Viral Persistence Induces Antibody Inflation without Altering Antibody Avidity

ABSTRACT

Antibodies are implicated in long-term immunity against numerous pathogens, and because of this property, antibody induction is the basis for many vaccines. Little is known about the influence of viral persistence on the evolving antibody response. Here, we examined the characteristics of antibody responses to persistent infection by employing the prototypic betaherpesvirus family member cytomegalovirus (CMV) in experimental mouse models. During the course of infection, mouse CMV (MCMV)-specific IgM and IgG responses are elicited; however, IgG levels gradually inflate in the persistent phase of infection while IgM levels are stably maintained. Whereas CD27-CD70 interactions are dispensable, the CD28/B7 costimulatory pathway is critical for the class switching of MCMV-specific IgM-to-IgG B cell responses, which corresponds to the CD28/B7-dependent formation of CD4⁺ T follicular helper cells (T_FH) and germinal center (GC) B cells. Furthermore, the initial viral inoculum dose dictates the height of the antibody levels during IgG antibody inflation and relates to the induction of long-lived plasma cells and memory B cells. Antibody avidity nonetheless is not altered after the establishment of viral persistence and occurs independently of the inoculum doses. However, repetitive challenge with intact viral particles, accompanied by increased GC reactivity, promotes the development of high-avidity IgG responses with neutralizing capacity. These insights can be used for the rational design of CMV-based vaccines aimed at inducing antibody responses.

IMPORTANCE Antibodies provide long-term protection to different pathogens. However, how antibody responses develop during persistent virus infection is not entirely clear. Here, we characterize factors that influence the virus-specific antibody response to persistent CMV. This study describes that during persistent infection, CMV-specific IgM antibody levels are stably maintained while IgG2b and IgG2c levels gradually inflate over time. In contrast, the IgG avidity remains similar after the establishment of viral persistence. The induction of T follicular helper cells and GC B cells requires CD4⁺ T cell help and CD28/B7 costimulation signals and is essential for the development of CMV-specific IgG antibody responses. Furthermore, neutralizing CMV-specific antibodies appear to develop late after infection, yet the neutralizing capacity can be improved upon repetitive viral challenge that is associated with increased GC reactivity. The results described here could inform the use of CMV-based vaccines and may help to understand how our immune system copes with this persistent virus.

INTRODUCTION

The maintenance of long-lived humoral responses after infection and vaccination is attributed to both long-lived plasma cells that continuously produce antibodies and to memory B cells that are able to form antibody-secreting cells after reexposure (1, 2). Antibodies can protect against numerous pathogens by direct neutralization and/or by supporting effector functions of immune cells (1, 3). Upon activation, B cells initially excrete antigen-specific IgM antibodies. This is followed by antibody isotype switching and affinity maturation when B cells receive the appropriate signals, including help signals by CD4⁺ T cells in germinal center (GC) reactions (4). During acute viral infections antibody levels increase, followed by a gradual decline once the antigen has disappeared. In the case of the appropriate induction of B cells leading to the generation of long-lived plasma cells, antibody levels eventually become stable and can mediate protection for many years.

Whereas memory B cells have self-renewal capacity in an antigen-dependent manner, long-lived plasma cells are thought to survive for decades (2). In the case of antigen persistence, as is the case in chronic infections, one could argue that antigenic boosting effects humoral immunity. How this impacts the kinetics of antibody levels and antibody avidity maturation is, however, largely unknown. Recently, vaccines based on persistent viruses such as cytomegalovirus have shown their value by inducing either long-lasting effector-memory T cell responses (5–7) or protective antibodies (8, 9), but many particulars of such vaccines remain to be determined.

To gain more insight into the determinants of antibody responses that develop during persistent virus infection or after challenge with vaccines based on persistent viruses, we used mouse cytomegalovirus (MCMV), a prototypic member of the betaherpesvirus family. We found that similar to so-called inflationary MCMV-specific T cell responses, which gradually increase to high frequencies (10), MCMV-specific IgG antibody levels inflate in the persistent phase of infection. MCMV-specific IgM antibody levels, however, remain relatively stable. Remarkably, this IgG antibody inflation is not accompanied by changes in antibody avidity after a single inoculum despite viral persistence. Instead, antibody avidity was amplified by repetitive challenge with virus and correlated with elevated GC reactivity. Moreover, we show that operational GC reactions and T follicular helper cell (T_FH) formation require the costimulatory CD28/B7 pathway while CD27/CD70 interactions are not critical.

