Antiviral CD19+CD27+ Memory B Cells Are Associated with Protection from Recurrent Asymptomatic Ocular Herpesvirus Infection

Abstract

Reactivation of herpes simplex virus 1 (HSV-1) from latently infected neurons of the trigeminal ganglia (TG) leads to blinding recurrent herpetic disease in symptomatic (SYMP) individuals. Although the role of T cells in herpes immunity seen in asymptomatic (ASYMP) individuals is heavily explored, the role of B cells is less investigated. In the present study, we evaluated whether B cells are associated with protective immunity against recurrent ocular herpes. The frequencies of circulating HSV-specific memory B cells and of memory follicular helper T cells (CD4⁺ T_fh cells), which help B cells produce antibodies, were compared between HSV-1-infected SYMP and ASYMP individuals. The levels of IgG/IgA and neutralizing antibodies were compared in SYMP and ASYMP individuals. We found that (i) the ASYMP individuals had increased frequencies of HSV-specific CD19⁺CD27⁺ memory B cells, and (ii) high frequencies of HSV-specific switched IgG⁺CD19⁺CD27⁺ memory B cells detected in ASYMP individuals were directly proportional to high frequencies of CD45R0⁺CXCR5⁺CD4⁺ memory T_fh cells. However, no differences were detected in the level of HSV-specific IgG/IgA antibodies in SYMP and ASYMP individuals. Using the UV-B-induced HSV-1 reactivation mouse model, we found increased frequencies of HSV-specific antibody-secreting plasma HSV-1 gD⁺CD138⁺ B cells within the TG and circulation of ASYMP mice compared to those of SYMP mice. In contrast, no significant differences in the frequencies of B cells were found in the cornea, spleen, and bone-marrow. Our findings suggest that circulating antibody-producing HSV-specific memory B cells recruited locally to the TG may contribute to protection from symptomatic recurrent ocular herpes. IMPORTANCE Reactivation of herpes simplex virus 1 (HSV-1) from latently infected neurons of the trigeminal ganglia (TG) leads to blinding recurrent herpetic disease in symptomatic (SYMP) individuals. Although the role of T cells in herpes immunity against blinding recurrent herpetic disease is heavily explored, the role of B cells is less investigated. In the present study, we found that in both asymptomatic (ASYMP) individuals and ASYMP mice, there were increased frequencies of HSV-specific memory B cells that were directly proportional to high frequencies of memory T_fh cells. Moreover, following UV-B-induced reactivation, we found increased frequencies of HSV-specific antibody-secreting plasma B cells within the TG and circulation of ASYMP mice compared to those of SYMP mice. Our findings suggest that circulating antibody-producing HSV-specific memory B cells recruited locally to the TG may contribute to protection from recurrent ocular herpes.

Keywords: B memory cells; asymptomatic herpes; ocular herpes; plasma cells; virus-specific B cell.

FIG 1

Circulating HSV-1 gD-specific memory B cell profile in asymptomatic and symptomatic herpes-infected individuals. PBMCs from asymptomatic and symptomatic HSV-1-infected individuals were stained for HSV-1 gD antigen-specific memory B cells. PBMCs were also treated with IL-2 and resiquimod for 5 days for polyclonal stimulation of memory B cells in plasma cells (antibody secreting cells [ASC]). The stimulated cells were then analyzed for HSV-1 gD-specific IgG/IgA ASC and total IgG/IgA ASC by ELISPOT. (A) FACS plot showing the gating strategy for HSV-1-specific memory B cells (CD19⁺CD27⁺ B cells) in PBMCs of asymptomatic (ASYMP) (Left) and symptomatic (SYMP) (Right) HSV-1-infected individuals. (B) Graph showing percentage of HSV-1-specific memory B cells (CD19⁺CD27⁺ B cells) in PBMCs of ASYMP and SYMP HSV-1-infected individuals. (C) Representative ELISPOT images for anti-HSV-1 IgG ASC (Left, top) and anti-HSV-1 IgA ASC (Left, bottom) from PBMCs of ASYMP and SYMP HSV-1-infected individuals and HSV-1 uninfected individuals (polyclonally stimulated for maturation of memory B cells to ASC). (D) Graph showing anti-HSV-1 IgG (Left, top) and IgA (Left, bottom) ASC; total IgG (Right, top) and total IgA (Right, bottom) ASC from PBMCs of ASYMP and SYMP HSV-1-infected individuals and HSV-1 uninfected individuals. Statistical analysis was done using Student's t test. NS, not significant.

FIG 2

Correlation of HSV-1-specific memory B cell and memory T follicular helper T cells in herpes-infected individuals. PBMCs from asymptomatic and symptomatic HSV-1-infected individuals were stained for switched memory B cells (CD19⁺CD27⁺IgD⁻) and HSV-1 gD-specific switched memory B cells. (A) FACS plot showing gating strategy for HSV-1-specific switched memory B cells (CD19⁺CD27⁺ B cells) in PBMCs of ASYMP (Left) and SYMP (Right) HSV-1-infected individuals. (B) Graph showing percentage of HSV-1-specific switched memory B cells (CD19⁺CD27⁺IgD⁻ B cells) in PBMC of ASYMP and SYMP HSV-1-infected individuals. (C) FACS plot showing gating strategy for T follicular helper cells (T_fh) (CD3⁺CD4⁺CXCR5⁺ T cells) and T follicular helper memory cells (T_fh memory) (CD3⁺CD4⁺CD45R0⁺CXCR5⁺ T cells) in PBMC of ASYMP (Left) and SYMP (Right) HSV-1-infected individuals. (B) Graph showing correlation of the percentage of HSV-1-specific switched memory B cells (CD19⁺CD27⁺IgD⁻ B cells) and T follicular helper memory cells (T_fh memory) (CD3⁺CD4⁺CD45R0⁺CXCR5⁺ T cells) in PBMCs of HSV-1-infected individuals. Statistical analysis was done using Student's t test. NS, not significant.

