Human Antibodies against Herpes Simplex Virus 2 Glycoprotein G Do Not Neutralize but Mediate Antibody-Dependent Cellular Cytotoxicity

Herpes simplex virus 2 (HSV-2) is a sexually transmitted infection affecting 491 million individuals globally. Consequently, there is a great need for both prophylactic and therapeutic vaccines. Unfortunately, several vaccine clinical trials, primarily employing the glycoprotein D of HSV-2 (gD-2), have failed. The immune protection conferred by human anti-HSV-2 antibodies in genital infection and disease remains elusive. It is well-known that gD-2 elicits cross-reactive neutralizing antibodies, i.e., anti-gD-2 antibodies recognize gD in HSV-1 (gD-1). In contrast, anti-glycoprotein G in HSV-2 (mgG-2) antibodies are exclusively type-specific for HSV-2. In this study, truncated versions of gD-2 and mgG-2 were recombinantly produced in mammalian cells and used for the purification of anti-gD-2 and anti-mgG-2 antibodies from the serum of five HSV-2-infected subjects, creating a pool of purified antibodies. These antibody pools were utilized as standards together with purified mgG-2 and gD-2 antigens in ELISA to quantitatively estimate and compare the levels of cross-reactive anti-gD-1 and anti-gD-2 antibodies, as well as anti-mgG-2 antibodies in sera from HSV-1+2-, HSV-2-, and HSV-1-infected subjects. The median concentration of anti-mgG-2 antibodies was five times lower in HSV-1+2-infected subjects as compared with cross-reactive anti-gD-1 and anti-gD-2 antibodies, and three times lower in HSV-2 infected subjects as compared with anti-gD-2 antibodies. The pool of purified anti-gD-2 antibodies presented neutralization activity at low concentrations, while the pool of purified anti-mgG-2 antibodies did not. Instead, these anti-mgG-2 antibodies mediated antibody-dependent cellular cytotoxicity (ADCC) by human granulocytes, monocytes, and NK-cells, but displayed no complement-dependent cytotoxicity. These findings indicate that antibodies to mgG-2 in HSV-2-infected subjects are present at low concentrations but mediate the killing of infected cells via ADCC rather than by neutralizing free viral particles. We, and others, speculate that Fc-receptor mediated antibody functions such as ADCC following HSV-2 vaccination may serve as a better marker of protection correlate instead of neutralizing activity. In an mgG-2 therapeutic vaccine, our findings of low levels of anti-mgG-2 antibodies in HSV-2-infected subjects may suggest an opportunity to enhance the immune responses against mgG-2. In a prophylactic HSV-2 mgG-2 vaccine, a possible interference in cross-reactive immune responses in already infected HSV-1 subjects can be circumvented.


