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. Author manuscript; available in PMC: 2013 Jul 29.
Published in final edited form as: J Immunol. 2011 Mar 25;186(9):5514–5521. doi: 10.4049/jimmunol.1002932

Circulating Human Antibody-Secreting Cells during Vaccinations and Respiratory Viral Infections Are Characterized by High Specificity and Lack of Bystander Effect

F Eun-Hyung Lee 1,*, Jessica L Halliley 1, Edward E Walsh 2, Andrew P Moscatiello 1, Brittany L Kmush 1, Ann R Falsey 2, Troy D Randall 3, Denise A Kaminiski 3, Richard K Miller 4, Iñaki Sanz 3
PMCID: PMC3726212  NIHMSID: NIHMS479987  PMID: 21441455

Abstract

Surges of serum antibodies after immunization and infection are highly specific for the offending antigen, and recent studies demonstrate that vaccines induce transient increases in circulating antibody-secreting cells (ASCs). These ASCs are highly enriched but not universally specific for the immunizing antigen, suggesting that a fraction of these ASCs could arise from polyclonal bystander stimulation of pre-existing memory cells to unrelated antigens. This model is proposed to explain maintenance of long-lived serological memory in the absence of antigen exposure. To test this model, we measure the ability of RSV and influenza virus infection or immunizations to influenza virus, tetanus toxoid, hepatitis B, and human papilloma virus to stimulate bystander memory cells specific for other major environmental antigens that represent a large fraction of the pre-existing memory B compartment. Bystander or non-specific ASC responses to RSV 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 antigens. Non-specific ASC responses in the healthy subjects were similar to frequencies in cord blood samples. In contrast, both vaccination and infection induce massive expansion of circulating antigen-specific ASCs without significant increases in the frequencies of ASCs against unrelated antigens. Hence, non-specific 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 ASC after antigenic challenge highlight the diagnostic value of interrogating ASCs as an ideal one-time-point diagnostic immune surrogate for serology during acute infection.

Keywords: Bystander, human, antibody-secreting cells, plasmablasts, influenza, RSV, infection, vaccine

Introduction

A long-standing immunological observation of major clinical consequence is that both vaccination and infection induce substantial rises in the levels of serum antibodies specific for the inciting antigens. Recent studies, including our own (1) (2), have demonstrated that these serological surges are mediated by a dramatic expansion of antigen-specific antibody secreting cells (ASCs) that are readily detected in the peripheral blood a few days after antigenic stimulation. Such antigen-specific ASC expansions have been demonstrated after vaccination with each antigen thus far tested including influenza, tetanus, diphtheria, and meningococcus (3–8) (9, 10). Of great significance for our understanding of serological memory, the antigenic specificity of early ASC expansions is consistent with the lack of rises of antibodies against unrelated antigens (11–13). Our recent demonstration that circulating ASC responses precede and correlate with serological responses also supports the notion that the characteristics of early ASC responses should reflect and predict the subsequent serological responses (1).

Interestingly, despite the tremendous enrichment (several hundred-fold) in antigen-specific ASC typically observed in early responses, a significant fraction (ranging from 10–80% depending on the specific antigen, timing, and individual donor), of all circulating ASC do not appear to produce antibodies against the immunizing antigen. This observation, suggestive of non-specific bystander stimulation, has been documented both by Elispot measurements and by generation of monoclonal antibodies (5, 7).

Nonetheless, the contribution of bystander, non-specific polyclonal stimulation of memory B cells to the homeostatic maintenance of both cellular and serological memory remains controversial. Several human studies argue against a bystander model (9, 11–13) and a mouse study shows that memory cells may not be needed to maintain the plasma cell compartment (14). By contrast, the potential contribution of non-specific polyclonal memory B cell activation to the maintenance of serological memory has been suggested by other relevant studies (5). The latter mechanism is also supported by elegant experiments showing that memory B cells in vitro efficiently differentiate into plasma cells through non-cognate, BCR-independent, TLR- or IFN-mediated stimulation (5, 15–18). It should be noted, however, that in vivo studies supporting non-specific bystander polyclonal activation have been limited in scope (5).

