chloromethyl reactive colorants such as (2,3,6,7-tetrahydro-9-bromomethyl-1H,5H-quinolizino(9,1-gh)-coumarin (CellTracker™ Violet BMQC), 7-amino-4-chloromethyl-coumarin (CellTracker™ Blue CMAC), 5-chloromethyl-fluorescein diacetate (CellTracker™ Green) and 5-chloromethylrhodamine (CellTracker™ Red) (all available from Life Technologies, Thermo Fisher Scientific) that stain the cells via reaction with cellular thiol groups. Cells can also be differentially labeled with one or more intracellularly-expressed fluorescent proteins including, but not limited to green fluorescent protein (GFP), mCherry, tdTomato, KeimaRed, yellow fluorescent protein (YFP), cyan fluoresent protein (CFP) as discussed for GFP in Chalfie et al., (1994) Science 263:802-805.
The exogenously-introduced fluorescent colorant provides a means by which each population of test cell antigens are distinguishable from each other by fluorescence. Fluorescence emission from the exogenously-provided cellular colorant of the test cell antigen-containing matrices is distinguishable from the fluorescence emission of the secondary antibodies discussed hereinafter, and fluorescence of any other material present in the assay.
In one preferred embodiment, the amount of immunoreaction is determined for each preadsorbed serum subsample that is combined with test cells, and human antibody binding to each population of test cells is detected by secondary labeling with anti-human antibodies that are admixed with test cells. A preferred label for the anti-human antibodies is a covalently-linked fluorescent compound whose fluorescence emission spectrum does not overlap with the fluorescence emission spectrum of the colorant used to differentially label (i) uninfected, (ii) HSV-1-infected, and (iii) HSV-2-infected antigen matrices. Illustrative covalently-linkable fluorescent dyes that can be conjugated to a secondary anti-human antibody include, but are not limited to, allophycocyanin (APC), phycoeryrthrin (PE), tetramethylrhodamine isothiocyanate (TRITC), and peridinin chlorophyll protein (PerCP).
It is preferred that any unreacted antibodies from the preadsorbed subsamples be separated from the immunoreaction products as previously discussed.
In addition to using a fluorescent label for the anti-human (secondary) antibodies, enzyme labels such as horseradish peroxidase (HRP), alkaline phosphatase and glucose oxidase can be covalently conjugated to the secondary antibodies as are often utilized in ELISA assays with an appropriate chromogenic substrate as are well known.
The anti-human (secondary) antibodies are themselves raised in an animal other than a human. Illustrative secondary antibodies include those raised in goats, donkeys, horses, rabbits, mice and rats. These anti-human antibodies preferably react with human Fc antibody portions.
On determining to which test cell antigens the antibodies present in each preadsorbed subsample portions bound, one can thereby ascertain whether the patient was infected with HSV-1, HSV-2, both or neither.
Any method of detecting immunofluorescence can be used to determine which, if any, of the preadsorbed subsamples bound to the test cell antigens including but not limited to fluorescent microscopy, a fluorescent plate reader, a flow cytometer, or a fluorescence-activated cell sorter. Preferably, a flow cytometer or FACS is utilized as such machines can measure both (1) the differential fluorescent color that indicated whether the test cell antigen-containing matrix were uninfected, HSV-1+, or HSV-2+, and the instrument simultaneously measures (2) a second fluorescent color that is indicative of the primary variable under study; namely, the amount of human antibody bound to uninfected versus HSV-1+ versus HSV-2+ test cell antigens.
The discussion hereinafter describes a particularly preferred assay that utilizes fixed cells as the test cell antigen-containing matrices.
The present invention also contemplates a serological assay kit for carrying out a before-described assay. An illustrative kit includes a) three separate vessels that separately contain one of i) cell antigens from uninfected cells in a physical matrix, (ii) cell antigens from HSV-1-infected cells in a same or different physical matrix, or (iii) cell antigens from HSV-2-infected cells in a same or different physical matrix from that of (i) or (ii).
A fourth vessel is also included. The fourth vessel contains a mixture of test cell antigens from cells uninfected by either HSV-1 or HSV-2, antigens from cells infected by HSV-1, and antigens from cells infected by HSV-2, each of those cell antigens linked to a second matrix that is the same or different from the first-named matrix. Each of those four vessels contains a sufficient amount of the recited ingredient to carry out at least one assay.
Instructions for carrying out an assay are also present in the kit. A contemplated kit is preferably provided as a container that holds the recited components.
The vessels of a contemplated assay kit are typically made of glass or a plastic to which the recited reagents adhere poorly such as to polyethylene glycol (PEG) coatings and coatings of polytetrafluoroethylene (PTFE).
The above-described serological assay kit further preferably includes a fifth vessel that contains labeled anti-human antibodies in an amount sufficient to carry out at least one assay. The label of the anti-human antibodies is preferably a fluorescent material whose fluorescence is distinguishable from the fluorescence of any other material present. It is also preferred that the mixture of fixed cells of the fourth vessel further include an exogenously-introduced fluorescent colorant by which each population of test cell antigen matrices is distinguishable from the others by the intensity of fluorescent emissions in a defined wavelength, and is also distinguishable from any other fluorescent species utilized in the assay.
In a preferred embodiment, the present invention is a serological assay for determining whether a subject is infected with HSV-1, HSV-2, both, or neither. The assay comprises the steps of dividing a serum sample obtained from a subject into at least three serum subsamples, preadsorbing the serum subsamples to at least three populations of fixed and permeabilized test cells, incubating the preadsorbed serum subsamples with a mixture of at least three populations of test cells in a suspension, incubating the serum subsamples with a detection antibody, and analyzing the cell-serum subsample admixture with a flow cytometer.
The one or more herpes simplex viruses are preferably selected from the group comprising herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2).
The at least three populations of fixed and permeabilized cells are preferably Vero cells, wherein a first population is uninfected, a second population is infected with HSV-1, and a third population is infected with HSV-2.
The at least three populations of test cells in suspension are preferably Vero cells, wherein a first population is uninfected and unlabeled, a second population is infected with HSV-1 and labeled with a low concentration of a first fluorescent molecule, and a third population is infected with HSV-2 and labeled with a high concentration of that first-noted fluorescent molecule. Preferably, the first fluorescent molecule is carboxyfluorescein diacetate, succinimidyl ester (CFSE).
The detection antibody is preferably an anti-IgG antibody, and anti-human IgG where the subject whose serum is assayed is human. The detection antibody is also preferably labeled with a second fluorescent molecule. The second fluorescent molecule should have a fluorescence emission spectrum that does not overlap with the fluorescence emission spectrum of the first fluorescent molecule used to label each population of test cells. A suitable second fluorescent molecule is allophycocyanin (APC), but many other fluorophores described herein are suitable as well.