MATERIALS AND METHODS

Mice and infection.

C57BL/6 mice were purchased from Charles River. Cd70^−/− (11), Cd80/86^−/− (12), and Cd70/80/86^−/− (13) mice, all on a C57BL/6 background, were bred in-house. Mice between 8 and 12 weeks of age were infected intraperitoneally (i.p.) with the indicated doses of MCMV-Smith, obtained from the American Type Culture Collection (Manassas, VA). Stocks were derived from salivary glands of infected BALB/c mice as described elsewhere (14). The viral load in mice was determined by quantitative PCR as described previously (15), and data are normalized to results for β-actin. All animals were maintained under specific-pathogen-free conditions at the animal facility in the Leiden University Medical Center (LUMC). All animal experiments were approved by the Animal Experiments Committee of LUMC (reference numbers 10227, 12006, and 13029) and performed according to the recommendations and guidelines set by LUMC and by the Dutch Experiments on Animals Act that serves as the implementation of the guidelines on the protection of experimental animals by the Council of Europe.

In vivo antibody use.

To deplete CD4⁺ T cells, mice received 150 μg of CD4-depleting antibody (GK1.5) i.p. prior to infection. The depletion of CD4⁺ T cells was maintained by the administration of 100 μg GK1.5 antibody once a week. For the blockade of costimulatory interactions during acute MCMV infection, mice received either 150 μg blocking CD70 antibody (clone FR70) or a combination of 200 μg blocking CD80 (B7.1) antibody (clone 16-10A1) and 200 μg blocking CD86 (B7.2) antibody (clone GL1) i.p. on days −1, 0, and 3 of MCMV infection.

Antibody detection by ELISA and antibody avidity assay.

Blood of mice was collected retro-orbitally. Upon brief centrifugation, serum was collected and stored at −20°C until further use. MCMV-specific antibody levels were determined by enzyme-linked immunosorbent assay (ELISA) as described previously (15), with minor alterations. In short, 96-well plates (Nunc MaxiSorp) were coated overnight at 4°C with tissue culture-derived MCMV-Smith in bicarbonate buffer (pH 9.6). Plates were incubated for 1 h at 37°C with blocking buffer (phosphate-buffered saline [PBS] containing 5% milk powder), followed by subsequent incubation with serum samples (diluted in PBS containing 1% milk powder) for 1 h at 37°C. Plates were washed with PBS containing 0.05% Tween, after which horseradish peroxidase (HRP)-conjugated IgM, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibodies (diluted in PBS with 1% milk powder) were incubated for 1 h at 37°C. To develop the plates, 50 μl of TMB (3,3′,5,5′-tetramethylbenzidine) (Sigma-Aldrich) was added to each well and incubated for 15 min at room temperature. To stop the reaction, 50 μl of stop solution (1 M H₂SO₄) was added. Plates were measured within 5 min at 450 nm using a microplate reader (model 680; Bio-Rad). To determine the avidity of the MCMV-specific antibodies, a serum dilution was used at which responses showed an optical density at 450 nm of 1. Plates were incubated with increasing concentrations of sodium thiocyanate (NaSCN) for 15 min, followed by washing with PBS containing 0.05% Tween and incubation with HRP-conjugated antibodies. The avidity of the MCMV-specific antibodies was determined by the ratio of the amount of antibodies bound after elution with different concentrations of NaSCN relative to the amount of antigen bound in the absence of NaSCN (16).

Quantification of antibody-secreting cells.