To formally address the question of bystander antigen specificity, we studied the ASC surge after immunization with four different vaccines to determine the relative contribution of cells specific for the corresponding antigen as compared to antigen unrelated responses against prevalent agents representing a substantial fraction of the pre-existing human memory B cell repertoire. Our results confirm the high specificity of the ASC response for the immunizing antigen and argue against universal stimulation of other memory cells in response to immunization. The vaccination results were also confirmed in over 20 adults with respiratory viral infections likely to induce more vigorous and broader responses than vaccines. Our results bear important clinical and immunological implications which will be discussed in detail in this manuscript.

METHODS

Subjects

A total of 97 adult subjects ages 20–96 years old (mean ± SD, 43 ± 19 years) were enrolled in this study during 2006–2009. Thirty-seven were men, and 60 were women. In addition, twelve cord blood samples were obtained. The 97 adult subjects included: 19 vaccinated healthy individuals; 28 asymptomatic healthy control subjects without current illness or significant medications; 28 additional asymptomatic healthy controls enrolled for the analysis of memory B cell frequencies; and 22 adults with confirmed RSV or influenza virus infections. All studies were approved by the University of Rochester and Rochester General Hospital Institutional Review Board.

Vaccinated subjects

We enrolled 19 subjects total. These 19 subjects, between the ages of 21 to 60 years (mean ± SD, 38 ± 17), received the following vaccines: Ten subjects each received the Trivalent Influenza Virus (TIV) 2008 (A/Brisbane/57/2007, A/Brisbane/10/2007, B/Florida/4/2006, Sanofi Pasteur, Swiftwater, PA), or (2009 A/Brisbane/57/2007, A/Brisbane/10/2007, B/Brisbane,) one hepatitis B (Merck, West Point, PA), or one human papilloma virus vaccinations (Merck, West Point, PA), and 7 subjects tetanus toxoid (Sanofi Pateur, Swiftwater, PA). Blood was drawn at 6–7 days post-vaccination. In one subject who received tetanus vaccination, blood was also obtained prior to and on days 5–9, 15 and 28 after immunization.

Subjects with Respiratory Infections

All subjects with respiratory viral infections were recruited from the Rochester General Hospital outpatient and inpatient facilities in Rochester, NY. Eleven adult subjects (age 50–94 years old) with respiratory symptoms such as fever, cough, sore throat, rhinorrhea and dyspnea who were RSV RT-PCR positive (19) were recruited during the winter of 2007–2008. In addition, 11 subjects (ages 21–96 years) with influenza-like illness were recruited in 2007–2009. Influenza B infection and pandemic (H1N1) influenza A infection were diagnosed by RT-PCR. From all patients with respiratory infections, nasal swabs and blood were drawn during the acute illness between days 2–11.

Healthy control subjects

Fifty-six healthy adults from 20–63 years old (36 ± 11 years) without concurrent infection or recent vaccination were enrolled as control asymptomatic healthy subjects during the spring and summer months of 2007–2009. Subjects were considered immunologically “healthy” as defined by a modified Senieur clinical survey (20). Half of the controls were randomly assigned to measure specificity of ASC, while 28 were used to measure memory B cells to common antigens.

Cord blood samples

Cord blood was isolated from 12 fresh placentas of healthy newborn deliveries at URMC during 2006–2009.

Reverse transcriptase-polymerase chain reaction for RSV and influenza viruses

The initial nasal swab samples were screened for the presence of RSV RNA by RT-PCR using a non-quantitative multiplex group A and B RSV-specific RT-PCR assay as previously described (21, 22). Influenza B RT-PCR was also performed on samples from selected individuals according to published methods (23) and pandemic 2009 H1N1 RT-PCR results were obtain using procedures provided by the Centers for Disease Control and Prevention, US (Lindstrom, personal communication).