The flow cytometry device can be any device capable of quantitatively measuring the fluorescence associated with individual antigen-containing test matrices of an appropriate diameter for the instrument, about 1 to about 20 microns. Examples of such appropriately sized antigen-containing test matrices include, but are not limited to, (1) live uninfected Vero cells, (2) fixed and permeabilized uninfected Vero cells, (3) live HSV-1-infected Vero cells, (4) fixed and permeabilized HSV-1-infected Vero cells, (5) live HSV-2-infected Vero cells, or (6) fixed and permeabilized HSV-2-infected Vero cells. Preferably, the cell sorting device is a flow cytometer, but a fluorescence-activated cell sorter (FACS) can-be used for the same purpose although generally such instruments are about 20-times more expensive and are thus reserved for the act of “sorting cells” (hence the name of the instrument) based on fluorescent intensity, as opposed to the more rudimentary task of measuring the fluorescent intensity associated with cells, which is generally performed with a flow cytometer.
Antibody-Binding to Virus-Infected Cells (ABVIC): A More Sensitive Method than ELISA to Measure Pan-HSV-2 IgG Antibodies-Two Cell Studies
As discussed herein, the presence of serum IgG antibodies that bind all HSV-2 antigens (pan-HSV-2 IgG) can be visualized by red fluorescent immunostaining of HSV-2 plaques in Vero cell monolayers (
This assay is referred to as the “ABVIC assay” because it measures antibody-binding to virus-infected cells. HSV-2-infected (HSV-2+) cells are labeled with a green fluorophore, CFSE, whereas uninfected (UI) cells lack this label, which permits the two cell populations to be differentiated in a flow cytometer (x-axis of
Test cell suspensions were incubated with serum from naïve mice or immunized mice, and the amount of IgG antibody bound to UI or HSV-2+ cells was detected via an anti-mouse IgG secondary antibody bearing a red fluorescent label (allophycocyanin; APC) (y-axis of
Antibody-capture ELISA and ABVIC were compared in a side-by-side manner to compare their relative sensitivity (
Two Cell Population ABVIC Assay Demonstrates that Two of Four “HSV-2 Indeterminate” Patients are Seronegative
Clinical serum samples have been obtained periodically from Terri Warren (Westover Heights Clinic) since 2011. Quorum IRB (Seattle, Wash.) and SIU School of Medicine's Springfield Committee for Research on Human Subjects both concluded the research was “exempt,” as only de-identified sera were evaluated. An analysis of human sera using the ABVIC assay is described, as follows.
Sera from n=3 seronegative individuals defined the background level of antibody-binding to HSV-2+ cells and UI cells (
The Illustrative Three Cell Population Type-Specific ABVIC Assay
Two modifications were employed to convert the two cell population ABVIC assay into the three cell population HSV type-specific ABVIC assay. These changes were: 1) serum preadsorption to UI cells, HSV-1+ cells, or HSV-2+ cells (
Regarding serum preadsorption, patients infected with HSV-1 and/or HSV-2 possess up to three populations of HSV antibodies: 1) type-common antibodies that bind HSV-1 and HSV-2 antigen proteins (Z's in
An enriched population of HSV-2-specific antibodies can be obtained by preadsorbing serum from a HSV-infected subject (human or other animal) to fixed HSV-1+ cells, which depletes type-common and HSV-1-specific antibodies (center column,
Regarding the three cell population assay, an optimized CFSE-labeling protocol was developed that yields populations of UI cells (no CFSE), HSV-1+ cells (CFSElo), and HSV-2+ cells (CFSEhi), which can be resolved in a flow cytometer (
A seronegative serum sample was incubated with test cells after preadsorption to each cell population. Following preadsorption to UI cells, negligible antibody binding to cells was noted (left graph,
Serum of a known HSV-2 seropositive individual was incubated with test cells after preadsorption to each cell population. Following preadsorption to UI cells, HSV-specific antibodies bound HSV-1+ and HSV-2+ cells to 10- and 15-fold higher levels than UI cells, respectively (left graph,
Indeterminate serum sample 3 discussed previously was incubated with test cells after separate subsample preadsorptions to each of the three cell populations. Following preadsorption to UI cells, HSV-specific antibodies bound HSV-1+ and HSV-2+ cells to 100- and 20-fold higher levels than UI cells, respectively (left graph,
These findings indicated that the patient who provided indeterminate serum sample 3 was infected with HSV-1, but not HSV-2 at the time the serum sample was obtained. Indeterminate serum sample 4 yielded equivalent results (not shown). Therefore, the three cell population type-specific ABVIC assay demonstrated that indeterminate serum samples 3 and 4 were both HSV-2 seronegative and HSV-1 seropositive.
HSV-2 indeterminate serum samples 1, 2, 3, and 4 represent four patients who could have been spared a great deal of anxiety and suffering if a better HSV serological assay were available to properly inform them that they were not infected with HSV-2 and thus could not transmit HSV-2 genital herpes to any sexual contacts.
Illustrative Three Cell Population Type-Specific ABVIC Assay is More Sensitive than HerpeSelect®
The HerpeSelect® assay (Quest Diagnostics, Inc.) is an antibody-capture ELISA that tests for the presence of antibodies specific for glycoprotein G of HSV-1 (gG-1) or HSV-2 (gG-2) [Whittington et al., 2001]. These are two of the most divergent HSV proteins known [Sanchez-Martinez et al., 1991; Roizman et al., 1984]. Patients infected with HSV-1 can mount an antibody response against 30 HSV-1 proteins including gG-1, and likewise HSV-2 infection can drive an antibody response against 30 HSV-2 proteins including gG-2 [Norrild et al., 1981; Gilman et al., 1981] (
A critical weakness of the HSV-2 HerpeSelect® assay is that it only tests for antibodies against gG-2, which represent 3-10% of an infected person's total repertoire of HSV-2-specific antibodies (
The Illustrative Three Cell Population Type-Specific ABVIC Assay is More Sensitive than Western Blot
The illustrative three cell population type-specific ABVIC assay is unique amongst HSV serological assays in that it tests for pan-HSV-type-specific antibodies, is internally controlled, and is based on thousands of replicate measurements. In the ABVIC assay, the level of a patient's IgG antibodies that bind thousands of HSV-2+ cells versus UI cells is measured, and these quantities are compared to those produced by a panel of control seronegative and HSV-2 seropositive sera.
Thus, the cutoff between “seronegative” and “seropositive” can be set to any level of statistical significance deemed appropriate (e.g., the probability that the patient who provided indeterminate serum sample 3 was HSV-2-seropositive was less than 1 in a million;
Detection of Human Antibody Binding to (i) Uninfected, (ii) HSV-1+, and/or (iii) HSV-2+ Test Cells
A. Immunofluorescent microscopy. It is possible to determine if a patient serum sample contains HSV-2-specific antibody by comparing its ability to bind uninfected cells versus virus-infected cells in the context of monolayers of Vero cells that are infected with a small amount of HSV-1 or HSV-2 virus that is allowed to form small foci of infection (a.k.a. “plaques”). In this embodiment of the type-specific ABVIC test, human antibody binding to virus-infected cells could be visualized with a fluorescent microscope (
1. HSV-1 plaques in a monolayer of mammalian cells;
2. HSV-2 plaques in a monolayer of mammalian cells;
3. uninfected (UI) antigen-, HSV-1+ antigen-, and HSV-2+ antigen-matrices.