Multiscreen-HA 96-well plates (Millipore) were coated overnight with MCMV in PBS at 4°C, subsequently washed with PBS, and blocked for 1 h with Iscove's modified Dulbecco's medium containing 8% fetal calf serum (FCS) at 37°C. A total of 2 × 10⁵ splenocytes or 8 × 10⁵ bone marrow cells of MCMV-infected mice were added per well and incubated for 5 h at 37°C. Plates were washed with PBS containing 0.05% Tween 20, followed by incubation overnight with HRP-conjugated IgG2c antibodies at 4°C. After subsequent washing, spots were visualized using TMB (Mabtech), and the reaction was stopped with tap water. The total number of antibody-secreting cells per organ was determined by dividing the absolute numbers of the organ by the amount of plated cells and multiplying this number by the amount of spots that were counted per well. CMV-specific memory B cells were determined as described previously (17), with some modifications. In short, a concentration range from 1.8 × 10⁵ to 2 × 10⁴ splenocytes of MCMV-infected mice were cultured in a flat-bottom 96-well plate for 6 days in the presence of irradiated feeder splenocytes of naive mice (1,200 rad), 0.4 μg/ml lipopolysaccharide (LPS), and 1 μg/ml phytohemagglutinin (PHA). After 6 days of culture, cells were washed, transferred to enzyme-linked immunosorbent spot (ELISPOT) assay plates, and developed as described above.

Antibody neutralization assay.

Different dilutions of serum of MCMV-infected mice were incubated for 45 min with 50 PFU of MCMV-Smith at room temperature. The virus-serum inocula subsequently were added to monolayers of M2-10B4 cells in 48-well plates. Cells were incubated for 1 h at 37°C, after which inocula were removed and cells covered in carboxymethyl cellulose-containing medium. After 5 days of incubation, cells were fixed with 25% formaldehyde and plaques were visualized using crystal violet solution.

Flow cytometry.

Splenocytes were obtained by mincing the tissue through a 70-μm nylon cell strainer (BD). Erythrocytes were lysed in a hypotonic ammonium chloride buffer. The antigen-specific T cell response was determined by MHC class I tetramers and intracellular cytokine staining as described previously (18). T cell restimulation was performed with MHC class I (i.e., M45_985–993, m139_419–426, M38_316–323, and IE3_416–423 [19])- and MHC class II (M25_409–423, m139_560–574, and m142_24–38 [20])-restricted peptides. Fluorescently conjugated antibodies were purchased by Affymetrix, BD Pharmingen, or BioLegend. Flow-cytometric acquisition was performed on a BD LSR II. Data were analyzed using FlowJo software (TreeStar).

Statistical significance.

The Mann-Whitney test was used to calculate the significance of viral titers. To evaluate significance between two groups the Student t test was used, and for more than two groups one-way analysis of variance (ANOVA) was used. Tukey's post hoc test was performed to correct for multiple comparisons. P values of <0.05 were considered significant.

RESULTS

MCMV-specific IgG antibodies inflate during persistent infection without altered antibody avidity.

We used MCMV as a model to determine antibody characteristics during a persistent virus infection and first examined if MCMV-specific antibodies provide protection upon reinfection. Following acute infection, IgG2b and IgG2c (the IgG2a equivalent in C57BL/6 mice) are the predominant isotypes that are produced (Fig. 1A), which is not uncommon after viral infection (21, 22). Serum of naive mice and of latent MCMV-infected mice was transferred to naive mice (200 μl per recipient) that subsequently were challenged with MCMV. An ∼10-fold diminished viral load was found in the liver and salivary glands of the mice that received serum containing MCMV-specific antibodies compared to the mice that received naive mouse serum (Fig. 1A), indicating that MCMV-specific antibodies have protective capacity even in cases of subordinate antibody titer (i.e., transfer of 200 μl of serum results in a much lower antibody titer in the recipient mouse than the donor). Consistent with the diminished viral load, MCMV-specific T cell responses also were reduced upon MCMV serum transfer and displayed a less activated phenotype, as evidenced by greater CD127^high and KLRG1^low expression (15) (Fig. 1B and C). Thus, although sterile immunity is not achieved under the conditions analyzed, the antibodies that are generated during MCMV infection can reduce viral titers upon challenge.

FIG 1.