ASC Elispot Assay

The frequency of antigen-specific ASCs was measured by ELISpot as previously described (24). Briefly, 96-well ELISpot plates (MAIPS4510 96 well) were coated overnight at 4°C in a humidified chamber with the following antigens: tetanus toxoid (1 Lf/mL Cylex Incorporated, Columbia, MD), TIV influenza Virus Vaccine (2007: A/Solomon Island/3/2006, A/Wisconsin/67/2005, B/Malaysia2506), (2008: A/Brisbane/57/2007, A/Brisbane/10/2007, B/Florida/4/2006), or (2009: A/Brisbane, A/Brisbane, B/Brisbane) (6μg/mL, Sanofi Pasteur Inc, Swiftwater, PA), Purified HA H1: New Caledonia/06/09, H1:Solomon Islands/03/06, H1:Brisbane/59/07, H3:Brisbane/10/07 or H7:A/Netherlands/219/03 (3μg/mL, Protein Sciences, Meriden CT), purified H1:2009 California (3μg/mL, Immune Technology Corp), purified NPA proteins (20μg/mL) (25), RSV F protein (26), recombinant surface hepatitis B antigen 10 ug/mL (Albevron, LLC, Fargo ND), recombinant Human Papilloma Virus vaccine (20 ug/mL, Gardasil, Merck), or anti-human IgG (5ug/mL, Jackson Immunoresearch, West Grove, PA). Bovine Serum Albumin 2% (BSA, MP Biomedicals, Solon, OH) in sterile PBS was used as an irrelevant antigen. Plates were incubated at 37°C for 18–20 hours with serially diluted numbers of PBMC. After incubation, wells were washed and bound antibodies were detected with alkaline phosphatase-conjugated anti-human IgG antibody (1μg/mL, Jackson Immunoreseach) for 2 hours and developed with VECTOR Blue, Alkaline Phosphatase Substrate Kit III (Vector Laboratories, Burlingame, CA). Spots in each well were counted using the CTL immunospot reader (Cellular Technologies Ltd). For analysis, background spots from wells without any capture antigen were subtracted from each well.

Enumerating memory B cells (memory B cell Elispot assay)

Total PBMC were cultured in vitro as previously described using protocols shown to induce differentiation of CD27+ memory B cells into antibody-secreting cells (17). Briefly, PBMC were stimulated with pokeweed mitogen (gift from Shane Crotty), IL-2 (10 pg/mL, Preprotech Inc.), and CpG (ODN 7909, 5′-TCG TCG TTT TGT CGT TTT GTC GTT -3′) (2.5 ug/mL, Oligos Etc.) for 6 days. ASC Elispot assays were performed as described above from the proliferated in vitro cultures. For cord bloods, total IgM was also measured using coating anti-human IgM 5 ug/mL (Invitrogen, Carlsbad, CA) and detected with alkaline phosphatase conjugated anti-human IgM 1ug/mL (Jackson Immuno Research, West Grove, PA).

Statistics Analysis

Comparison of the ASC frequencies between in vitro proliferation of memory B cells or direct ex vivo frequencies of cord blood and healthy adult subjects were performed using the Mann-Whitney 2-tailed unpaired T tests, as variances were not assumed to be equal. Similar analyses were used to compare direct ex vivo ASC frequencies of non-specific antigens in subject during vaccination or infection to healthy controls.

RESULTS

Adult memory B cell compartment contains high frequencies of cells specific for universally exposed antigens

Our studies were designed to test the specificity of the ASC response after immunization or infection and the degree of expansion of cells specific for prevalent but unrelated antigens both as a proof of specificity and a measure of potentially universal bystander effect. We reasoned that if the latter mechanism makes a consistent and substantial contribution to maintaining serological memory, that immunization with one antigen should trigger an expansion of cells specific for other unrelated antigens that represent a significant frequency of the pre-existing memory compartment. To test this hypothesis, we chose a small number of important antigens (included tetanus, hepatitis B, RSV, and influenza virus), because nearly all adults have had a history of exposure to these antigens either through childhood vaccination or infection (27–30). All adults have had exposure to RSV and influenza virus (27), and 81% and 100% of our subjects reported having received hepatitis B and tetanus vaccines respectively.

The frequency of circulating memory B cells specific to two of these antigens, RSV and tetanus, was measured in a sample of healthy adults using the memory B cell Elispot assay. Memory B cells to tetanus were readily detected in all 16 adults with frequencies ranging from 0.52 to 40 per 1000 total IgG (mean 8.5 ± 12.5/ 1000 total IgG) (figure 1A). Memory B cells to RSV were observed in 27/28 subjects with frequencies ranging 0–52 per 1000 total IgG (mean 4.9 ± 10 cells/1000 total IgG) (figure 1B). The subject without memory RSV B cells had evidence of memory B cells to tetanus suggesting that all healthy adults tested had memory B cells to either RSV and/or tetanus. As expected, memory IgG B cell frequencies to RSV and tetanus were not detected in 7 cord blood samples (figure 1A+B). Therefore, functional memory B cells against both RSV and tetanus are readily detectable from healthy adults. These data indicate that bystander stimulation would be likely to impact these responses and that the contribution of bystander stimulation to the maintenance of serological memory should be measurable in most healthy subjects.