FIG 3

Anti-HSV-1 IgG and IgA antibody levels and neutralizing antibody titer in ASYMP and SYMP herpes-infected individuals. Serum samples from asymptomatic and symptomatic HSV-1-infected individuals were used for an estimation of anti-HSV-1 gD antibodies by ELISA. (A) Graphs showing levels of anti-HSV-1 IgG antibody level (Left) and anti-HSV-1 IgA antibody level (Left) in serum of ASYMP and SYMP herpes-infected individuals. (A) Graph showing anti-HSV-1 neutralizing antibody titer in serum of ASYMP and SYMP herpes-infected individuals. Statistical analysis was done using Student's t test. NS, not significant.

FIG 4

Memory B cell profile in PBMCs, spleen, and bone marrow of ASYMP and SYMP HSV-1-reactivated mice. For this experiment, the corneas of B6 mice were infected with HSV-1 McKrae (1 × 10⁶ PFU/eye) by scarification, and virus reactivation was provoked at day 35 p.i. in latently infected mice using 60 s corneal UV-B irradiation. At day 6 postreactivation, mice were categorized into ASYMP or SYMP depending on disease occurrence. ASYMP and SYMP mice were euthanized and immune cells from peripheral blood, spleen, and bone marrow were collected for flow cytometry staining for memory B cells. (A) FACS plot showing representative plots for memory B cells (B220⁺CD73⁺ B cells) and the subsets (B220⁺CD80⁺PD-L2⁺ B cells) in spleen (Left) and bone marrow (Right) of ASYMP and SYMP infected mice. (B) Graph showing percentage of memory B cells (B220⁺CD73⁺ B cells) and the subsets (B220⁺CD73⁺CD80⁺PD-L2⁺ B cells) in spleen (left) and bone marrow (Right) of ASYMP and SYMP infected mice. (C) Representative ELISPOT images for anti-HSV-1 IgG ASC (Left) and total IgG ASC (Right) from PBMCs, spleen, and bone marrow from ASYMP and SYMP infected mice (polyclonally stimulated with mouse polyclonal B cell activator (IL-2 and resiquimod) for maturation of memory B cells to ASC). Graph showing anti-HSV-1 IgG (Left) and total IgG (Right) ASC from PBMCs of ASYMP, SYMP HSV-1-infected mice. Statistical analysis was done using Student's t test. NS, not significant.

FIG 5

Plasma cell profile in PBMC, spleen, bone marrow, and TG of ASYMP and SYMP HSV-1-infected mice. For this experiment, the corneas of B6 mice (n = 6) were infected with HSV-1 McKrae (1 × 10⁶ PFU/eye) by scarification, and virus reactivation was provoked at day 35 p.i. in latently infected mice using 60 s corneal UV-B irradiation. At day 6 postreactivation, mice were categorized into ASYMP or SYMP depending on disease occurrence. ASYMP and SYMP mice were euthanized, and immune cells from peripheral blood, spleen, and bone marrow were collected for flow cytometry staining for plasma cells. (A) Representative plots for plasma B cells (CD138⁺ B cells) in PBMC, spleen, BM, and TG (Right) of ASYMP and SYMP mice is shown. Graph showing percentage of plasma B cells (CD138⁺ B cells) in PBMC, spleen, PBMC, and TG (Right) of ASYMP and SYMP mice. (B) Representative FACS plot showing HSV-1-specific plasma B cells (HSV-1 gD⁺CD138⁺ B cells) in PBMC, spleen, PBMC, and TG (Left) of ASYMP and SYMP mice. Graph showing percentage of HSV-1-specific plasma B cells (HSV-1 gD⁺CD138⁺ B cells) in PBMC, spleen, PBMC, and TG (Right) of ASYMP and SYMP mice.

FIG 6

T follicular helper (T_fh) and T follicular helper memory (T_fh memory) cell profile in spleen of ASYMP and SYMP HSV-1-infected mice. B6 mice (n = 6) were infected with HSV-1 McKrae (1 × 10⁶ PFU/eye) by scarification, and at day 35 p.i., reactivation was done by 60 s corneal UV-B irradiation. Mice were categorized as ASYMP/SYMP and euthanized at day 6 postreactivation. Spleen cells were collected for flow cytometry staining of T_fh (CD3⁺CD4⁺CXCR5⁺PD-1⁺ cells) and T_fh memory (CD3⁺CD4⁺CD44⁺CXCR5⁺PD-1⁺ cells). (A) Representative FACS plots for T_fh cells (CD3⁺CD4⁺CXCR5⁺PD-1⁺ cells) are shown in the top panel, and T_fh memory cells (CD3⁺CD4⁺CD44⁺CXCR5⁺PD-1⁺ cells) are shown in bottom panels for ASYMP (Left) and SYMP (Right) infected mice. (A) Graph showing percentage of T_fh cells (top) and T_fh memory cells (bottom) in spleen of ASYMP and SYMP infected mice is shown.

FIG 7

B cell ligand and cytokine level in serum of asymptomatic and symptomatic herpes. Asymptomatic (n = 20) and symptomatic (n = 20) patient serum samples were assayed for cytokines involved in B cell development. Graph showing serum level of APRIL (B cell proliferation inducing ligand), BAFF (B cell-activating factor), and IL-10 (B regulatory cell cytokine) (A) as well as IL-21 (involved in expansion and differentiation of plasma cells), IL-7 (B cell development cytokine), and TNF-β (B cell development cytokine) by Luminex. Statistical analysis was done using Student's t test. NS, not significant.