Protective properties of MCMV-specific antibodies. (A) C57BL/6 mice were infected with 1 × 10⁴ PFU MCMV. After 1.5 years, immune serum was obtained and transferred to naive mice that subsequently were infected with 1 × 10⁴ PFU MCMV. Serum of naive mice was used as a control. Six days postserum transfer, viral load was determined in the liver and the salivary glands by qPCR. (B) The magnitude of the MCMV-specific CD8⁺ T cell response was determined in the spleen by intracellular cytokine staining upon restimulation with the indicated MHC class I-restricted peptides. (C) The cell surface expression of CD127 and KLRG1 on splenic M45 tetramer⁺ CD8⁺ T cells (n = 5 mice per group; *, P < 0.05).

We next determined the kinetics of MCMV-specific IgM antibodies and the predominant IgG2b and IgG2c isotypes. In the acute phase of infection, high levels of MCMV-specific IgM antibodies were detected in the serum. After acute infection, IgM levels declined but remained clearly detectable throughout the chronic phase of infection (Fig. 2A), a phenomenon that is not observed after infection with viruses causing only acute infection (23). Another striking feature we observed was that MCMV-specific IgG2b and IgG2c antibody levels gradually accumulated with time (Fig. 2A and B). This particular inflation of MCMV-specific IgG2b and IgG2c antibodies is reminiscent of certain inflationary MCMV-specific T cell responses that progressively accumulate in the chronic phase of infection (10).

FIG 2.

MCMV-specific IgG antibodies inflate during persistent infection while antibody avidity remains stable. (A) Mice were infected with 1 × 10⁴ PFU MCMV. The MCMV-specific antibody levels are shown for the indicated serum dilution. (B) MCMV-specific IgG2c antibody titers in sera are depicted as means ± standard errors of the means (SEM) from days 8, 30, and 100 postinfection. (C) The elution profile of MCMV-specific IgG2c antibodies is shown as means ± SEM. (D) The percentage of antibodies bound to MCMV in the presence of 1 M NaSCN relative to the amount of antigen bound in the absence of NaSCN is shown. Data are pooled from 3 independent experiments (n = 4 mice per group; *, P < 0.05).

To examine if the avidity of these inflating antibodies varies with time, an antibody avidity assay was performed with increasing concentrations of sodium thiocyanate (NaSCN), which accordingly disrupts the antigen-antibody bond. The avidity of MCMV-specific antibodies was relatively low at day 8 postinfection, as ∼50% of the antibodies were eluted upon incubation with 1 M NaSCN (Fig. 2C and D). However, 30 days postinfection the avidity of MCMV-specific IgG2c antibodies was increased, as only 20% of the total MCMV-specific IgG2c antibodies could be eluted with 1 M NaSCN. Throughout the ensuing persistent phase of MCMV infection, the avidity of the MCMV-specific antibodies did not further increase compared to the avidity detected at day 30 postinfection. Similar results were obtained with the IgG2b isotype (data not shown). Thus, the levels of the MCMV-specific IgG2b and IgG2c antibodies inflate in chronic MCMV infection, but this is not accompanied by differences in antibody avidity during viral persistence.

MCMV-specific IgG antibody responses are dependent on CD28/B7-driven CD4⁺ T cell responses.