A specific example of such a test is illustrated in
Hence, the difference in “mean red fluorescent intensity” (ΔMFI) is what one's eye notes that tells one this individual must possess “HSV-specific antibody,” and this is precisely the same quantity that is being compared in the more quantitative, flow-cytometry-based variation of the type-specific ABVIC test. In the images of
Specifically, serum of a patient who was known to be HSV-2-seropositive was used to validate that these three sets of reagents were useful to test for the presence of HSV-2-specific antibodies. Starting at the top of the panels shown in
When this patient's serum sample was diluted 1:2,000 and was preadsorbed to a matrix of uninfected (UI) cell antigens, it still contained a mixture of HSV-type common antibodies and HSV-2-specific antibodies that collectively bound both (i) HSV-1 plaques shown on the left and (ii) HSV-2 plaques shown on the right. Hence, UI cell antigen-preadsorbed patient serum was insufficient to determine if this individual was infected with HSV-1 and/or HSV-2 (
When this patient's serum sample was diluted 1:2,000 and was preadsorbed to a matrix of HSV-1-infected (HSV-1+) cell antigens, HSV-type common antibodies were depleted out but HSV-2-specific antibodies remained. Hence, the enriched population of HSV-2-specific antibodies only poorly bound to HSV-1 plaques on the left, but bound strongly to HSV-2 plaques shown on the right. The HSV-1 cell antigen-preadsorbed patient serum was sufficient to determine that this individual was HSV-2 seropositive, which is prognostic for an underlying HSV-2 infection (
Finally, when this patient's serum sample was diluted 1:2,000 and was preadsorbed to a matrix of HSV-2-infected (HSV-2+) cell antigen, HSV-type common antibodies and HSV-2-specific antibodies were depleted out. If this patient were infected with HSV-1, and thus were HSV-1 seropositive, HSV-1-specific antibodies present would remain. However, what is observed in the final panel is that the HSV-2-preadsorbed serum did not possess antibodies that bound HSV-1 plaques on the left to a level higher than HSV-2 plaques shown on the right. Hence, this HSV-2 cell antigen-preadsorbed patient serum was sufficient to determine that this individual was HSV-1 seronegative (
In
B. Flow cytometry. One can determine if a patient serum sample contains HSV-2-specific antibodies by comparing its ability to bind uninfected cells versus virus-infected cells in the context of suspensions of uninfected, HSV-1-infected, and HSV-2-infected mammalian cells. In this embodiment of the type-specific ABVIC assay, human antibody binding to virus-infected cells can be quantitatively measured using a flow cytometer (
Such an experimental system can be used to measure “HSV-2-specific antibody” using the three elements of:
1. HSV-1-infected mammalian cells;
2. HSV-2-infected mammalian cells; and
3. Three populations of uninfected (UI) test cell antigen-, HSV-1-infected test cell antigen-, and HSV-2-infected test cell antigen-matrices.
A specific example of such a test is illustrated in
In this particular embodiment of the ABVIC assay, a patient's antibody binding to the test cell suspension containing fixed and permeabilized UI cells, HSV-1+ cells, and HSV-2+ cells was measured using “allophycocyanin (APC)-conjugated goat-anti-human IgG” antibody, which produces a far-red color that is measured in the FL4 channel of a flow cytometer (y-axes in each sub-panel in
The data in
There are three important quantitative features that are unique to the flow cytometry-based embodiment of the type-specific ABVIC test and these are, as follows:
1. Estimates of “HSV-specific antibody” level are based on the difference in mean fluorescent intensity (ΔMFI) in the FL4 channel (y-axis) between n about 20,000 UI cells vs n about 20,000 HSV-1+ cells vs n about 20,000 HSV-2+ cells, which provides a high degree of confidence in quantitative estimates of HSV-specific antibody abundance in a patient's blood;
2. UI cells provide an internal control that defines the background of the assay, and hence the assay is insensitive to patients whose blood possesses antibodies that cause a higher background signal, which is a major variable that confounds the HerpeSelect® assay and Herpes Western Blots, and likely accounts for at least 50% of “Indeterminate” results that mislead many people to the erroneous conclusion that they are HSV-2 infected/HSV-2 seropositive; and
3. defining the mean and standard deviation of the ΔMFIHSV-1 associated with HSV-1+ cells (MFIHSV-1-MFIUI) and the mean and standard deviation of the ΔMFIHSV-2 associated with HSV-2+ cells (MFIHSV-2-MFIUI) creates the opportunity for statistical analysis of the probability that a given patient is HSV-1-seronegative or HSV-2 seronegative based on where their own ΔMFIHSV-1 or ΔMFIHSV-2 values fall on the normal distribution of ΔMFIHSV-1 or ΔMFIHSV-2 values for control HSV-seronegative samples (
For individuals who are HSV-1 seropositive, the control “UI preadsorbed” serum sample (left panel in
Based on subsequent statistical considerations (
For the individuals who are HSV-2 seropositive, the control “UI preadsorbed” serum sample (left panels in
For the individual who is HSV-1-seropositive and HSV-2 seropositive, the control “UI preadsorbed” serum sample (left panel in
Preadsorption of Human Antibodies to Uninfected, HSV-1+, or HSV-2+ Antigen Matrices.
A. Preadsorption to CNBr-Activated Sepharose® 4B Matrix.
Examples of the use of cyanogen-bromide (CNBr)-activated Sepharose® 4B (GE Healthcare Life Sciences) as an UI, HSV-1, or HSV-2 cell antigen matrix for the preadsorption step in the type-specific ABVIC test are shown in
B. Preadsorption to Fixed Vero Cells Attached to a Solid Matrix.
Examples of the use of fixed and permeabilized Vero cells as an UI, HSV-1, or HSV-2 cell antigen matrix for the preadsorption step in the type-specific ABVIC test are shown in
Fixed test cells or cell antigen matrices that are (i) uninfected, (ii) HSV-1+, and/or (iii) HSV-2+ are stable over time. The concept of fixation, as the term implies, involves “fixing” a biological tissue into a form that does not decay, and is thus stable over time. This is the basis of embalming humans for funeral preparations, which was practiced in ancient Egypt to produce preserved mummies. The use of fixatives such as formaldehyde, methanol, ethanol, acetone, etc. has been commonplace in biology since the 19th century. In studies in the inventor's laboratories, (1) suspensions of fixed uninfected, HSV-14-, or HSV-2+ Vero test cells or (2) uninfected, HSV-1+, or (iii) HSV-2+ cell antigen matrices are stable at 4° C. for at least 1 month.