Introduction
Herpes simplex virus 2 (HSV-2) infects the genital mucosa and establishes a life-long infection in the sensory ganglia.Following a primary infection, HSV-2 may reactivate resulting in genital lesions or more commonly asymptomatic shedding of the virus.HSV-2 is wide-spread, with an estimated 491.5 million people aged 15-49 years infected globally, giving a worldwide prevalence of 13.2% in 2016 [1].The same study estimated the annual incidence to be 23.9 million infections.HSV-2 infection also lead to recurrent meningitis, severe neonatal infection, and significantly increases the risk of acquiring HIV [2].Given this epidemiological situation there is a need for the development of both therapeutic and prophylactic vaccines.However, several clinical trials have yielded discouraging results.
The primary target in these trials has been HSV-2 glycoprotein D (gD-2) due to its essential role for cell entry and the ability to induce neutralizing antibodies in HSV-infected subjects.For instance, Chiron's adjuvanted gB-2/gD-2 prophylactic vaccine showed high levels of neutralizing antibodies but had an overall vaccine efficacy of only 9% [3].Glaxo-SmithKline (GSK) also tested an adjuvanted gD-2 in a prophylactic clinical vaccine trial in HSV-1-and HSV-2-negative women, which showed no protection against HSV-2 infection but did provide partial protection against HSV-1-induced disease and infection [4].This protection correlated with the level of anti-gD-2 antibodies in ELISA but not with the cellular responses [5].Additionally, serum from gD-2-vaccinated subjects also showed neutralizing activity against HSV-1 [6][7][8].
In a recent report, the antibody profiles were investigated after vaccination with an HSV-2 replication-defect vaccine HSV529 in HSV-1-and HSV-2-negative vaccine recipients and compared with the antibody responses in naturally HSV-2-infected subjects.Using a random peptide display library and serum antibodies, the two most enriched epitopes were located within the defined immunodominant epitope region of mgG-2, both after vaccination and after natural HSV-2 infection [16].
In an effort to produce an HSV-2 vaccine we have shown that mgG-2 together with adjuvant induced protection against genital and neurological disease in a mouse vaccination genital challenge model where anti-mgG-2 antibodies presented antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) [17].A recombinantly produced truncated version of the mgG-2 (EXCT4-mgG-2) also induced protection in the mouse vaccination model with different adjuvants [18].However, although promising results have been described for several animal vaccination models, the results have only been partially predictive of the outcome in clinical trials.
We recently described that mgG-2 promotes virus release from the surface of infected cells by interaction with glycosaminoglycan mimicking oligosaccharides [19].The function of anti-EXCT4-mgG-2 antibodies in human HSV-2 infection remains unknown.In this study, we first estimated the concentrations of cross-reactive anti-gD-1, anti-gD-2, and anti-mgG-2 antibodies in HSV-1+2-, HSV-2-, and HSV-1-infected subjects.Second, we estimated the levels of neutralizing activity of pools of purified anti-gD-2 and anti-mgG-2 antibodies, and finally, we evaluated whether purified human anti-EXCT4-mgG-2 antibodies present ADCC and/or CDC activity.

Sera from HSV-1-Infected and HSV-Negative Subjects
Ten consecutive samples were collected from the pool of sera submitted to the Clinical Virological Laboratory at Sahlgrenska University Hospital for routine testing (Table 1).Samples were selected based on an end-point titer of ≥3200 to an in-house HSV-1/2 cross-reactive sodium deoxycholate-solubilized HSV-1-infected membrane preparation in ELISA [20].Additionally, serological diagnosis of HSV-1 infection was confirmed by HerpeSelect1 ELISA IgG, and by CLIA LiaisonXL HSV1 IgG.Sera from the HSV-1-infected subjects were negative to Helix pomatia lectin purified mgG-2, negative in HerpeSelect2 ELISA IgG, and negative in LiaisonXL HSV2 IgG.Information on clinical symptoms of the HSV-1-infection was not available, but none of the sera were analyzed specifically because of symptoms associated with genital HSV infection.Sera from three HSV-1-and HSV-2negative subjects were included as controls and were negative to the HSV-1/2 in-house antigens and in the HSV-1 and HSV-2 type-specific assays.

Production of EXCT4-mgG-2 and Coupling to an Immunosorbent Column
The EXCT4-mgG-2 construct codes for the entire extracellular region (EX) including residues alanine 345 -aspartic acid 649 , the entire cytoplasmic (C) region including residues alanine 671 -aspartic acid 698 , and four residues in the transmembrane region (T4) including residues valine 667 -alanine 670 (HSV-2 strain 333, GenBank accession number LS480640).EXCT4-mgG-2 was expressed and purified as described earlier [18].Briefly, the coding sequence was subcloned into pEE12.4using Chinese hamster ovary (CHO)-K1 cells provided with the GS expression system (Lonza, Cambridge, United Kingdom).The protein was purified over an anion exchange column Hiprep16/10QFF (Pharmacia, Uppsala, Sweden) followed by a Sephacryl S-400 high resolution 16/60 gel filtration column (Pharmacia, Uppsala, Sweden).The target protein was detected with an apparent molecular weight of 80-85 kDa in Western blot and the purity was estimated to be ≥80% based on SDS-PAGE and Imperial blue staining [18].The antigen was coupled to an epoxy-activated Sepharose 6B column (Cytiva, Life Sciences, Uppsala, Sweden) as described [15].