To examine factors that influence antibody inflation and avidity during persistent viral infection, we first aimed to identify the signals that are critical for GC-related processes, such as isotype switching. In this respect, help signals provided by CD4⁺ T cells are shown to be crucial for inducing class switching of antibodies. Upon the depletion of CD4⁺ T cells, the acute MCMV-specific IgM responses as well as the maintenance of the IgM levels were not much affected (Fig. 3A). IgG2b and IgG2c responses were, however, severely hampered, also at late times postinfection. To identify the molecular interactions that provide the help signal, we specifically focused on the role of costimulatory pathways involving the Ig superfamily member CD28 and its ligands B7.1 (CD80) and B7.2 (CD86) (here referred as B7) and the TNFR family member CD27 and its ligand CD70, because Cd80/86^−/− and Cd70^−/− mice have reduced MCMV-specific CD4⁺ T cell responses (Fig. 3B) (18, 24). No differences were found in the MCMV-specific IgM response during acute and persistent infection in all mice devoid of B7- and/or CD70-mediated costimulatory signals (Fig. 3C), suggesting T cell costimulation is not required for the initiation and maintenance of virus-specific IgM antibody responses. In contrast, B7-mediated costimulation was crucial for the development of MCMV-specific IgG2b and IgG2c responses throughout the course of infection (Fig. 3C). Despite the diminished MCMV-specific effector CD4⁺ T cell response upon CD70 abrogation, comparable IgG2b and IgG2c levels were found in wild-type (WT) and Cd70^−/− mice. Mice deficient in both CD70 and B7 molecules (Cd70/80/86^−/−) had a defect in antibody inflation comparable to that of Cd80/86^−/− mice, indicating that the CD28/B7 pathway has a dominant effect on the development of antibody responses. This particular effect of CD28/B7 costimulation coincided with lower levels of MCMV-specific CD4⁺ T cells in Cd80/86^−/− mice than in Cd70^−/− mice (Fig. 3B). Furthermore, MCMV-specific antibody isotype switching was not observed when mice were deprived of B7-mediated signals in the acute phase of infection but did occur when CD70 signals were abrogated (Fig. 3D). These data show that the B7-mediated signals are required at the beginning of infection.

FIG 3.

CD4 help and B7-mediated costimulation are critical for development of MCMV-specific IgG antibodies. (A) WT and CD4⁺ T cell-depleted mice were infected with 1 × 10⁴ PFU MCMV, and at days 8 and 30 postinfection the MCMV-specific antibody response was determined by ELISA. (B) WT and costimulation-deficient (i.e., Cd70^−/−, Cd80/86^−/−, and Cd70/80/86^−/−) mice were infected with 1 × 10⁴ PFU MCMV, and the MCMV-specific CD4⁺ T cell response was determined in the spleen 8 and 30 days postinfection by intracellular cytokine staining upon restimulation with the indicated MHC class II-restricted peptides. (C) The MCMV-specific antibody response in WT and costimulation-deficient mice in time is shown. (D) WT mice and costimulation-deficient mice were infected with 5 × 10⁴ PFU MCMV. CD70- and B7-mediated interactions were blocked in WT mice from day −1 to day 3 by administration of blocking antibodies. MCMV-specific IgG2c levels were determined in the serum 30 days postinfection. All bar graphs represent means ± SEM. Data are shown from one representative experiment of 3 independent experiments (n = 4 mice per group; *, P < 0.05).

We next aimed to explore the mechanisms underlying the dependence of IgG antibody responses on the CD28/B7 pathway. Most strikingly and fully consistent with the phenotype was the virtual absence of the T_FH subset (CXCR5⁺ PD1⁺) and of the further differentiated GC-associated T_FH cells (CXCR5⁺ GL7⁺) in the Cd80/86^−/− mice, while Cd70^−/− mice still had an induction of these cells (Fig. 4A to C). Moreover, upon the abrogation of B7- but not of CD70-mediated costimulation, a huge reduction in GC B cells, identified as B220⁺ CD19⁺ CD95⁺ GL7⁺, was observed at day 15 postinfection (Fig. 4A and D). In line with this, diminished plasma cells characterized by B220⁺ CD19⁺ CD138⁺ IgD⁻ (Fig. 4A and E) and no splenic MCMV-specific IgG2c-secreting cells were detected in Cd80/86^−/− and Cd70/80/86^−/− mice (Fig. 4F). Together, these data indicate that the development of MCMV-specific IgG antibody responses is fully dependent on B7-mediated activation of CD4⁺ T cell helper subsets and, in particular, of the T_FH subset.

FIG 4.

GC reactions in CMV infection are dependent on B7-mediated interactions. WT and costimulation-deficient mice were infected with 1 × 10⁴ PFU MCMV. (A) Representative flow cytometry plots of splenic cell populations at day 15 post-MCMV infection. Depicted are T_FH (PD-1⁺ CXCR5⁺) and GC T_FH (GL-7⁺ CXCR5⁺) gated on CD4⁺ CD62L⁻ cells as well as GC B cells (GL7⁺ FAS⁺) and plasma cells (CD138⁺ IgD⁻) gated on B220⁺/CD19⁺ cells. Numbers indicate percentages of positive cells within the gated population. (B to D) Total number of T_FH, GC T_FH, and GC B cells in the spleen at day 15 postinfection. (E) The percentage of plasma cells within the splenic B cell population at day 15 postinfection. (F) The number of MCMV-specific IgG2c-secreting cells was determined in the spleen 15 days postinfection using ELISPOT assay. All bar graphs represent means ± SEM. Data are shown from one representative experiment of 3 independent experiments (n = 4 mice per group; *, P < 0.05).