The uninfected (UI) control antibody test is sufficient to distinguish HSV-seronegative from HSV-seropositive, but does not differentiate whether a person is infected with HSV-1, HSV-2, or both. The leftmost column of panels in
The presence of HSV-1-specific antibody in a subject's serum permits calculation of the probability that the subject was HSV-1 seronegative, and thus one could infer that a person is HSV-1 seropositive if their probability of being HSV-1 seronegative is less than 0.5%. Data that supports these points are presented in
The presence of HSV-2-specific antibody allows us to calculate the probability a person is HSV-2 seronegative, and thus we may infer a person is HSV-2 seropositive if their probability of being HSV-2 seronegative is less than 0.5%. Data that support these points are presented in
The Type-Specific ABVIC Asay combines the (i) uninfected control test, (ii) HSV-1-specific antibody test, and the (iii) HSV-2-specific antibody test. Data that support these points are presented in
The Type-Specific ABVIC Assay is highly quantitative and permits statistical interpretation of the probability that a person is HSV-1 and/or HSV-2 seropositive. Data that supports these points are presented in
The quantitative and statistical power of the Type-Specific ABVIC Assay allows the test to resolve Indeterminate Test Results of Herpes Western Blot tests. Data that support these points are presented in
HSV-1: The probability that patient 17A is HSV-1 seronegative is 61%.
HSV-2: The probability that patient 17A is HSV-2 seronegative is 79%.
Conclusion: Patient 17A is HSV-1 seronegative and HSV-2 seronegative.
HSV-1: The probability that patient 17B is HSV-1 seronegative is 77%.
HSV-2: The probability that patient 17B is HSV-2 seronegative is 41%.
Conclusion: Patient 17B is HSV-1 seronegative and HSV-2 seronegative.
HSV-1: The probability that patient 17C is HSV-1 seronegative is 3%.
HSV-2: The probability that patient 17C is HSV-2 seronegative is 85%.
Conclusion: Patient 17C is HSV-1 equivocal and HSV-2 seronegative.
HSV-1: The probability that patient 18A is HSV-1 seronegative is 0.4%.
HSV-2: The probability that patient 18A is HSV-2 seronegative is 45%.
Conclusion: Patient 18A is weakly HSV-1 seropositive and HSV-2 seronegative.
HSV-1: The probability that patient 18B is HSV-1 seronegative is less than 0.01%.
HSV-2: The probability that patient 18B is HSV-2 seronegative is 8%.
Conclusion: Patient 18B is HSV-1 seropositive and HSV-2 seronegative.
HSV-1: The probability that patient 18C is HSV-1 seronegative is less than 0.01%.
HSV-2: The probability that patient 18C is HSV-2 seronegative is 30%.
Conclusion:. Patient 18C is HSV-1 seropositive and HSV-2 seronegative.
General Development
Development of the Two Cell Population ABVIC (Antibody Binding to Virus-Infected Cells) Assay
Introduction
There exists a need for a correlate of immunity to herpes simplex virus 2 (HSV-2) that can be used to differentiate whether a HSV-2 vaccine elicits robust or anemic protection against genital herpes.
It has been suggested that past difficulties in identifying a clinically useful correlate of immunity to HSV-2 may have stemmed from a failure to identify the correct parameter of the T-cell response that controls HSV-2 in vivo [Rouse and Kaistha, 2006]. However, there is a second possibility. Most attempts to identify a correlate of immunity to HSV-2 have focused on monovalent (gD-2) or bivalent (gB-2+gD-2) subunit vaccines that present less than 3% of HSV-2's 40,000 amino-acid proteome to the immune system [Shlapobersky et al., 2012; Bernstein et al., 2010; Bourne et al., 2005; Bernstein, 2005; Bourne et al., 2003; Khodai et al., 2011; Bernstein et al., 2011; Allen et al., 1990; Weir et al., 1989; Kuklin et al., 1997; Manickan et al., 1995; Eo et al., 2001; Natuk et al., 2006; Orr et al., 2007; Karem et al., 1997; Brans and Yao, 2010; Meigner et al., 1988]. This approach does not consider HSV-2's full complement of antigens; at least 20 viral proteins are known targets of the human B- and T-cell response to HSV-2 [Hosken et al., 2006; Laing et al., 2010; Gilman et al., 1981]. Therefore, it was postulated that a correlate of immunity might be more readily identified if: 1) animals were immunized with a polyvalent immunogen such as a live virus; and/or 2) the magnitude of the vaccine-induced immune response was gauged in terms of the IgG antibody response to all of HSV-2's antigens (pan-HSV-2 IgG).
The current study was initiated to test these predictions. A novel, flow cytometry-based assay was developed to measure pan-HSV-2 IgG levels. Using this assay, 117 naïve and immunized animals were analyzed to compare pre-challenge serum levels of pan-HSV-2 IgG to two measures of protection against HSV-2. Pre-challenge pan-HSV-2 IgG levels and protection against HSV-2 were compared in mice and/or guinea pigs immunized with a gD-2 subunit vaccine, wild-type HSV-2, or one of several attenuated HSV-2 ICP0− viruses (0Δ254, 0Δ810, 0ΔRING, or 0ΔNLS). These six HSV-2 immunogens elicited a wide range of pan-HSV-2 IgG levels spanning an about 500-fold range. For 5 of the 6 immunogens tested, pre-challenge levels of pan-HSV-2 IgG quantitatively correlated with reductions in HSV-2 challenge virus shedding and increased survival frequency following HSV-2 challenge. Collectively, the results suggest that pan-HSV-2 IgG levels may provide a simple and useful screening tool for evaluating the potential of a HSV-2 vaccine candidate to elicit protection against HSV-2 genital herpes.
Materials and Methods
Mice and guinea pigs were handled in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. This study was approved by the Southern Illinois University School of Medicine Laboratory Animal Care and Use Committee, and was performed as described under approved protocol 205-08-019.
Vero cells and U2OS cells were obtained from the American Type Culture Collection (Manassas, Va.), and ICP0-complementing L7 cells were kindly provided by Neal Deluca (University of Pittsburgh; Samaniego et al., 1998). All cells were propagated in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 100 U/ml penicillin G, and 100 mg/ml streptomycin, hereafter referred to as “complete DMEM.” Wild-type HSV-2 MS (ATCC) was propagated and titered on Vero cells. The HSV-2 ICP0− mutant viruses used in this study (HSV-2 0Δ810, 0Δ254, and 0ΔRING: Halford et al., 2010) were propagated in U2OS cells and titered in ICP0-complementing L7 cells.