Production of gD-2 and Coupling to an Immunosorbent Column
The entire extracellular region of gD-2 (amino acids 26-342) was produced by recombinant technique in CHO-K1 cells as described [21], kindly provided by A.M. Harandi.The protein was purified using a HiTrap Chelating HP column (GE Healthcare, Freiburg, Germany).The target protein was detected with an apparent molecular weight of 42 kDa in Western blot and the purity was estimated to be 95% based on SDS-PAGE and Imperial blue staining.The gD-2 antigen was coupled to a cyanogen bromide-activated Sepharose 4B gel according to manufacturer's instructions (Cytiva Life Sciences, Uppsala, Sweden).

Production of Pools of Purified Human Antibodies Used for Standard Curves and for Functional Assays
To create a standard for polyclonal antibody reactivity to EXCT4-mgG-2 and to gD-2 antigens, five sera from symptomatically HSV-2-infected subjects were used.To increase the yield of antibodies and include different antibody specificities, sera were selected presenting high titers of anti-mgG-2 antibodies to the in-house Helix pomatia lectin purified mgG-2, and high titers of anti-gD-2 antibodies to an in-house HSV-1/2 cross-reactive sodium deoxycholate-solubilized HSV-1-infected membrane preparation (titers ≥ 6400).The serum samples were classified as HSV-2 positive and HSV-1 negative by Helix pomatia lectin purified mgG-2 ELISA, HerpeSelect1 IgG, HerpeSelect2 IgG ELISA (Focus Technologies, Cypress, CA, USA), and by CLIA LiaisonXL HSV1 IgG and HSV2 IgG (Diasorin S.p.A, Saluggia, Italy).The serum samples (1.2 mL each) were pooled and diluted 1:4 in Trisbuffered saline (TBS) to a total volume of 24 mL.The EXCT4-mgG-2 and the gD-2 affinity columns were equilibrated with 30 mL TBS, and 6 mL of diluted serum samples were applied to each of the columns and recirculated for 1 h.After washing, antibodies were eluted with 0.1 M glycine-HCl pH 2.8 and neutralized with 1 M Tris-HCl pH 8.0.Purified samples were thereafter pooled.IgG-antibody concentrations were measured with a Human IgG ELISA kit (Ramcom A/S, Stockholm, Sweden).Flow-through from the EXCT4-mgG-2 immunosorbent column, used for purification of anti-EXCT4-mgG-2 antibodies, was unreactive to in-house Helix pomatia lectin purified mgG-2, and flow-through from the gD-2 immunosorbent column, used for purification of anti-gD-2 antibodies, was negative for inhouse HSV-1/2 cross-reactive sodium deoxycholate-solubilized HSV-1-infected membrane preparation in ELISA (OD values < 0.2), suggesting a complete acquisition of antigenspecific antibodies.
Purified antigens were coated onto Maxisorp microtiter plates (Nalge Nunc International Corporation, Rochester, NY, USA) at a concentration of 1.0 µg/mL in carbonate buffer (pH 9.6) at 4 • C. The in-house cross-reactive HSV-1/2 antigen was coated at a 1:1000 dilution, and the mgG-2 peptide at a concentration of 5 µg/mL.Prior to use, the plates were washed and blocked with 2% skim milk in phosphate-buffered saline (PBS) for 30 min at 37 • C.
Pooled purified anti-EXCT4-mgG-2 and anti-gD-2 antibodies were evaluated at a 1:100 dilution resulting in a final concentration of 5 µg/mL.An HSV-negative serum was used as control, defining the cut-off as the mean of four wells plus two standard deviations (SD).
For the evaluation of cross-reactivity of IgG antibodies in other herpes viruses to the EXCT4-mgG-2 and gD-2 antigens, seven HSV-negative sera containing IgG antibodies to cytomegalovirus, Epstein-Barr virus (Alinity, Abbot Laboratories, Chicago, IL, USA), human herpes virus 6 (Vidia spol.s r.o., Vestec, Czech Republic), and recombinantly produced glycoprotein E of varicella zoster virus in an in-house ELISA [23], were selected for evaluation.These sera were tested at a 1:100 dilution in ELISA, and the cut-off was defined using the same HSV-negative control serum as described for the purified antigens above.
From the endpoint titer created by the standard curves, an optical density (OD) interval of ±0.3 was defined from the inflection point to ensure linearity between the OD value and the concentration.When necessary for individual serum samples, the dilution was adjusted so the OD value was within the defined OD interval.
Standards of the pools of purified antibodies were included on each plate in two-fold dilutions.Each serum was evaluated simultaneously for both antigens.The plates were incubated for 1.5 h at 37 • C.After washing, alkaline phosphates labeled goat anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at a 1:1000 dilution served as the conjugate, and P5994-25T tablets (Sigma-Aldrich, Stockholm, Sweden) dissolved in a DEA buffer served as the substrate.OD values were read in a spectrophotometer at 405 nm, and the concentrations of antibodies were calculated from the standard curves.The cut-off for positive samples was defined as 2 µg/mL.Intra-and inter-assay coefficients of variation in the ELISA were calculated using two sera from HSV-2-infected subjects in five positions on the same Maxisorp microtiter plate and on three separate plates at different occasions.

