Persistence of Virus-Specific Antibody after Depletion of Memory B Cells

Abstract

Humoral immunity is a major component of the adaptive immune response against viruses and other pathogens with pathogen-specific antibody acting as the first line of defense against infection. Virus-specific antibody levels are maintained by continual secretion of antibody by plasma cells residing in the bone marrow. This raises the important question of how the virus-specific plasma cell population is stably maintained and whether memory B cells are required to replenish plasma cells, balancing their loss arising from their intrinsic death rate. In this study, we examined the longevity of virus-specific antibody responses in the serum of mice following acute viral infection with three different viruses: lymphocytic choriomeningitis virus (LCMV), influenza virus, and vesicular stomatitis virus (VSV). To investigate the contribution of memory B cells to the maintenance of virus-specific antibody levels, we employed human CD20 transgenic mice, which allow for the efficient depletion of B cells with rituximab, a human CD20-specific monoclonal antibody. Mice that had resolved an acute infection with LCMV, influenza virus, or VSV were treated with rituximab starting at 2 months after infection, and the treatment was continued for up to a year postinfection. This treatment regimen with rituximab resulted in efficient depletion of B cells (>95%), with virus-specific memory B cells being undetectable. There was an early transient drop in the antibody levels after rituximab treatment followed by a plateauing of the curve with virus-specific antibody levels remaining relatively stable (half-life of 372 days) for up to a year after infection in the absence of memory B cells. The number of virus-specific plasma cells in the bone marrow were consistent with the changes seen in serum antibody levels. Overall, our data show that virus-specific plasma cells in the bone marrow are intrinsically long-lived and can maintain serum antibody titers for extended periods of time without requiring significant replenishment from memory B cells. These results provide insight into plasma cell longevity and have implications for B cell depletion regimens in cancer and autoimmune patients in the context of vaccination in general and especially for COVID-19 vaccines. IMPORTANCE Following vaccination or primary virus infection, virus-specific antibodies provide the first line of defense against reinfection. Plasma cells residing in the bone marrow constitutively secrete antibodies, are long-lived, and can thus maintain serum antibody levels over extended periods of time in the absence of antigen. Our data, in the murine model system, show that virus-specific plasma cells are intrinsically long-lived but that some reseeding by memory B cells might occur. Our findings demonstrate that, due to the longevity of plasma cells, virus-specific antibody levels remain relatively stable in the absence of memory B cells and have implications for vaccination.

Keywords: B cell responses; antibodies; immune memory; plasma cells; viral immunity.

FIG 1

Acute viral infections generate durable antibody responses. Mice were infected with influenza (A), LCMV (B), or VSV (C) and virus-specific antibody titers in the serum were measured by ELISA. Data (mean and SEM) from a representative experiment (n = 5 mice) are shown.

FIG 2

Efficient depletion of B cells in hCD20tg mice by rituximab. hCD20tg mice were infected with influenza virus, LCMV, or VSV, and weekly treatment with rituximab (red) or PBS (black) was initiated on day 62 postinfection. (A) Experimental design. (B to D) The frequency of B cells (B220⁺ CD19⁺ CD3⁻) among peripheral blood mononuclear cells (PBMCs) was monitored by flow cytometry at the indicated times after infection with LCMV (B), Influenza (C), or VSV (D). Treatment initiation and period are indicated by vertical dotted lines and gray shading, respectively. (E) Representative ELISPOT shows LCMV-specific IgG⁺ memory B cells in the spleen of LCMV immune mice 295 days after treatment initiation with either rituximab or PBS. Graph shows the total number of LCMV-specific IgG⁺ memory B cells in the spleen. Dotted line indicates the limit of detection. (F and G) Graphs show the total number of Influenza-specific (F) and VSV-specific (G) IgG⁺ memory B cells in the spleen at the end of the treatment period. Dotted horizontal lines indicate the limit of detection. Data (mean and SEM) from a representative experiment (n = 2 to 6 mice per group) are shown.

FIG 3

Examining the durability of virus-specific antibody after depletion of B cells. (A to F) Virus-specific antibody responses were assessed in the serum of rituximab-treated (red) and PBS-treated (black) hCD20tg mice immune to LCMV (A), influenza (B and D), or VSV (C and E). (A to C) Virus-specific IgG was measured by ELISA. (D) Hemagglutination inhibition (HAI) titers of influenza immune mice. (E) VSV neutralization titers in VSV immune mice. Treatment initiation and period are indicated by vertical dotted line and gray shading, respectively. Horizontal dotted line indicates the limit of detection. Data (mean and SEM) from a representative experiment (n = 4 to 6 mice per group) are shown. (F) Combined estimated half-life of virus-specific antibody responses measured in panels A to E are shown for both phases of the biphasic decline.

FIG 4

Analysis of virus-specific IgG⁺ plasma cells in the bone marrow of rituximab-treated mice. Virus-specific plasma cell numbers in the bone marrow of rituximab- (red) or PBS- (black) treated hCD20tg mice were quantified by ELISPOT. (A) Number of LCMV-specific IgG-producing bone marrow (BM) plasma cells per femur after 185 and 295 days of continuous treatment. (B) Number of influenza virus-specific BM plasma cells per femur after 342 days of continuous treatment. (C) Number of VSV-specific BM plasma cells per femur after 253 days of continuous treatment. Data (mean and SEM) from one experiment with n = 2 to 6 mice per group are shown. One-way ANOVA with Tukey’s multiple comparison test (A) and unpaired two-sided t test (B and C) was used for analyses with P values being indicated.