A retrospective analysis of serum obtained two years earlier was performed in the current study (
Female Hartley guinea pigs were obtained at an average weight of 250 g from Charles River (Wilmington, Mass.). On Day 0, guinea pigs were anesthetized by i.p. administration of xylazine (5 mg/kg) and ketamine (30 mg/kg), and were immunized via right, rear footpad injection of 100 μl containing: 1) complete DMEM (naïve); 2) 2×106 pfu HSV-2 0ΔNLS; 3) 2 ×106 pfu HSV-2 MS; or 4) 5 μg recombinant glycoprotein D-2 (gD-2) antigen+20 μg monophosphoryl lipid A (Avanti Polar Biolipids)+Imject® alum adjuvant (Thermo Scientific). The gD-2 antigen was expressed from a baculovirus vector [Nicola et al., 1996] and has been used as a vaccine antigen in numerous studies [Bernstein et al., 2010; Bernstein et al., 2011; Halford et al., 2011]. The details of purification of this His-tagged gD-2 protein are described elsewhere [Halford et al., 2011]. Guinea pigs immunized with HSV-2 MS received 1 mg/ml oral acyclovir in their drinking water between Days 0 and 20 post-immunization to limit viral pathogenesis; 100% of guinea pigs survived their primary exposure to HSV-2 MS without developing overt signs of disease. Guinea pigs received an equivalent immunization in their left, rear footpads on Day 30 (per design shown in
All guinea pigs were challenged with HSV-2 MS on Day 90, as follows. Prior to viral inoculation, guinea pigs were anesthetized by i.p. administration of xylazine (5 mg/kg) and ketamine (30 mg/kg). Naïve and immunized guinea pigs were vaginally challenged with wild-type HSV-2 MS by: 1) first clearing the mucus plug from the vagina with a cotton swab; 2) twirling a second cotton swab inside the vaginal vault to further dry the walls of the vagina; and 3) instilling the vaginal vault with 40 μl complete DMEM containing 2×106 pfu of HSV-2 MS.
Viral titers in the vaginal vault of challenged guinea pigs were determined at 8 hours post-challenge (eclipse phase) and on Days 1, 2, 3, 4, 6, and 8 post-challenge by inserting and twirling a swab in the vaginal vault of guinea pigs, and transferring the tip into 0.4 ml complete DMEM. Viral titers were determined as described above. Guinea pigs were monitored daily, and animals that exhibited severe perivaginal ulceration were euthanized at the earliest possible time. The perivaginal region of all guinea pigs was photographed on Day 7 post-challenge. Surviving guinea pigs were euthanized on Day 30 post-challenge.
Female strain 129 mice were obtained at 6- to 8-weeks of age from Charles River (Wilmingtion, Mass.). On Days 0 and 30, n=10 mice were anesthetized by i.p. administration of xylazine (7 mg/kg) and ketamine (100 mg/kg), and were immunized via right and left rear footpad injection, respectively, of 50 μl containing 106 pfu HSV-2 0ΔNLS. On Day 85, n=5 HSV-2 0ΔNLS-immunized mice were sacrificed to harvest HSV-2 antiserum, and n=5 age-matched, naïve mice were sacrificed to harvest naïve serum. On Day 90, naïve mice received an adoptive transfer of 0.25 ml pooled HSV-2 antiserum or 0.25 ml pooled naïve serum. Immediately following adoptive transfer, these n=10 naïve mice were anesthetized by i.p. administration of xylazine (7 mg/kg) and ketamine (100 mg/kg), and were challenged with 100,000 pfu per eye of HSV-2 MS. Likewise, n=5 mice immunized with HSV-2 0ΔNLS (on Days 0 and 30) were anaesthetized and challenged at the same time with 100,000 pfu per eye of HSV-2 MS. HSV-2 MS shedding was monitored in these mice as described elsewhere [Halford et al., 2011].
High-binding EIA 96-well plates (Costar, Corning, N.Y.) were coated overnight (about 18 hours) at 4° C. with 100 μl per well of sodium carbonate buffer (pH 9.6) containing 0.2 μg per ml total HSV-2 antigens. Total HSV-2 antigen was isolated from HSV-2 infected Vero cells, as follows: five 100-mm dishes of Vero cells (8 million cells per dish) were inoculated with 3 pfu per cell of HSV-2 MS and incubated at 37° C. for 16 hours. Culture medium was aspirated from dishes, cells were rinsed with 5 ml PBS per dish, and cells were covered in 2 ml of sodium carbonate buffer (pH 9.6) per dish and frozen at −80° C. HSV-2 cell lysates were thawed and clarified by low-speed centrifugation to remove cell debris. The clarified supernatant had a protein concentration of 10 μg/ml, and was frozen in 0.2 ml aliquots. For each 96-well plate to be coated with HSV-2 antigen, a single aliquot of HSV-2 total antigen was diluted 1:50 (0.2 μg per ml) and used to coat a high-binding EIA plate. After overnight (about 18 hours) coating with total HSV-2 antigen, wells were blocked for 2 hours with 400 μl of 2% dry milk dissolved in phosphate-buffered saline (PBS)+0.02% Tween-20 (polyoxyethylene-20-sorbitan monolaurate), hereafter referred to as PBS-T buffer.
Each serum sample to be tested was diluted 2.5:250 in PBS+1% fetal bovine serum+0.02% Tween-20. After discarding blocking buffer from ELISA plates, duplicate 100-μl samples of diluted serum were added to total HSV-2 antigen-coated wells and were incubated for 2 hours.
ELISA plates were rinsed three times with an excess of PBS-T buffer prior to the addition of 100 μl secondary antibody diluted 1:1500 in PBS-T buffer; the secondary antibody was alkaline phosphatase-conjugated goat anti-mouse IgG Fc fragment (Rockland Immunochemicals, Gilbertsville, Pa.). After allowing 1 hour, secondary antibody was rinsed from plates seven times with PBS-T buffer, and 200 μl of p-nitrophenyl phosphate substrate (Sigma Chemical Co., St. Louis, Mo.) was added to each well, and colorimetric development (OD405) was measured after a 30-minute incubation at room temperature. The quantitative relationship between abundance of log (pan-HSV-2 IgG) (x) and OD405 (y) was defined using a 0.33-log dilution series of HSV-2 antiserum and a hyperbolic tangent-based standard curve (
as described elsewhere [Halford et al., 2010; Halford et al., 2005a].
Single-cell suspensions of a mixture of HSV-2+ cells and uninfected (UI) cells were generated, as follows. Twelve 100-mm dishes were seeded with 7×106 Vero cells per dish in complete DMEM, and six dishes were inoculated 6 hours later with 3 pfu per cell of HSV-2 MS. HSV-2+ Vero cells were harvested 12 hours after inoculation, and UI Vero cells were harvested in parallel at the same time.
