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. 2015 Feb 6;10(2):e0116091.
doi: 10.1371/journal.pone.0116091. eCollection 2015.

Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are Among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

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Free PMC article

Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are Among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

Joshua J Geltz et al. PLoS One. .
Free PMC article

Abstract

Virion glycoproteins such as glycoprotein D (gD) are believed to be the dominant antigens of herpes simplex virus 2 (HSV-2). We have observed that mice immunized with a live HSV-2 ICP0- mutant virus, HSV-2 0ΔNLS, are 10 to 100 times better protected against genital herpes than mice immunized with a HSV-2 gD subunit vaccine (PLoS ONE 6:e17748). In light of these results, we sought to determine which viral proteins were the dominant antibody-generators (antigens) of the live HSV-2 0ΔNLS vaccine. Western blot analyses indicated the live HSV-2 0ΔNLS vaccine elicited an IgG antibody response against 9 or more viral proteins. Many antibodies were directed against infected-cell proteins of >100 kDa in size, and only 10 ± 5% of antibodies were directed against gD. Immunoprecipitation (IP) of total HSV-2 antigen with 0ΔNLS antiserum pulled down 19 viral proteins. Mass spectrometry suggested 44% of immunoprecipitated viral peptides were derived from two HSV-2 infected cells proteins, RR-1 and ICP8, whereas only 14% of immunoprecipitated peptides were derived from HSV-2's thirteen glycoproteins. Collectively, the results suggest the immune response to the live HSV-2 0ΔNLS vaccine includes antibodies specific for infected cell proteins, capsid proteins, tegument proteins, and glycoproteins. This increased breadth of antibody-generating proteins may contribute to the live HSV-2 vaccine's capacity to elicit superior protection against genital herpes relative to a gD subunit vaccine.