Neutralization Assay without or with Complement
GMK-AH1 cells were employed for the viral plaque reduction assay.Pools of purified anti-EXCT4-mgG-2 or anti-gD-2 antibodies, with two-fold dilutions of antibodies at a start dilution of 10 µg/mL, were evaluated without or with complement.An HSV-negative serum was used as complement source at a final concentration of 2.5%.Antibody samples were mixed with 150 HSV-2 (strain 333) plaque-forming units and incubated for 1 h at 37 • C and transferred to the cells.A methylcellulose overlay was applied, and the plaques were counted after 72 h.An inactivated HSV-negative serum and an inactivated HSV-2-positive serum were used as controls at 1:100 dilution.The lowest concentrations of antibodies which reduced the number of plaques by 50% (NT 50 ) were calculated based on the inactivated HSV-negative serum control.

Antibody-Dependent Cellular Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC)
Blood granulocytes were purified from buffy coats from a blood donor using dextran sedimentation and a Ficoll-Paque gradient centrifugation, resulting in 93-97% purity [24].Erythrocytes were disrupted by hypotonic lysis, and granulocytes were washed and resuspended in a Krebs-Ringer solution with glucose and kept on ice.Monocytes and NK-cells were prepared from peripheral blood mononuclear cells (PBMC) using the BD Imag™ Human Monocyte and NK Cell Enrichment Sets (BD Biosciences, Stockholm, Sweden).
BHK-21-cells were infected with the HSV-2 strain 333 at a multiplicity of infection of four and used as target cells for both ADCC and CDC.After an 18 h infection, cells were dispersed using EDTA-buffer supplemented with 0.025% trypsin and washed four times in TBS supplemented with 2% inactivated fetal calf serum (IFC).After washing, cells were diluted in Eagle's minimal essential medium supplemented with 10% IFC (EMEM).Cell counts were adjusted to 10 6 cells/mL and labeled with Calcein AM (Thermo Fisher Scientific, Uppsala, Sweden) [25].Cells were washed in EMEM and adjusted to 10 5 cells/mL for ADCC and to 2 × 10 5 cells/mL for CDC.An HSV-2-positive serum, an HSV-negative serum, and a pool of purified anti-EXCT4-mgG-2 antibodies were diluted in EMEM.
For the ADCC-assay granulocytes, monocytes, NK-cells, and PBMC were used as effector cells, with an HSV-2-positive serum as the control.Assays were performed in a Ubottom 96-well microtiter plate (Nunc, Roskilde, Denmark).Serum and the pool of purified anti-EXCT4-mgG-2 antibodies, which was used for standards in the ELISA and for the neutralization assay, were tested in duplicate, where each well contained 50 µL target cells mixed with 50 µL diluted HSV-2-positive serum (positive control) or anti-EXCT4-mgG-2 antibodies.Optimal effector:target (E:T) ratios were set to 100:1 for granulocytes, 50:1 for PBMC and monocytes, and 5:1 for NK-cells.The cut-off for positive signal for pool 1 of purified anti-EXCT4-mgG-2 antibodies was set to 5% specific cytotoxicity based on the mean value of the fluorescence intensity from E:T wells + 2 SD.
Fresh PBMCs were used in the preparation of the monocytes and NK-cells and derived from the same donor.Two different blood donors were used for preparation of granulocytes.Each run included four wells containing only E:T cells and medium, and three wells containing target cells and 2% Triton-100 (max release).Cells and antibodies were incubated at 37 • C in 5% CO 2 for 6 h.
For the CDC assay, the target cells, the HSV-2-positive serum, and purified anti-EXCT4-mgG-2 antibodies, were mixed as described above.Three wells with target cells plus complement and three wells with only target cells for maximum release were included.Plates were incubated for 1 h in 5% CO 2 followed by addition of 10% human HSV-negative serum as source of complement and finally incubated for another 2 h.Additionally, Helix pomatia lectin purified anti-mgG-2 antibodies [15] from five HSV-2-positive sera were used.
For both ADCC and CDC assays, 75 µL of the supernatants from each well were transferred to a 96-well flat bottom microtiter plate (Nunc, Roskilde, Denmark).Fluorescence intensity was measured using FLUOstar Omega (BMG Labtech, Ortenberg, Germany), with excitation of 485 nm and emission of 520 nm.The mean values from two wells (serum and antibodies), from three wells (maximum release), and from four wells (E:T) were used for calculation of ADCC.In a similar way, the mean value from two wells (serum and antibodies), and from three wells (target cells plus complement and maximum release) were used for calculation of CDC.