Both cell populations were dispersed by aspirating culture medium, rinsing each dish with 5 ml PBS, and adding 2 ml PBS+5 mM ethylene diamine tetraacetic acid (EDTA) pH 8.0. It should be noted that PBS+5 mM EDTA was sufficient to cause Vero cells to lift and detach from one another without the use of trypsin. In the case of HSV-2+ cells, the PBS+5 mM EDTA solution was supplemented with 1 μM carboxyfluorescein diacetate, succinimidyl ester (CFSE; Anaspec, Fremont, Calif.) to label HSV-2+ cells with a green fluorophore.
Cells were incubated at room temperature on a rocking platform for 10 minutes until cells began to lift, and were then dispersed by trituration with the aid of a P-1000 pipettor. All dispersed UI cells were placed in a single 50-ml conical, and all dispersed HSV-2+ cells were placed in a second 50-ml conical, and both were centrifuged at 200× g for 5 minutes to pellet cells. Supernatants were decanted, cell pellets were resuspended in 12 ml PBS, and an equal volume of 2× fixative (7.4% formaldehyde+4% sucrose) was added.
Cells were incubated in 1× fixative for 20 minutes, centrifuged, and resuspended in 24 ml of 90% methanol to permeabilize the cells. After a 10 minute incubation, cells were centrifuged, resuspended in PBS+3% fetal bovine serum (PBS-F), and cell clumps were removed by passage through a 40 μM, nylon mesh cell strainer (BD Biosciences, San Jose, Calif.) followed by passage through a 25-gauge needle.
Cell density in single-cell suspensions of UI Vero cells and CFSE-labeled HSV-2+ cells was determined, and UI cells and HSV-2+ cells were combined in an approximate 2:1 ratio. Cells were centrifuged, resuspended at a concentration of 1.25×106 cells per ml in PBS-F-Ig block solution (i.e., PBS-F supplemented with 20 μg/ml each of donkey γ-globulin, goat γ-globulin, and human γ-globulin; Jackson Immunoresearch Laboratories, Inc., West Grove, Pa.).
Aliquots of UI and HSV-2+ cells (400 μl; 500,000 cells) were placed in 1.7 ml microfuge tubes, and 2 μl of 1:30 diluted serum was added to each cell suspension to achieve a net serum dilution of 1:6,000. Cells were incubated at room temperature for four hours on a LabQuake® rotisserie hybridization rotator to keep cells in suspension by rotation (Barnstead International, Dubuque, Iowa), and primary antibody was removed by two, sequential 1.25 ml PBS-F rinses, where a swinging bucket centrifuge was used to pellet cells and rinse supernatant was aspirated.
To enumerate the amount of IgG antibody bound to HSV-2+ versus UI cells, cells were incubated with a 1:1,000 dilution of APC-conjugated goat-anti mouse IgG Fc fragment or APC-conjugated donkey anti-guinea pig IgG (H+L) (Jackson Immunoresearch Laboratories, Inc.). After an 1-hour incubation, excess secondary antibody was removed by three, sequential 1.25 ml PBS-F rinses.
Cells were resuspended in a total volume of 0.2 ml PBS-F and analyzed by two-color flow cytometry in the FL1 and FL4 channels of an Accuri™ C6 flow cytometer using CFlow software (Accuri Cytometers, Inc., Ann Arbor, Mich.). On average, 125,000 events were recorded per sample; specifically, the flow cytometer was set to record events until 25,000 single HSV-2+ cells were included in the data set. Pan-HSV-2 IgG levels in each serum sample were calculated based on the difference in mean fluorescent intensity (ΔMFI) of 25,000 HSV-2+ cells versus ˜50,000 UI cells (
Unless otherwise specified, all values presented are the mean±standard error of the mean (sem) of replicate samples. Viral titers were determined by microtiter plaque assay and were statistically analyzed on a logarithmic scale (e.g., log [pfu/vagina]). Infectious virus was not detectable in some ocular or vaginal swabs of well-immunized animals. In such events, the sample was assigned a value of 8 pfu per swab (i.e., the lower-limit of detection of the assay), such that all samples could be analyzed on a logarithmic scale. The significance of differences in multiple group comparisons was compared by one-way analysis of variance (ANOVA) followed by Tukey's post hoc t-test using GraphPad Instat™ v3.10 software (GraphPad Software, Inc., La Jolla, Calif.). The significance of difference between two groups was performed using the “t-test assuming equal variances” function of Microsoft Excel. The significance of differences in survival frequency was determined by Fisher's Exact Test using freely available online software (Preacher and Briggs, 2001).
All data were statistically analyzed using logarithmic values. Linear regression analysis was performed by the method-of-least-squares using the “regression” analysis function in Microsoft Excel, and was used to calculate the goodness-of-fit (r2-value) and the probability (p) that the y-variable did not change as a function of the x-variable.
The coefficient-of-variance values reported in
Results
The presence of serum IgG antibodies that bind total HSV-2 antigens (pan-HSV-2 IgG) may be qualitatively tested by immunofluorescent staining of HSV-2 plaques in fixed Vero cell monolayers (
Suspensions of about30% HSV-2+ cells and about 70% UI cells were incubated with serum from naïve mice or HSV-2-immunized mice, and were fluorescently labeled with allophycocyanin (APC)-anti-mouse IgG Fc fragment secondary antibody. Antibody-labeled cells were analyzed by 2-color flow cytometry (
Mouse serum levels of “pan-HSV-2 IgG” antibody were estimated based on the difference in mean fluorescence intensity (ΔMFI) between HSV-2+ cells versus UI cells. The resulting ΔMFI-value associated with each serum sample was normalized to a “fold-increase above background” by the following calculation: ΔMFItest sample÷average ΔMFInaïve sera. When this approach was applied, sera from n=6 naïve mice were estimated to possess pan-HSV-2 IgG levels that were 1.0±0.2 times background (
Flow cytometry-based measurements of pan-HSV-2 IgG abundance were compared to two more traditional assays; namely, a HSV-2 neutralization assay and an antibody-capture ELISA. For this comparison, an antiserum dilution series was constructed by diluting mouse HSV-2 antiserum into naïve serum in 0.33-log increments spanning a 4,640-fold range. The use of naïve mouse serum as a diluent ensured that serum protein concentration (e.g., IgG) remained constant while HSV-2 specific antibodies were selectively diluted out in 0.33-log increments.