Figure 1.

Figure 1

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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.

Direct ex vivo antibody-secreting cell specificity after vaccination

After immunization, recently proliferated ASCs typically appear in the circulation on day 4 and peak between day 5–8 with secondary exposure (1). Accordingly, ASC Elispots were performed with un-stimulated PBMCs to assess the frequency of both antigen-specific and nonspecific responses 6–7 days after vaccination. As expected, a brisk expansion of total IgG ASC (5–10 fold from baseline) was observed after immunization with 4 different antigens (influenza, tetanus, hepatitis B and HPV). Strikingly, in all cases, we readily detected significant frequencies of ASCs reactive with the corresponding vaccine but no responses above background levels to any of the other 4 antigens (figure 2A & B). For example, increased influenza-specific ASC after TIV are shown with undetectable ASC to the unrelated antigens. Similar specificities were detected with ASCs against tetanus, hepatitis B, and human papilloma virus (HPV) antigens although the magnitude of the responses was variable (figure 2A & B). Tetanus responses were particularly striking as nearly all ASCs are antigen-specific accounting for 97% of the total IgG frequencies. Hepatitis B and HPV vaccination showed similar results but lower frequencies as compared with influenza or tetanus. Importantly, the non-specific bystander ASC responses in adults to universally exposed RSV protein are not detectable with influenza, tetanus, hepatitis B, and HPV vaccination. This data is representative of lack of bystander ASC responses to at least one of the non-specific antigens in an additional 9 and 5 patients receiving influenza or tetanus vaccine respectively. The non-specific RSV ASC frequencies during immunization were similar to asymptomatic responses (p=0.08). In addition, ASCs to these 5 antigens could not be detected in an asymptomatic healthy adult control (figure 2A & B).

Figure 2.

Figure 2

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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).

The lower percentage of antigen-specific ASC responses to Hepatitis B and HPV vaccines may be due to several reasons. First, fewer antigen epitopes may be exposed when the proteins are coated onto the PVDF membrane in the ASC Elispot assay. Second, all healthy asymptomatic adult subjects have low levels of total IgG ASC responses circulating at steady-state. Small rise of ASC numbers in response to the vaccine above this steady-state total IgG responses may demonstrate only a small proportion of the total IgG frequencies but may represent a large fraction of the total IgG frequencies that proliferated due to the vaccine.

Since one of the adults tested did not have evidence of memory B cells to RSV (figure 1A), the lack of non-specific ASC responses could have been related to a deficiency of memory B cells to these antigens in the vaccine recipients. Therefore, memory B cells were measured prior to immunization from the blood of this subject prior to influenza vaccination, and despite memory B cell frequencies of 6, 4, 0.2, and 0.2 per 1000 IgG producing cells for RSV, tetanus, hepatitis B, and HPV respectively, direct ex vivo ASC specificities were not detectable. Hence, the lack of bystander ASCs in the blood after vaccination was not due to a deficiency of circulating memory B cells to these individual antigens.

Direct ex vivo antibody secreting cell specificity in asymptomatic healthy controls

Direct ex vivo ASC specificities could not be detected in 28 asymptomatic adult subjects without a recent history of infection or vaccination. The mean frequencies for ASC to TIV, RSV F, tetanus, hepatitis B and HPV were 2.4 ± 3.7, 0.8 ± 1.2, 0.2 ± 1.2, 0.0 ± 1.0, and 0.4 ± 1.2 spots/106 PBMC respectively (figure 3A). The total IgG ASC frequencies are also much lower at steady state (mean ± SD, 134 ± 118 spot/106 PBMC) than following the surges during vaccination or acute infection consistent with other studies (31).

Figure 3.

Figure 3

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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.