Statistics
The distribution of the antibody concentrations in sera from HSV-1+2-and HSV-2infected subjects was tested using the D'Agostino-Pearson and the Shapiro-Wilk normality tests (GraphPad Prism, Gothenburg University, Sweden).As the concentrations of anti-EXCT4-mgG-2 and anti-gD-2 antibodies in the HSV-2-infected cohort were not normally distributed, the nonparametric two-tailed Mann-Whitney test was used for comparison.A p value of <0.05 was considered significant.

Specificity of Purified anti-EXCT4-mgG-2 and anti-gD-2 Antibodies
Recombinant EXCT4-mgG-2 and gD-2 antigens were produced in CHO-K1 cells and coupled to an immunosorbent column for purification of antibodies from the serum of five HSV-2-infected subjects.Specificity of pooled purified antibodies and seven HSV-negative sera containing IgG to other herpes viruses were evaluated to EXCT4-mgG-2 and gD-2 antigens with in-house ELISAs and in the commercial type-specific assays (Table 2).
Table 2. Reactivity of purified anti-EXCT4-mgG-2, anti-gD-2 antibodies (Abs), and HSV-negative sera.Pools of purified Abs, produced from serum of five HSV-2 infected subjects, and HSV-negative sera, containing IgG antibodies against other herpes viruses, were evaluated to different antigens.Reactivity was evaluated with in-house ELISA and in the commercial HSV-1 and HSV-2 type-specific assays (HerpeSelect and LiaisonXL).For the in-house ELISA, the cut-off was defined as the mean value of four wells + 2 SD using an HSV-negative serum, and reactivity was classified as: − negative (OD values < 0.3), + clearly positive (OD values between 2.5-3.0), and ++ highly positive (OD values ≥ 3.5).Results from the commercial assays were classified as negative or positive.
The pool of anti-EXCT4-mgG-2 antibodies demonstrated specific reactivity to Helix pomatia lectin purified mgG-2 antigen and to the mgG-2 peptide, positive in HerpeSelect2 IgG (Focus Technologies, Cypress, CA, USA), and positive in CLIA LiaisonXL HSV2 IgG (Diasorin S.p.A, Saluggia, Italy), while negative to an HSV-1/2 cross-reactive sodium deoxycholate-solubilized HSV-1-infected membrane preparation, negative in HerpeSelect1 IgG, negative in CLIA LiaisonXL HSV1 IgG, and negative to the gB-2 antigen and to two gC-2 antigens, as well as to the recombinantly produced gD-2 antigen (Table 2).As a positive control, the pool of anti-EXCT4-mgG-2 antibodies presented a mean OD value of 2.7 to purified EXCT4-mgG-2 antigen.Similar reactivity was observed to Helix pomatia lection purified mgG-2 antigen and to the mgG-2 peptide and classified as clearly positive with mean OD values between 2.5 and 3.0.
The pool of anti-gD-2 antibodies was reactive to the cross-reactive HSV-1 antigen, but negative in the commercial type-specific assays (gG1 and gG2 antigens), negative to Helix pomatia lectin purified mgG-2 antigen, negative to the mgG-2 peptide, and negative to the gB-2 and gC-2 antigens (Table 2).As a positive control, the pool of anti-gD-2 antibodies presented a mean OD value of 3.6 to purified gD-2 antigen.
The results obtained from testing seven HSV-negative sera, which contained IgG antibodies to cytomegalovirus, Epstein-Barr virus, human herpes virus 6, and varicellazoster virus, showed OD values below 0.2 for specified antigens (Table 2).Based on these findings, it can be concluded that the purified anti-EXCT4-mgG-2 and anti-gD-2 antibodies exhibit high specificity.