HSV-2 antiserum neutralized the infectivity of HSV-2 between dilutions of 1:21 and 1:1,000, and exhibited little to no neutralizing activity at 1:2, 150 or greater dilutions (
HSV-2 antibody abundance in the antiserum dilution series was evaluated by antibody-capture ELISA using lysates of HSV-2-infected Vero cells as a coating antigen. Antibody capture-ELISA yielded significant conversion of para-nitrophenylphosphate substrate (OD405) at serum dilutions between 1:100 and 1:100,000 (
HSV-2 antibody abundance in the antiserum dilution series was evaluated by a novel, flow cytometry-based assay (
All three assays yielded parallel estimates of pan-HSV-2 antibody abundance, but the flow cytometry-based assay was the most sensitive. Specifically, the flow assay had a lower limit-of-detection of 1:6,000,000 relative to HSV-2 antiserum, whereas the HSV-2 neutralization assay and antibody-capture ELISA had lower limits of 1:2, 100 and 1:100,000, respectively (
Pan-HSV-2 IgG Correlates with Protection Against Ocular HSV-2 Challenge in Mice
A retrospective analysis was performed on n=48 serum samples derived from mice used in a previously published ocular HSV-2 challenge experiment (
The design of the original experiment is reviewed. Five of 6 groups of mice were inoculated in the right eye with culture medium (naïve controls) or 100,000 pfu per right eye of the HSV-2 ICP0− mutant viruses HSV-2 0ΔNLS, 0Δ810, 0Δ254, or 0ΔRING (
Pre-challenge levels of pan-HSV-2 IgG in the immunization groups were determined and rank-ordered (
Regression analysis was applied to determine if pre-challenge pan-HSV-2 IgG levels correlated with reduced HSV-2 shedding after ocular challenge. The null hypothesis predicted that the best-fit linear regression model (y=b+mx) for these 48 matched datum pairs would have a slope (m) of 0 (
The goodness-of-fit (r2) value for the best-fit linear regression model was 0.65, which reflected the fact that the observed level of HSV-2 shedding in many mice did not conform perfectly to the quantity predicted by the equation y=3.35−0.56x (black line in
The frequency with which immunized mice survived ocular HSV-2 challenge was plotted as a function of pre-challenge pan-HSV-2 IgG levels (
A test was conducted to determine if flow cytometry measurement of pan-HSV-2 IgG levels offered any practical advantage relative to antibody-capture ELISA. To this end, the same mouse serum samples considered above were re-analyzed by antibody-capture ELISA using HSV-2-infected cell lysates as coating antigen. A 0.33-log dilution series of HSV-2 antiserum was used to precisely define the sigmoidal relationship between OD405 absorbance values and log (pan-HSV-2 IgG) levels using a hyperbolic tangent equation (
ELISA-based estimates of log (pan-HSV-2 IgG) correlated with decreased ocular HSV-2 shedding (black line in
The relative sensitivity of ELISA versus flow cytometry estimates of pan-HSV-2 IgG was graphically analyzed. ELISA estimates of log (pan-HSV-2 IgG) were plotted on the x-axis, whereas the corresponding flow cytometry estimates were plotted on the y-axis (
Pan-HSV-2 IgG Correlates with Protection Against Vaginal HSV-2 Challenge in Mice
A second, retrospective analysis was performed on mouse serum derived from a previously published experiment (
The design of the original experiment is reviewed. Mice were immunized on Days 0 and 30 in their right and left rear footpads, respectively, with: 1) culture medium (naïve controls); 2) 2.5 μg green fluorescent protein (GFP) adjuvanted with alum and 10 μg MPL; 3) 2.5 μg gD-2306t [Nicola et al., 1996] adjuvanted with alum and 10 μg MPL; 4) 106 pfu HSV-2 0ΔNLS; or 5) 106 pfu wild-type HSV-2 MS where acyclovir was used to limit the pathogenesis of the primary exposure to MS (
Pan-HSV-2 IgG levels in the immunization groups were determined and rank-ordered (
Regression analysis was applied to determine if pre-challenge pan-HSV-2 IgG levels correlated with reduced HSV-2 shedding after vaginal challenge. The null hypothesis predicted that the best-fit linear regression model for these 50 matched datum pairs would have a slope (m) of 0 (
Pan-HSV-2 IgG Correlates with Protection Against Vaginal HSV-2 Challenge in Guinea Pigs
A third, prospective analysis was performed to determine if pre-challenge pan-HSV-2 IgG levels varied in proportion to protection against HSV-2 in a species other than mice. To address this question, groups of n=5 guinea pigs were immunized on Days 0 and 30 in their right and left rear footpads, respectively, with: 1) culture medium (naïve); 2) 5 μg gD-2 adjuvanted with alum and 20 μg MPL; 3) 2×106 pfu HSV-2 0ΔNLS; or 4) 2×106 pfu of wild-type HSV-2 MS where acyclovir was used to restrict the pathogenesis of the primary exposure to MS (
Regression analysis was applied to determine if pre-challenge pan-HSV-2 IgG levels in guinea pigs correlated with reduced HSV-2 shedding after vaginal challenge. The null hypothesis predicted that the best-fit linear regression model for these n=19 matched datum pairs would have a slope (m) of 0 (
Regarding disease progression, naïve guinea pigs uniformly developed florid perivaginal disease and had to be sacrificed on or before Day 11 post-challenge (
The results of vaginal HSV-2 challenge experiments in mice and guinea pigs was compared (
High levels of pan-HSV-2 IgG antibodies correlated with robust protection against HSV-2 MS challenge in mice immunized with several live HSV-2 vaccines. A final experiment was conducted to determine if adoptive transfer of HSV-2 antiserum recapitulated the level of protection against HSV-2 observed in mice immunized with the HSV-2 0ΔNLS virus.
To this end, strain 129 mice (n=10) were immunized in their right and left rear footpads with 106 pfu of HSV-2 0ΔNLS on Days 0 and 30, respectively. On Day 85, five immunized mice were sacrificed to collect HSV-2 antiserum, and naïve serum was harvested at this time from age-matched controls. On Day 90, naïve mice received an adoptive transfer of 0.25 ml pooled naïve serum or HSV-2 antiserum (n=5 per group), and were then challenged with 100,000 pfu per eye of HSV-2 MS. Likewise, n=5 mice immunized with HSV-2 0ΔNLS were also challenged with 100,000 pfu per eye of HSV-2 MS.
Ocular shedding of HSV-2 MS was compared. On Day 1 post-challenge, mice treated with naïve serum shed an average 3,000 per eye of HSV-2 MS, whereas mice treated with HSV-2 antiserum shed an average 16-fold less HSV-2 and this difference was significant (
Adoptive transfer of HSV-2 antiserum delayed, but did not prevent, the progression of HSV-2-induced pathogenesis. Specifically, 100% of naïve serum-treated mice succumbed to ocular HSV-2 challenge on Days 7 or 8 post-challenge (
Two of 5 HSV-2 antiserum-treated mice survived ocular HSV-2 challenge, and as a group these mice survived for 19±5 days post-challenge (
Specifically, 100% of HSV-2 antiserum-treated mice developed overt periocular fur loss and disease between Days 10 and 14 post-challenge, and 60% of these mice succumbed to challenge (
Discussion
The current study demonstrates that bloodstream levels of pan-HSV-2 IgG antibody in vaccinated mice and guinea pigs correlated with protection against HSV-2. It has not been determined in this study if other components of the adaptive immune response would also correlate with vaccine-induced protection against HSV-2. For example, HSV-2-specific T-cell frequency [Laing et al., 2010; St leger et al., 2011; Posavad et al., 2010] or anti-HSV-2 IgA abundance in the vaginal mucosa [Tirabassi et al., 2011] may provide better correlates of immunity for a HSV-2 vaccine. However, it should be noted that the utility of a correlate of immunity is not dependent on its role in mediating protection. Rather, a correlate of immunity is a screening tool whose utility lies solely in its ability to gauge the magnitude of vaccine-induced protection against a microbial pathogen. It remains to be determined if pan-HSV-2 IgG levels would be useful in gauging HSV-2 vaccine efficacy in human clinical trials.