The initial viral inoculum dose affects antibody levels but not antibody inflation or avidity.

Given that the initial viral inoculum dose influences memory T cell inflation (15), we determined if the viral dose impacts MCMV-specific antibody inflation as well. In mice infected with either a low (10¹ PFU) or a high (10⁴ PFU) viral inoculum dose of MCMV, differences in antibody levels were determined. In low-dose-infected mice, IgM responses at day 8 were less pronounced than those of high-dose-infected mice, but in the chronic phase of infection similar levels persevered (Fig. 5A). MCMV-specific IgG2b and IgG2c levels were diminished upon a low-dose infection at all times, but antibody inflation still occurred comparable to that of high-dose infection (Fig. 5A). The differences in antibody levels were reflected by distinct numbers of plasma cells (Fig. 5B), the occurrence of IgG2c-secreting cells (Fig. 5C), and increased numbers of splenic GC B cells (Fig. 5D). Also in persistent CMV infection, more MCMV-specific, IgG2c-secreting, long-lived plasma cells were detected in the bone marrow upon a high-dose infection (Fig. 5E), but no IgG2c-secreting plasma cells were detected in the spleen in both low- and high-dose-infected mice (data not shown). However, MCMV-specific IgG2c-secreting memory B cells persisted at higher levels in the spleen in high-dose- versus low-dose-infected mice (Fig. 5F). Thus, the initial viral inoculum dose impacts the number of long-lived plasma cells and memory B cells that are maintained during chronic infection, thereby impacting the amount of IgG antibodies that are present in the serum. IgG antibody inflation, however, occurs despite differences in the initial viral inoculum.

FIG 5.

Impact of viral inoculum dose on antibody levels but not on antibody avidity. Mice were infected with either a low dose (10¹ PFU) or a high dose (10⁴ PFU) of MCMV. (A) The levels of MCMV-specific antibodies within the serum. (B) The total number of plasma cells identified by CD138⁺/IgD⁻ status in the spleen at day 8 postinfection. Cells are gated on B220⁺/CD19⁺ cells. (C) The amount of IgG2c-secreting cells in the spleen determined by ELISPOT assay at day 8 postinfection. (D) The number of GC B cells identified by Fas⁺/GL-7⁺ at day 8 postinfection. (E) The total number of IgG2c-secreting long-lived plasma cells determined in the bone marrow by ELISPOT assay at day 35 postinfection. (F) The total number of MCMV-specific IgG2c memory B cells determined by ELISPOT assay at day 35 postinfection. (G) The elution profile of MCMV-specific IgG2c antibodies is shown as means ± SEM. (H and I) Mice were infected either with 10³ PFU, 10⁴ PFU, or 10⁵ PFU MCMV, and the MCMV-specific antibody levels were determined in the serum 1.5 years postinfection. (I) The elution profile of MCMV-specific IgG2c antibodies is shown as means ± SEM. All bar graphs represent means ± SEM (n = 4 mice per group; *, P < 0.05).

Although the viral dose had a major impact on the antibody levels, no differences were found in antibody avidity between low- and high-dose-infected mice (Fig. 5G). Moreover, when mice were infected with three different doses of MCMV, ranging from 10³ to 10⁵ PFU, the effect of the initial viral inoculum dose again was reflected only in the differences in the IgG2b and IgG2c antibody levels (Fig. 5H), while no differences in the avidity of the MCMV-specific antibodies were detected (Fig. 5I). Together, these data show that the initial viral inoculum dose impacts the antibody levels during antibody inflation but not antibody avidity.

Repetitive viral challenge stimulates the development of neutralizing CMV-specific antibodies and improves antibody avidity.