Neutralization Activity
The concentrations of pools of purified anti-EXCT4-mgG-2 and anti-gD-2 antibodies, which reduced the number of HSV-2 plaques by 50% without and with complement, are listed in Table 3.For the anti-EXCT4-mgG-2 antibodies no neutralization activity was observed (>10 µg/mL).The pool of anti-gD-2 antibodies presented neutralization activity at all concentrations evaluated both without and with complement.A-C) Quantification of antibodies in clinical sera.Anti-EXCT4-mgG-2 antibodies, c reactive anti-gD-1 and gD-2 antibodies in sera of HSV-1+2-infected, HSV-2-infected, HSV-1-infe and HSV-negative subjects were evaluated using purified EXCT4-mgG-2 and gD-2 antigen ELISA.Pools of purified anti-EXCT4-mgG-2 and anti-gD-2 antibodies with known concentra were included for generation of standard curves.For statistical analyses the Mann-Whitney parametric test was used.The median values are marked with a horizontal line.

Neutralization Activity
The concentrations of pools of purified anti-EXCT4-mgG-2 and anti-gD-2 antibo which reduced the number of HSV-2 plaques by 50% without and with complement listed in Table 3.For the anti-EXCT4-mgG-2 antibodies no neutralization activity was  A-C) Quantification of antibodies in clinical sera.Anti-EXCT4-mgG-2 antibodies, crossreactive anti-gD-1 and gD-2 antibodies in sera of HSV-1+2-infected, HSV-2-infected, HSV-1-infected and HSV-negative subjects were evaluated using purified EXCT4-mgG-2 and gD-2 antigens in ELISA.Pools of purified anti-EXCT4-mgG-2 and anti-gD-2 antibodies with known concentrations were included for generation of standard curves.For statistical analyses the Mann-Whitney nonparametric test was used.The median values are marked with a horizontal line.Table 3. Viral plaque reduction assay in GMK-AH1 cells of pools of purified anti-EXCT4-mgG-2 and anti-gD-2 antibodies (Abs) from five symptomatically HSV-2-infected subjects.HSV-2 (150 plaque forming units, PFUs) was mixed with two-fold dilutions of antibodies with a start dilution of 10 µg/mL without (C − ) or with complement (C + ).An HSV-negative serum was used as complement source at a final concentration of 2.5%.The mean PFUs from two experiments without complement and from three experiments with complement are indicated.An inactivated HSV-negative control serum, a 1:100 dilution, presented a mean value of 75 PFUs without complement, and 70 PFUs with complement, and used for calculation of NT 50% .An inactivated HSV-2-positive control serum, at a 1:100 dilution, presented no PFUs both without and with complement, and used as positive control.

ADCC
Purified anti-EXCT4-mgG-2 antibodies were titrated in two-fold serial dilution, starting at 9 µg/mL for granulocytes, and at 3 µg/mL for monocytes and NK-cells.The cut-off for granulocytes was set to a concentration of 1.8 µg/mL of purified antibodies, the cut-off for monocytes was 0.015 µg/mL and 0.05 µg/mL for NK-cells (Figure 2A-C).For the highest antibody concentrations evaluated, lower ADCC was observed for monocytes and NK-cells, suggesting a pro-zone phenomenon.
An HSV-2-positive serum and an HSV-negative serum for granulocytes, were used at a 1:400 dilution and evaluated for the three effector cells.For granulocytes the mean value for the positive control was 18% (SD, 9), and no ADCC was observed with the HSV-negative control.The mean value of the positive control for monocytes was 25% (SD, 8.4), and for NK-cells 20% (SD, 3), (Figure 2D).

