Circulating human antibody-secreting cells during vaccinations and respiratory viral infections are characterized by high specificity and lack of bystander effect

Abstract

Surges of serum Abs after immunization and infection are highly specific for the offending Ag, and recent studies demonstrate that vaccines induce transient increases in circulating Ab-secreting cells (ASCs). These ASCs are highly enriched but not universally specific for the immunizing Ag, suggesting that a fraction of these ASCs could arise from polyclonal bystander stimulation of preexisting memory cells to unrelated Ags. This model is proposed to explain maintenance of long-lived serological memory in the absence of Ag exposure. To test this model, we measure the ability of respiratory syncytial virus and influenza virus infection or immunizations to influenza virus, tetanus toxoid, hepatitis B Ag, and human papillomavirus to stimulate bystander memory cells specific for other major environmental Ags that represent a large fraction of the preexisting memory B compartment. Bystander or nonspecific ASC responses to respiratory syncytial virus and tetanus could not be detected above the background levels in healthy adults, despite the presence of circulating memory B cells specific for the corresponding Ags. Nonspecific ASC responses in the healthy subjects and cord blood samples were similar. In contrast, both vaccination and infection induce massive expansion of circulating Ag-specific ASCs without significant increases in the frequencies of ASCs against unrelated Ags. Hence, nonspecific stimulation of memory B cells is unlikely to contribute to the mechanisms of long-term serological memory against major human pathogens. Additionally, high specificity of circulating ASCs after antigenic challenge highlights the diagnostic value of interrogating ASCs as an ideal single-time-point diagnostic immune surrogate for serology during acute infection.

Figure 1

Memory IgG B cell frequencies in healthy adults and cord blood. For these experiments, PBMC were stimulated in vitro for 6 days, then replated for ASC Elispot assays as described in the methods. (A) Tetanus-specific memory B cell frequencies in 16 adults compared to 7 cord bloods. (B) RSV-specific memory B cell frequencies in 29 adults compared to 7 cord bloods.

Figure 2

High specificity of the ASC Elispot assay is consistently detected in subjects after immunization for the corresponding antigens with the absence of cross-reactivity to non-specific pathogens. For these experiments, ASC Elispot assays were performed directly ex vivo without in vitro proliferation. (A) Representative wells of the Elispot assay. The sample in each row is representative blood from 4 subjects with indicated vaccination. Bottom row is representative of an asymptomatic healthy subject at steady state. Columns represent ASC assays specific for the following antigens: Trivalent inactivated influenza vaccine (TIV), Respiratory syncytial virus fusion protein (RSV F), tetanus toxoid (Tet), hepatitis B vaccine, and human papilloma virus vaccine (HPV). Peripheral blood lymphocytes (PBMCs) were incubated at 300,000 cells/well directly ex vivo (without stimulation). Spots were detected with anti-human IgG. Total IgG ASCs are shown in the far right column. Numbers below each well indicate represent mean ± SD of triplicate wells. Numbers in parenthesis indicate percentage of antigen-specific to total IgG spots. ND, not done. (B) Quantitation of data based on the assay in (A).

Figure 3

Baseline antigen-specific ASC from PBMCs of 28 healthy human subjects at steady state (A) and 9 cord blood samples (B). ASC assays specific for trivalent influenza vaccine (TIV), RSV F, tetanus, hepatitis B, human papilloma virus (HPV) and total IgG. Boxes show mean ± standard deviation (SD). For these experiments, ASC Elispot assays were performed directly ex vivo without in vitro proliferation.

Figure 4

Kinetics of tetanus and bystander (RSV F, influenza, hepatitis B, HPV)-specific ASC frequencies from one subject prior to and after tetanus vaccination on days 5–9, 15, and 30. For these experiments, ASC Elispot assays were performed directly ex vivo without in vitro proliferation.

Figure 5

ASC specificity in the blood against influenza, RSV F, tetanus, hepatitis B, HPV and total IgG during acute respiratory viral infections: (A) Wells of 300,000 PBMC plated from blood of patients with acute infection with influenza A virus (top panel) and Respiratory Syncytial Virus (bottom panel). (B) ASC specificity for RSV, influenza and total IgG in 11 patients with acute influenza A 2009 pandemic H1N1 and B infections and (C) 11 patients with acute RSV infection. *represents comparison of ASC responses to RSV and influenza excluding the patient with dual infection in figure 7. For these experiments, ASC Elispot assays were performed directly ex vivo without in vitro proliferation.

Figure 6

Cross-reactivity of direct ex vivo circulating ASC responses in one patient with confirmed the 2009 pandemic H1N1 infection. HA epitopes for H1: California 2009, H1: New Caledonia, H1: Solomon Islands, H1: Brisbane, H3 Brisbane, H7: Netherlands, and NP_A are shown. ASC assays were performed directly ex vivo without in vitro proliferation.

Figure 7

Patient with circulating ASCs specific for both RSV and influenza as enumerated in figure 5B. Patient with dual RSV and influenza virus B infection confirmed by nasopharyngeal PCR. ASC Elispot assays were performed directly ex vivo without in vitro proliferation.