Conflict of interest statement

Competing Interests: William Halford is a co-author on United States Patent Number 8,802,109, which describes the uses of herpes simplex virus mutant ICP0 in the design of a live-attenuated HSV-2 vaccine strain. This does not alter the authors’ adherence to all PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Western blot analysis to screen for candidate antibody-generating proteins of the live HSV-2 0ΔNLS vaccine.
Representative Western blots of (UI) uninfected Vero cells or cells inoculated with 2.5 pfu/cell of HSV-1 KOS or HSV-2 MS incubated with 1:20,000 dilutions of serum from (A) mock-immunized mice (naïve) or mice immunized with (B) gD-2 + alum/MPL adjuvant, (C) HSV-2 0ΔNLS (ICP0 -) virus, or (D) an acyclovir-restrained HSV-2 MS infection (MS+ACV). Red diamonds (1–9) denote the positions of HSV-2 proteins most commonly targeted by mouse IgG antibodies, and the open arrow denotes the MW of gD-2.
Figure 2
Figure 2. Virus-specific antibody binding to target HSV-2 proteins as a function of MW: gD-2 versus HSV-2 0ΔNLS antiserum.
Three-dimensional line graphs summarize the relative intensity of IgG binding to HSV-2 proteins (y-axis) as a function of protein MW (x-axis) based on (A) sera from n = 5 gD-2-immunized mice (primary data shown in S1 Fig.) or (B) sera from n = 5 HSV-2 0ΔNLS-immunized mice (primary data shown in S1 Fig.). Red diamonds (1–9) denote positions of HSV-2 proteins commonly targeted by mouse IgG antibodies, and open arrows denote the MW of gD-2.
Figure 3
Figure 3. Cycloheximide-release analysis segregates candidate HSV-2 0ΔNLS antigens by IE, E, or L expression kinetics.
(A and B) Western blot of Vero cells that were uninfected (UI) or were inoculated with 5 pfu per cell of HSV-2 0ΔRING or wild-type HSV-2 MS. Virus-infected cells were treated with cycloheximide (CHX) for 10 hours followed by 7 hours of treatment with actinomycin D (ActD; lanes 1 and 6); acyclovir (ACV; lanes 2 and 7); or no drug (VEH; vehicle; lanes 3 and 8). HSV-2 0ΔRING and HSV-2 MS-infected cells that were not drug-treated (lanes 4 and 9) were included as a control, and were harvested at 17 hours p.i. (A) Two-color analysis of HSV-2 proteins and GFP-tagged ICP0 (expressed by HSV-2 0ΔRING) labeled with 1:20,000 mouse α-0ΔNLS antiserum (red signal) and 1:5,000 rabbit α-GFP antiserum (green signal). (B) Grayscale representation of mouse IgG (in 0ΔNLS antiserum) binding to HSV-2 proteins.
Figure 4
Figure 4. Western blot analysis of purified HSV-2 virions segregates candidate HSV-2 0ΔNLS antigens into infected cell proteins versus virion proteins.
Representative Western blots of (UI) uninfected Vero cells, total HSV-2-infected cell proteins (MOI = 2.5), or sucrose-gradient-purified HSV-2 virions incubated with 1:20,000 dilutions of serum from (A) a mock-immunized mouse (naïve) or mice immunized with (B) gD-2 + alum/MPL adjuvant, (C) HSV-2 0ΔNLS, or (D) an acyclovir-restrained HSV-2 MS infection (MS+ACV). Red diamonds (1–9) denote the positions of viral proteins in total HSV-2-infected cell samples most commonly targeted by mouse IgG antibodies.
Figure 5
Figure 5. Immunoprecipitation-mass spectrometry (IP-mass spec) analysis as a tool to screen antibody specificities in HSV-2 0ΔNLS antiserum.
(A-B) IP-mass spec experiment #1. Uninfected Vero cell proteins (UI Ag) or HSV-2 MS-infected cell proteins (HSV-2 Ag) were resuspended in a NP40-based buffer containing 150 mM NaCl and were incubated with 2% naïve mouse serum or 2% mouse 0ΔNLS-antiserum for 2 hours followed by overnight incubation with Protein A/G agarose beads. (A) Coomassie-blue stained polyacrylamide gel of immunoprecipitates formed by HSV-2 Ag + mouse 0ΔNLS antiserum versus three negative-control immunoprecipitation reactions. Black arrows denote three protein species pulled down by 0ΔNLS antiserum that were not present in controls. (B) Identity of proteins excised from the gel (panel A), as determined by MALDI-TOF mass spectrometry. (C-D). IP-mass spec experiment #2. (C) Coomassie-blue stained polyacrylamide gel of immunoprecipitates formed by HSV-2 MS-infected cell proteins (HSV-2 Ag) following incubation with 1% mouse 0ΔNLS-antiserum and Protein A/G agarose beads. The entire lane of the gel was analyzed by MALDI-TOF mass spectrometry after being cut into 18 equivalent sized slices (denoted by boxes 1–18); slice-by-slice mass spectrometry identification results for the five most abundant HSV-2 proteins are shown