CDC
The same HSV-2-positive serum used in the ADCC-assay was used in a 1:400 dilution and presented 30% specific cell lysis, Figure 2F.The cut-off for positive CDC was defined for ADCC as the mean value for target + complement +2 SD and set to 10%.Pool 1 of the purified anti-EXCT4-mgG-2 antibodies and Helix pomatia lectin purified anti-mgG-2 antibodies [15] were evaluated at the concentrations of 6 µg/mL, 1.5 µg/mL, and 0.19 µg/mL, and presented no CDC.The rationale to also use Helix pomatia lectin purified anti-mgG-2 antibodies was that mouse antibodies, after vaccination [17], presented CDC.Similarly, no CDC was detected, Figure 2F.

Discussion
The role of anti-HSV antibodies in immunity is often debated, with focus on whether HSV-2 antibodies can protect against HSV-2 infection.In a prophylactic clinical trial using adjuvanted gD-2 antigen (GSK), the vaccine did not protect against HSV-2-induced genital disease or infection [4].However, anti-gD-2 antibodies, but not T cell responses, partially protected against genital HSV-1 disease and infection.These data challenge the notion that the cell-mediated immunity is most important in controlling genital HSV-1/2 disease and infection [26].
In this study, the EXCT4-mgG-2 and the gD-2 proteins were produced with recombinant technique in CHO-K1 cells and used for the purification of anti-EXCT4-mgG-2 and anti-gD-2 antibodies from symptomatically HSV-2-infected subjects.Using standard curves of a pool of purified antibodies, we were able to estimate the concentrations of specific antibodies in sera of HSV-infected and in HSV-negative subjects.A limitation with the study is that we only purified anti-gD-2 antibodies from HSV-2-infected subjects.We can therefore not estimate the levels of type-specific anti-gD-1 antibodies in HSV-1+2-and HSV-1-infected subjects.Such an estimation could be possible if also gD-1 antigen is produced, and anti-gD-1 antibodies are purified.Thus, in this study, the total amounts of anti-gD-1 antibodies in sera of HSV-1+2-and HSV-1-infected subjects are probably underestimated, (Figure 1B).
The concentrations of anti-EXCT4-mgG-2 antibodies were significantly lower as compared with cross-reactive anti-gD-1 and anti-gD-2 antibodies in sera of HSV-1+2-and HSV-2-infected subjects.The low levels of anti-EXCT4-mgG-2 in serum were somewhat surprising as Wang et al. identified that the most enriched antibody-binding epitopes were localized within mgG-2 both in HSV-1-and HSV-2-negative subjects after HSV529 vaccination, as well as for HSV-2-naturally-infected subjects [16].However, using peptides for epitope mapping is known to be biased towards linear epitopes [27].The results may be explained by the fact that mgG-2 contains linear epitopes which are easily detected with peptide display libraries [16,28], using pepscan analysis with peptides coupled to a cellulose support [15], or with branched peptides [14].Furthermore, antibody-binding to full-length mgG-2 was possible to block with peptides comprising the predicted epitopes in a competitive ELISA which support that the mapped epitopes are linear [15].The presence of anti-mgG-2-antibodies has been used for several years as a type-specific marker of infection in serodiagnosis of HSV-2 infection.These observations were confirmed in this study, as sera of HSV-1-infected subjects presented no or negligible binding to EXCT4-mgG-2 antigen (Figure 1A).
Although mgG-2 is an HSV-2 type-specific protein and used in serodiagnosis of HSV-2 infection, this antigen has diagnostic limitations.For instance, false negative reactivity using a single mgG-2 based assay has been described in recurrent HSV-2-PCR-positive subjects [29].These findings may at least partly be explained by the low concentrations of anti-EXCT4-mgG-2 antibodies.Approximately 10% of sera of HSV-1+2-and HSV-2-infected subjects presented concentrations of anti-EXCT4-mgG-2 antibodies below 10 µg/mL.Another possibility might be that not all anti-EXCT4-mgG-2 antibodies were captured to the immunosorbent column.In such a case, the pool of anti-EXCT4-mgG-2 antibodies, used as a standard for estimation of the amounts of antibodies in sera, could be affected.However, the flow-through from the column was negative to a different mgG-2 antigen (Helix pomatia lectin purified mgG-2), supporting a complete acquisition of antibodies.
We measured neutralization activity of purified antibodies as a functional aspect of anti-HSV-2 antibodies.Earlier studies have shown that HSV neutralization antibodies are mostly directed to gD, and in some extent to gB, for both symptomatic and for asymptomatic HSV-infected subjects [10,11].A pool of anti-gD-2 antibodies presented neutralization activity both without and with complement at low concentrations (Table 3), while a pool of anti-EXCT4-mgG-2 antibodies presented no neutralization activity.Similarly, a lack of neutralization activity was shown in anti-mgG-2 monoclonal antibodies [30,31], in hyperimmune serum after vaccination with mgG-2 in mice [17], as well as in rabbit anti-mgG-2 hyperimmune serum [32].
We have described earlier that anti-mgG-2 antibodies, elicited after vaccination with Helix pomatia lectin purified full-length mgG-2, in a mouse genital challenge model present CDC and ADCC by macrophages and NK-cells.We show in this study for the first time that purified human anti-EXCT4-mgG-2 antibodies present ADCC by granulocytes, monocytes, NK-cells, and with PBMC.A limitation of the study is that the relative contribution of the anti-EXCT4-mgG-2 antibodies to the total ADCC activity cannot be estimated as the concentrations and specificities of other HSV-2 antibodies exerting ADCC in HSV-2 sera were not determined.Such an evaluation needs further studies, for instance, by a comparison of purified anti-gD-1, anti-gD-2 antibodies and antibodies to other HSV proteins from sera