To determine the development of MCMV binding antibodies that are able to neutralize the virus, in vitro neutralization assays were performed with serum of recently and latently infected mice. Neutralizing antibodies were below the detection limit in low- and high-dose-infected mice during the first months postinfection (Fig. 6A). Nevertheless, neutralizing antibodies eventually were detected in high-dose-infected mice. We next examined if the level of neutralizing antibodies could be elevated by repetitive exposure to MCMV. We administered a dose of 5 × 10⁴ PFU MCMV every week in the same mice the following year. The levels of the MCMV-specific antibodies were elevated to some extent in mice receiving MCMV repetitively (Fig. 6B). Moreover, mice that were exposed to intact CMV particles every week developed antibodies with a higher avidity (Fig. 6C). The levels of neutralizing antibodies was consistently higher in the group of mice that received MCMV every week (Fig. 6D). Notably, these effects coincided with larger amounts of GC B cells in the spleen (Fig. 6E and F). These data show that repetitive challenge with intact virus compared to a single inoculum promotes avidity maturation and development of neutralizing antibodies.

FIG 6.

Repetitive antigen exposure increases the avidity and neutralization capacity of MCMV-specific antibodies. (A) Mice were infected with either a low dose (10¹ PFU) or a high dose (10⁴ PFU) of MCMV. At the indicated times the neutralization capacity of the MCMV-specific antibodies was determined. The table indicates the neutralizing antibody titer expressed as the reciprocal of the serum dilution at which 50% of the total PFU was impaired. (B to F) Mice were infected with a single dose of 5 × 10⁴ PFU MCMV or received a weekly dose of 5 × 10⁴ PFU MCMV for 1 year. (B) The levels of MCMV-specific antibodies in the serum at 1 year postinfection. (C) The elution profile of MCMV-specific IgG2c antibodies in the presence of NaSCN is shown as means ± SEM. (D) The percentage of neutralization for each serum dilution is shown as means ± SEM. (E) Representative plots show cell surface expression of GL7 and FAS gated on splenic B220⁺/CD19⁺ cells. Numbers indicate the percentage of GC B cells within the total B cell gate. (F) Total amount of GC B cells in the spleen. All bar graphs represent means ± SEM (n = 7 mice per group; *, P < 0.05).

DISCUSSION

Memory T cell inflation is found in response to certain viral infections, most strikingly after persistent cytomegalovirus infection, and is characterized by the accumulation and maintenance of functional effector-memory CD8⁺ T cells (10). Here, we found that virus-specific IgG2b and IgG2c antibodies also inflate during persistent MCMV infection, and that the degree of antibody inflation relates to the dose of the initial viral inoculum. In other persistent infections, such as chronic lymphocytic choriomeningitis virus (LCMV) (25, 26) or herpes simplex virus infection (27), virus-specific antibodies also accumulate with time, albeit to various degrees. However, the serum levels of these virus-specific IgG antibodies eventually decline. Longitudinal follow-up of HCMV-infected individuals indicates that HCMV-specific antibodies with different specificities expand over time (28, 29), suggesting similar mechanisms underlying antibody inflation. Gradual antibody accumulation likely is related to viral persistence, as it is not observed in various acute infections with influenza virus (30), vesicular stomatitis virus (31), and LCMV Armstrong (32).

Consistent with other studies in mice and humans (33–39), we found that immune sera of mice latently infected with CMV provide protection from a new CMV infection. It should be noted that we transferred 200 μl of serum from donor to recipient mice, which is actually an underestimation of the protective capacity. Whether these protective effects are mediated via neutralizing antibodies that limit cell-to-cell spread (39) and viral dissemination (35) or via nonneutralizing MCMV-binding antibodies is unclear. Neutralizing CMV-specific antibodies were detectable rather late in infection and the titers of these neutralizing antibodies were low, which is consistent with other low-cytopathogenicity viruses (40). Repetitive administration of viral particles, however, did improve the neutralization capacity of MCMV-specific antibodies.