Direct ex vivo ASC specificity in cord blood samples

In cord bloods, the mean frequencies of direct ex vivo ASC frequencies to TIV, RSV F, tetanus, hepatitis B and HPV were 0.0 ± 0.7, 0.1 ± 1.2, 0.0 ± 0.3, 0.1 ± 0.3, and 0.1 ± 0.6 spots/106 PBMC respectively (figure 3B). Spontaneous total IgG frequencies were much lower in the cord bloods (mean ± SD, 5.7 ± 6.7 spots/106 PBMC) as compared to the blood of adults. Viability of the cord blood cells was assured in all samples with trypan blue exclusion (>99%) and/or total memory IgM responses (>1000/106 cord blood mononuclear cells).

Notably, no statistical differences were found between the spontaneous antigen-specific ASC frequencies of cord bloods and adult samples for TIV, RSV F, tetanus, hepatitis B and HPV (p= 0.06, 0.44, 0.58, 0.90, 0.52 respectively). The ASC frequencies to influenza in asymptomatic adults and cord blood samples were 2.4 ± 3.7 and 0.0 ± 0.7 spot/106 PBMC, respectively and thus it is possible that this difference could have reached statistical significance with a larger sample size. Importantly, this difference was not noted with the other 4 antigens suggesting that it was not a universal phenomenon. One explanation could be the complex nature and large numbers of different influenza antigens in the trivalent influenza vaccine since purified individual hemagglutinin proteins did not give the same responses (data not shown). Collectively, these data suggests that memory B cells do not affect the occasional low-level spontaneous production of antigen-specific ASC to the above-mentioned antigens.

Kinetics of bystander ASC responses

It is possible that the kinetics of bystander responses may be different from antigen-specific responses. Therefore, we measured both ASC to tetanus (antigen-specific) and non-relevant antigens (influenza, RSV, hepatitis B, and HPV) on days 0, 5–9, 15 and 28 in one subject (figure 4). Again, we found no ASC responses to influenza, RSV, hepatitis B and HPV above background levels of the asymptomatic healthy subjects (p= 0.24, 0.85, 0.06, 0.72 respectively). This lack of bystander ASCs at these additional time points suggests that timing does not affect ASC specificities.

Figure 4.

Figure 4

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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.

Bystander and specific ASC responses during acute viral infections

Compared with inactivated or purified protein vaccines, live virus infections are likely to generate a more vigorous, sustained and complex response, and may stimulate the generation of non-specific ASC from bystander memory B cells due to TLR and cytokine-mediated mechanisms (5, 32). Yet, circulating ASCs detected during acute respiratory viral infections also possessed very high specificity and recognized only the pathogens of exposure. For instance, in the blood of a patient with influenza infection, only ASCs reacting to influenza proteins, but not to control antigens were detectable (figure 5A). Similar findings were noted with RSV infection with detection of only RSV-specific ASCs on day 2 of symptom onset. Specificities to the bystander antigens (influenza, tetanus, hepatitis B, and HPV) were not detectable above background responses.

Figure 5.

Figure 5

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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.

These findings were consistent in all 11 patients with acute influenza virus infection on day 4–11 after symptom onset (mean ± SD 7 ± 2 days). Five had influenza B infection in 2007, and six had pandemic H1N1 infection in the spring of 2009. All 11 patients had influenza-specific ASC in the blood at a single time point during the acute illness and undetectable frequencies of RSV-specific ASC responses (figure 5B). The same was true regarding ASC specificity during acute RSV infections on day 2–10 (mean ± SD 7 ± 2 days). All 11 patients (6 outpatients and 5 inpatients) with acute RSV infection had only RSV-specific ASCs detected. Ten of the 11 had undetectable influenza-specific ASCs (figure 5C).

ASC responses in patients with acute 2009 pandemic influenza and influenza B infections were detected using the 2009 and 2007 trivalent influenza vaccine respectively. Mouse studies demonstrated that antibodies produced in response to primary infection with 2009 H1N1 virus were cross-reactive with older H1N1 influenza viruses (33). The nucleoprotein of the 2009 virus had nearly 95% conserved amino acid sequences to seasonal H1N1 viruses. To be sure cross-reactive H1:Brisbane and NPA ASC responses could be measured using TIV as the antigen, we measured ASC responses directly ex vivo (no proliferation) to individual influenza proteins in several subjects with 2009 pandemic H1N1 infection. We detected ASC responses to influenza NPA antigens, H1:2009 California, H1: New Caledonia, H1: Solomon Islands, H1: Brisbane, but not to H3 or H7 which are closely related HA proteins (figure 6). Hence, the H1:Brisbane and NP antigens contained in the TIV preparation were actual antibody epitopes that were contained in the 2009 H1N1 virus.