in S3 Fig. (D) Number of peptide matches per positively identified HSV-2 protein. A total of 14,729 peptides were identified by mass spectrometry as being derived from 19 HSV-2 proteins that met our inclusion criteria, which were that a “positive identification” should (1) contribute >1% to the total pool of positive HSV-2 peptides (i.e., >147 peptides); (2) have >30% of its peptides recovered from 3 consecutive gel slices at the protein’s expected MW (e.g., S3 Fig.); (3) have >25% of its protein sequence represented were detected by the mass spectrometer, and should (4) yield 10 or more unique peptides. Seventy-two percent of the positive HSV-2 peptides in immunoprecipitates were derived from the 5 most dominant proteins identified; namely, RR-1, ICP8, VP1–2, VP5, and gB.
Figure 6
Figure 6. Two-color Western blot: mouse HSV-2 0ΔNLS antiserum versus rabbit antisera against HSV-2 glycoproteins B, C, and D.
Western blots of (UI) uninfected Vero cells, total HSV-2-infected cell proteins (MOI = 2.5), or sucrose-gradient-purified HSV-2 virions were incubated with a 1:20,000 dilution of mouse HSV-2 0ΔNLS antiserum (green signal = mouse IgG) and 1:10,000 dilutions of rabbit antisera specific for (A) HSV-2 gB, (B) HSV-2 gC, or (C) HSV-2 gD (red signal = rabbit IgG).
Figure 7
Figure 7. Western blot analysis of HSV gD-antigen-deletion mutants: effect on antibody-binding targets of gD-2 antiserum versus HSV-2 0ΔNLS antiserum.
Western blots of (UI) uninfected Vero cells or cells inoculated with 5 pfu/cell of HSV-1 KOS, a HSV-1 ΔgD virus (KOS-gD6), HSV-2 MS, or a HSV-2 ΔgD virus (HSV-2 ΔgD-BAC) incubated with 1:20,000 dilutions of serum from mice immunized with (A) gD-2 + alum/MPL adjuvant or (B) HSV-2 0ΔNLS. Red diamonds (1–9) denote the positions of viral proteins most commonly targeted by mouse IgG antibodies.
Figure 8
Figure 8. Flow cytometric analysis of HSV gD-antigen-deletion mutants: effect on antibody-binding targets of gD-2 antiserum versus HSV-2 0ΔNLS antiserum.
Three-population cytometric analysis comparing IgG antibody-binding to a mixture of uninfected (UI) Vero cells versus Vero cells inoculated with HSV-2 ΔgD-BAC or HSV-2 MS. Each cell population was dispersed, differentially labeled with 0, 0.45, or 6 μM CFSE, fixed, permeabilized, and combined for antibody staining and flow cytometry. (A—D) Mixed populations of test cells were incubated with 1:6,000 dilutions of serum from (A) a naïve mouse or a mouse immunized with (B) gD-2 + alum/MPL, (C) HSV-2 0ΔNLS, or (D) an acyclovir-restrained HSV-2 MS infection (MS+ACV). Pan-HSV-2 IgG binding (y-axes) was detected using APC-labeled goat anti-mouse IgG secondary, and was measured in three gates (dashed columns) at the center of the CFSE-negative, CFSE lo, and CFSE hi populations to compare IgG binding to UI cells, HSV-2 ΔgD+ cells, versus HSV-2 MS+ cells, respectively. E. Mean ± sem of pan-HSV-2 IgG levels in n = 5 mice per immunization group, as measured by i. the increase in mean fluorescent intensity (ΔMFI) of IgG bound to HSV-2 MS+ cells relative to UI cells, and ii. the ΔMFI of IgG bound to ΔgD+ cells relative to UI cells.
Figure 9
Figure 9. Cell lines expressing epitope-tagged HSV-2 antigens: relative abundance of gD-, ICP8-, RR-1-, and VP5-specific antibodies in HSV-2 0ΔNLS antiserum.
Western blots of (UI) uninfected Vero cells, cells inoculated with 2.5 pfu/cell of HSV-2 MS, or Vero cell lines that stably express the following, recombinant HSV-2 proteins: gD-FLAG, ICP8-FLAG, RR-1-FLAG, or VP5-FLAG incubated with 1:20,000 dilutions of serum from (A) naïve mice or (C, E) mice immunized with (C) gD-2 + alum/MPL adjuvant or (E) HSV-2 0ΔNLS. Following incubation with mouse serum, blots were rinsed and re-probed with mouse α-FLAG antibody to validate the relative amount of FLAG-tagged HSV-2 protein on each blot. (B, D, F) Normalized amount of IgG antibody bound to recombinant gD, FLAG, RR-1, or VP5 on blots incubated with (B) naïve mouse serum (n = 3), (D) gD-2 antiserum (n = 3), or (F) 0ΔNLS antiserum (n = 6). Levels of bound IgG antibody were normalized to account for blot-to-blot variance in the relative amount of each target based on the relative amount of α-FLAG antibody that bound each recombinant protein. In panel F, ‘**’ denotes that IgG antibody in 0ΔNLS antiserum bound RR-1 to significantly greater levels than gD, ICP8, or VP5 (p<0.001, one-way ANOVA and Tukey’s post-hoc-t-test).

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