CMV-specific memory B cells that are adoptively transferred also have protective capacity (36), indicating that antibodies and memory B cells together form the humoral immune response. We show that MCMV infection elicits both MCMV-specific long-lived plasma cells and memory B cells, and both are found in greater numbers after high-dose than low-dose inoculum. Consequently, IgG levels are higher after high-dose infection, yet IgG inflation seems to occur after both high- and low-dose infection. Furthermore, IgM levels are equally stably maintained after low- and high-dose infection. Long-term IgM maintenance usually is not observed after acute infection (23) but recently has been reported for responses to chronic bacteria and bacterium-associated polysaccharides (41–43). Whether the observed IgG inflation and IgM maintenance is directly connected to a gradual change of the antibody-producing population driven by the persistence of antigen and/or to the induction of long-lived antigen-independent antibody-producing cells remains to be explored.

Avidity maturation of CMV-specific antibodies occurred within a month after the initial viral inoculum and remained stable afterwards. This phenomenon is actually used as a diagnostic tool to identify a recent infection with human cytomegalovirus (HCMV) (44). Remarkably, we observed that avidity maturation during the persistent phase of infection still can increase but only by reexposure to intact viral particles, which was accompanied by increased GC activity. In this respect, it is of interest to note that a study in humans with virosomal vaccines containing functional viral envelope glycoproteins demonstrated that the avidity of antibodies can be improved upon booster vaccination, suggesting that multiple encounters with intact rather than replicating virus is sufficient for avidity maturation (45).

If avidity maturation during viral persistence indeed is driven by newly induced GC reactions requiring intact viral particles, one could argue that the establishment of viral persistence after a single inoculum does not lead to numbers of viral particles sufficient to sustain GC reactions, while reexposure does. However, such reexposure does not lead to incremental changes in IgM levels, as occurs with primary infection, which may be related to preexisting immunity preventing the activation of IgM-producing plasmablasts. In mice, replicating virus at late time points postinfection can be observed in salivary glands, but whether the viral production at this site contributes to the observed antibody inflation remains to be examined. Nevertheless, it has been observed that following intraglandular MCMV infection the salivary gland can operate as a mucosal inductive site for isotype-switched IgG⁺ B cells (46). Moreover, studies by the Reddehase laboratory showed that in other tissues, such as lungs, viral replication is abortive, as only the expression of (immediate) early genes are observed, which nonetheless can lead to stimulating inflationary T cells (47).

MCMV-specific IgG2b and IgG2c levels were strongly decreased upon the abrogation of B7-mediated costimulation but were intact in the absence of CD70-driven costimulation. Upon influenza virus infection, similar virus-specific IgG levels also are found between WT and CD27^−/− mouse infection, but a compensatory role for CD27 signaling is found in the absence of CD28 costimulation (30). We did not observe such a compensatory role, but this may relate to a stronger dependence of the induced IgG responses on CD28/B7 costimulation in the MCMV model. In contrast to positive or neutral effects, in both acute and chronic LCMV infection, CD70 interactions have been described to have a negative effect on B cell responses (48, 49). As opposed to the low-level persistence of MCMV, LCMV persistence is accompanied by high-level replication and the induction of the profound expression of costimulatory molecules (13). Apparently, such high levels could lead to adverse effects of CD27/CD70 signaling on B cell responses and resembles findings in CD70 transgenic mice constitutively expressing CD70, in which deleterious effects on B cells occur (50). In agreement with numerous other studies (51–53), a prominent role of CD28-B7 interactions for inducing proper T and B cell responses was found. We found that B7-mediated signals are clearly implicated in the induction of T_FH, but direct effects on B cell responses also might be of importance, for example, via enhancing the survival of CD28⁺ bone marrow-resident plasma cells (54).

CMV-based vectors have shown promising results in diverse infectious and cancer models, but the success of these vaccines is considered T cell mediated (5, 55, 56). Recently, it has been shown that CMV vectors encoding additional antigens also can induce protective anti-melanoma or anti-tetanus toxin antibodies (8, 9). Thus, CMV-based vectors also are promising in settings were antibodies can mediate protection. The results described here could help further support the use of CMV-based vaccines and may help to understand how our immune system copes with this persistent virus.

Funding Statement

The funder had no role in study design, data collection and interpretation, or the decision to submit the work for publication.