Figure 1 .
Figure 1.(A-C) Quantification of antibodies in clinical sera.Anti-EXCT4-mgG-2 antibodies, c reactive anti-gD-1 and gD-2 antibodies in sera of HSV-1+2-infected, HSV-2-infected, HSV-1-infe and HSV-negative subjects were evaluated using purified EXCT4-mgG-2 and gD-2 antigen ELISA.Pools of purified anti-EXCT4-mgG-2 and anti-gD-2 antibodies with known concentra were included for generation of standard curves.For statistical analyses the Mann-Whitney parametric test was used.The median values are marked with a horizontal line.

Figure 1 .
Figure 1.(A-C) Quantification of antibodies in clinical sera.Anti-EXCT4-mgG-2 antibodies, crossreactive anti-gD-1 and gD-2 antibodies in sera of HSV-1+2-infected, HSV-2-infected, HSV-1-infected and HSV-negative subjects were evaluated using purified EXCT4-mgG-2 and gD-2 antigens in ELISA.Pools of purified anti-EXCT4-mgG-2 and anti-gD-2 antibodies with known concentrations were included for generation of standard curves.For statistical analyses the Mann-Whitney nonparametric test was used.The median values are marked with a horizontal line.

Figure 2 .
Figure 2. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of purified anti-EXCT4-mgG-2 antibodies (Abs).Pool 1 and pool 2 of anti-EXCT4-mgG-2 antibodies were each purified from five different HSV-2-infected sera.Pool 1 antibodies were evaluated for ADCC by human granulocytes (A), monocytes (B), and NK-cells (C).The dashed horizontal lines mark the cut-off which was defined as mean reactivity of target and effector cells without antibodies plus 2 SD.The dashed vertical lines in (A-C) define the cut-off concentrations of antibodies.An HSV-2-positive serum was diluted 1:400 and used as positive control for the different effector cells including an HSV-negative control serum for granulocytes (D).Total ADCC from peripheral blood mononuclear cells (PBMC), with an HSV-2-positive and with an HSV-negative serum, at 1:400 dilutions, and for comparison pool 1 and pool 2 of purified anti-EXTC4-mgG-2 antibodies, used at concentration of 3 µg/mL, were evaluated (E).Mean values from pool 1 were calculated from duplicate wells in two experiments (A-D) and from three experiments (E), except for pool 2 antibodies which was evaluated in one experiment.An HSV-2-positive serum, and pool 1 of purified anti-EXCT4-mgG-2 antibodies, and Helix pomatia lectin (HPA) purified anti-mgG-2 antibodies, in three concentrations, were evaluated in CDC in two experiments (F).The bars represent mean values +/− SD (A-C) and +SD (D-F).