Figure 6.

Figure 6

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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 NPA are shown. ASC assays were performed directly ex vivo without in vitro proliferation.

One patient with an RSV-specific ASC response also had a substantial expansion of influenza-specific ASCs from the same blood sample (figure 7). These findings suggested two possibilities: a unique massive bystander response or a simultaneous RSV and influenza virus infection. Additional nasopharyngeal PCR tests revealed a co-infection with influenza B (and not influenza A) virus confirming the ASC specificities. In conclusion, similar to the vaccine responses, ASCs during an acute respiratory viral infection are highly specific to only the pathogen of recent exposure and when dual specificities are noted the results are due to true co-infections.

Figure 7.

Figure 7

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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.

DISCUSSION

Collectively, our data show that the massive expansion of ASCs detected in the blood after vaccination is highly enriched for antigen-specific clones and that bystander ASC specificities to unrelated antigens are not detected above background responses. Notably, a bystander ASC response was not observed with immunization despite the near universal presence of memory B cells to the antigens tested. Similarly, during acute viral infections, only pathogen-specific responses to the exposed microorganism are detected, with the lack of ASCs with unrelated specificities. Since measuring specificities to all previously exposed antigens is not possible, we chose a few universally exposed antigens such as tetanus, RSV and influenza virus to serve as surrogates.

These results begin to address the cellular underpinning of the maintenance of long-lived antibody memory. Several in vitro studies including our work also demonstrate the proliferation of memory B cells with non-cognate polyclonal activation presumably through TLR or cytokine-related activation in vitro culture systems (17). In this study, from a collection of healthy adults, RSV-specific memory B cell frequencies ranged from 0–52/ 1000 IgG producers (mean 5/1000 total IgG). If equal bystander proliferation occurred during acute influenza viral infections, expected RSV-specific ASC frequencies would reach 52–572 RSV-specific ASC /106 PBMC when total IgG ASC reached 1000– 11,000 per 106 PBMC as demonstrated in virus infected patients in this study. Our results do not reveal bystander RSV ASC levels close to those frequencies.

The low percentages of influenza- and RSV-specific ASC responses (37.4 ± 26.4% and 15.6 ± 6.4 % of total IgG ASC respectively) during these acute infections are concerning since a large fraction of the ASCs are unaccountable. However, several possible explanations may account for this result. Firstly, loss of epitopes may occur when proteins bind to the PVDF membranes in the ASC elispot assay. Secondly, for influenza infections, there may be poor matching of the antigens in the TIV vaccine to the strains that causing the infection. Thirdly and most important, not all viral proteins were coated on the ASC elispot well. For example, RSV has 8 structural proteins which are highly immunogenic for antibody responses but we coated with only RSV F protein. Despite low percentage of antigen-specific responses to total IgG responses, the lack of bystander responses to antigens with known ample memory B cell frequencies is consistent in during vaccination or respiratory viral infection.

Non-specific ASC responses using monoclonal antibody generation, after influenza vaccine have been shown (7). Both specific (71% or 61/86 antibodies) and non-influenza specific (29%) monoclonal antibodies were generated from circulating ASCs after influenza vaccination (7). However, those authors also remark that the specificities to non-influenza protein are likely to be explained by multiple causes such as technical errors introduced in cloning, specificities to denatured non-vaccine components (7), or even long-lived plasma cells released from the bone marrow (34) proposing strong possibilities for the lack of bystander responses.

The implications of the bystander concept is controversial and one may argue that despite high memory B cell frequencies, the activation of unrelated memory B cells occurs at extremely low levels relative to the antigen-specific frequencies (5). This phenomenon does not appear to be the case since the data demonstrate similar frequencies of spontaneous antigen-specific ASC in the adult blood (which contains high frequencies of memory B cells) and cord blood samples (that contain no memory IgG B cells). Although no statistical differences were found in ex vivo ASC responses to all 5 antigens between the adult and cord bloods, a p value = 0.06 for influenza specific ASC responses raises the possibility that influenza antigens may be maintained by this model. However, this trend was not seen with RSV, tetanus, Hep B, or HPV antigens suggesting that a universal model for bystander proliferation for plasma cell maintenance could not be applied to all antigens. More than likely, influenza antigens from formalin-inactivated vaccine preparation may yield higher background responses since individual influenza protein preparations did not yield this background response. Thus, more than likely, extremely low levels of antigen-specific ASC directly ex vivo of 0.1 to 1 spot/106 PBMC are the technical limits of the Elispot assay sensitivity and these low frequencies are difficult to reproduce.