The relevance of humoral versus cell-mediated immunity in vaccine-induced protection against HSV-2 remains incompletely defined. What is evident from decades of studies dating back to Oakes, 1975 is that adoptively transferred anti-HSV antibodies or B-cells alone are not sufficient to prevent peripheral HSV-1 infection from progressing to fatal disease in immunodeficient nude or SCID mice [Nagafuchi et al., 1979; Halford et al., 2005b]; whereas, adoptively transferred T-cells are sufficient to allow immunodeficient animals to survive peripheral infection with low virulence strains of HSV-1 [Nagafuchi et al., 1979; Halford et al., 2005b]. Moreover, T-cells play a direct role in controlling HSV-1 and HSV-2 infections in sensory ganglia [Divito et al., 2006; Khanna et al., 2003; Knickelbein et al., 2008; Theil et al., 2003; Liu et al., 2000; Simmons and Tscharke, 1992; Zhu et al., 2007]. Thus, vaccine-induced protection against HSV-2 will almost certainly be dependent upon the T-cell response to HSV-2 antigens [Koelle and Corey, 2008; Johnston et al., 2012; Laing et al., 2012; Dudek and Knipe, 2006; Morrison, 2002].
Complete, vaccine-induced protection against HSV-2 genital herpes lesions will most likely be dependent upon a balanced B-cell (antibody) and T-cell response to HSV-2's antigens. Two lines of evidence support this hypothesis. First, SCID mice reconstituted with both B- and T-cells control HSV-1 infection significantly more rapidly than SCID mice reconstituted with T-cells alone (
Against this background, a logical function for anti-HSV-2 antibodies would be to serve as the first line of adaptive immune defense that triggers the pro-inflammatory events (e.g., complement cascade) that promote the rapid recruitment of T-cells into virus-infected tissues at the portal of HSV-2 entry (e.g., the vagina).
Previous attempts to identify correlates of immunity to HSV-2 have focused on immune responses to the immunogens under study; namely, gB and/or gD [Shlapobersky et al., 2012; Bernstein et al., 2010; Bourne et al., 2005; Bernstein, 2005; Bourne et al., 2003; Khodai et al., 2011; Bernstein et al., 2011; Natuk et al., 2006; Chentoufi et al., 2010]. These approaches do not consider HSV-2's full complement of antigens. At least 20 viral proteins are known targets of the human B- and T-cell response to HSV-2 [Hosken et al., 2006; Laing et al., 2010; Gilman et al., 1981]. Such glycoprotein-focused studies have not adequately considered that viral antigens other than gB-2 and gD-2 may also contribute to immunity to HSV-2.
Glycoprotein-centric correlates of immunity suggest that gB-2 and/or gD-2 subunit vaccines should be sufficient to prevent HSV-2 genital herpes in humans [Bernstein et al., 2010; Bourne et al., 2005; Bernstein, 2005; Bourne et al., 2003]. This prediction has not been borne out by the data from human clinical trials spanning the last 23 years [Belshe et al., 2012; Stanberry et al., 2002; Straus et al., 1997; Corey et al., 1999; Straus et al., 1994; Mertz et al., 1990]. The pan-HSV-2 IgG metric is a more realistic correlate of immunity because it weighs the relative abundance of IgG antibodies against all of HSV-2's antigens, and thus is not contingent upon an assumption that the immune response to 1 or 2 specific proteins will necessarily provide an accurate gauge of immunity to HSV-2.
The results of the current two cell study demonstrate that immunization with a gD-2 vaccine elicits a significant pan-HSV-2 IgG antibody response and a significant reduction in vaginal HSV-2 shedding (
Several HSV-2 vaccine-challenge studies have attempted to measure protection against HSV-2 in terms of disease scores, survival, or weight gain after HSV-2 challenge [Khodai et al., 2011; McClements et al., 1996; Pyles et al., 2002]. Non-parametric statistics (i.e., disease and survival) or tangential parameters (i.e., weight gain) are likely weak measures of the primary variable under study, protection against HSV-2. In contrast, reductions in HSV-2 challenge virus shedding are a precise measure of protection against HSV-2, and vary over an about 500-fold range. The use of this robust measure of protection allowed linear regression analysis to be applied in the current study to determine if increased pan-HSV-2 IgG levels (x) correlated with protection against HSV-2 (y), as gauged by reductions in ocular or vaginal HSV-2 shedding (
Linear regression analysis is one of the most powerful statistical tools available to determine if a correlation exists between two variables. It is believed that the current study is the first to apply regression analysis to detect a correlation between a parameter of the adaptive immune response and protection against HSV-2. This innovation was critical to the success of the current study. The ability to detect a correlation between two parameters by regression analysis is dependent on three variables. Variable 1 is the number of matched x, y datum pairs in the data set. Variable 2 is the precision of measurements of the x- and y-variables. Variable 3 is the range of Δx and Δy over which a correlation may be observed.
Regarding Variable 2, the flow cytometric assay introduced herein improved the precision and sensitivity of estimates of pan-HSV-2 IgG levels (
Regarding Variable 3, if the current study had focused exclusively on one vaccine modality such as the HSV-2 0ΔNLS vaccine, then the observed range of pan-HSV-2 IgG levels (Δx) would have been too narrow (about 5-fold) to detect a meaningful correlation (
The current studies illustrate that the disclosed demonstrates that in vaccinated mice and guinea pigs, the pan-HSV-2 IgG antibody response to several vaccines varies in proportion to protection against HSV-2. It is possible that this same approach may provide a useful screening tool in human clinical trials of a HSV-2 vaccine. Based on the results, a HSV-2 vaccine formulation that elicits the most potent and durable pan-HSV-2 IgG antibody response in humans should elicit the greatest protection against HSV-2 genital herpes. However, the proposed utility of pan-HSV-2 IgG as a potential correlate of vaccine-induced protection against HSV-2 remains to be tested in humans. Therefore, it will be of interest to test this prediction in coming years, and determine if pan-HSV-2 IgG levels provide a useful correlate of vaccine-induced protection against HSV-2 in humans.
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This application claims priority from application Ser. No. 61/917,584 that was filed on Dec. 18, 2013, whose disclosures are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US14/70915 | 12/17/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61917584 | Dec 2013 | US |