The specificity of the recently blasted ASCs found in the blood after antigen exposure could be utilized to identify the “antigen” or pathogen causing illness. We have recently demonstrated this concept with acute RSV infections (2). Currently available immune assays preclude their routine use for diagnosing acute illness. For example, IgM serology offers low diagnostic yields with frequent false positives (35), and a single IgG level is not helpful in diagnosing secondary respiratory infections in adults. A single elevated serum antibody titer to multiple antigens could reflect several possible scenarios: (1) a new encounter with the pathogen; (2) persistence of long-lived bone marrow antibody-secreting cells (ASCs) producing antibodies demonstrating infection, which may have occurred long ago; (3) sustained production of antibodies due to persistence of chronic infections; or (4) the presence of acute co-infection by more than one organism. Therefore, serum antibodies require longitudinal changes to distinguish among new acute infections, exacerbations of chronic infections, and past pathogen exposures. These ambiguities could be resolved by examination of circulating ASC specificity.

This pathogen-specific ASC found in the blood as we demonstrate in this paper could function as a serologic surrogate with similarly high specificities of antibodies, but with one major advantage: the pathogen-specific ASCs require only a single time point during the acute illness. Additionally, the ASC assay may also have greater benefits over the single IgM serology since the assay detects high-affinity IgG or IgA antibodies. Evidence of bystander proliferation and differentiation of memory B cells to ASC would undoubtedly complicate the application of the test by “falsely” elevating the level of ASC with antigenic specificities unrelated to the current infection. However, this study demonstrates that minimal non-specific ASC response during respiratory viral infections occurs. Clearly, further evaluation is needed to determine the kinetics of the circulating ASC during acute viral infections before it can be utilized as a diagnostic test, but the lack of bystander non-specific ASCs detected in this assay demonstrates its potential.

In conclusion, we demonstrate high specificity of recently blasted ASCs after four different vaccines and two respiratory viral infections, and that bystander ASC responses are not observed. Whether this finding can be translated to all infections including bacterial and fungal pathogens needs further investigation. Clearly, active respiratory viral infections do not appear to significantly increase the frequency of circulating ASCs against other antigens. Although serum antibodies narrate a tale of a patient’s life history of pathogen exposure, the circulating ASCs can be instructive of only the most recent microbial exposure and may be an ideal measure for a novel diagnostic assay for acute infections.

Acknowledgments

Supported by: K23 AI67501, U01AI045969, ACE-Rochester-project1-ARRA:AI056390-06S2, HHSN266200500030C (N01-AI50029)

We would like to thank Deanna Maffett, Patricia Hennessey, and Mary Criddle for enrolling the patients in this study, MaryAnn Formica for performing the PCR assays and serology and Tim Mosmann for his kind advice.

Abbreviations

ASC

Antibody secreting cell

PBMC

peripheral blood mononuclear cells

TIV

trivalent inactivated influenza vaccine

Footnotes

Conflicts of Interests: Dr. Lee has research grants from Trellis Biosciences, Inc. Dr. Sanz has done consulting work for Genetech and Biogen. Dr. Falsey has done consulting work for AstraZeneca, Medimmune, Sanofi Pasteur, and Wyeth. Dr. Walsh has research grants from GSK and Sanofi Pasteur and has consulted for Astra Zeneca. Drs. Walsh and Falsey have research grants from GlaxoSmithKline and Sanofi Pasteur. Dr. Miller has research grants from OrthoClinical Diagnostics and is the Dysmorphologist and a member of the Scientific Advisory Board for the National Ribavirin Pregnancy Registry. Jessica Halliley, Andrew Moscatiello, and Brittany Kmush have no conflicts of interest. Drs. Randall and Kaminiski also have no conflicts of interest.

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