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. Author manuscript; available in PMC: 2014 Mar 15.
Published in final edited form as: J Immunol. 2013 Feb 11;190(6):2692–2701. doi: 10.4049/jimmunol.1202333

Long-term antibody memory induced by synthetic peptide vaccination is protective against Streptococcus pyogenes infection and is independent of memory T-cell help

Manisha Pandey 1, Michelle N Wykes 2, Jon Hartas 1, Michael F Good 1, Michael R Batzloff 1
PMCID: PMC3594626  NIHMSID: NIHMS432534  PMID: 23401589

Abstract

Streptococcus pyogenes (group A streptococcus; GAS) is a leading human pathogen associated with a diverse array of mucosal and systemic infections. Vaccination with J8, a conserved region synthetic peptide derived from the M-protein of GAS and containing only 12 amino acids from GAS, when conjugated to DT, has been shown to protect mice against a lethal GAS challenge. Protection has been previously shown to be antibody-mediated. J8 does not contain a dominant GAS-specific T-cell epitope. The current study examined long-term antibody memory and dissected the role of B and T-cells. Our results demonstrated that vaccination generates specific memory B-cells and long-lasting antibody responses. The memory B-cell response can be activated following boost with antigen or limiting numbers of whole bacteria. We further show that these memory responses protect against systemic infection with GAS. T-cell help is required for activation of memory B-cells but can be provided by naïve T-cells responding directly to GAS at the time of infection. Thus, individuals whose T-cells do not recognize the short synthetic peptide in the vaccine will be able to generate a protective and rapid memory antibody response at the time of infection. These studies significantly strengthen previous findings, which showed that protection by the J8-DT vaccine is antibody-mediated and suggest that in vaccine design for other organisms the source of T-cell help for antibody responses need not be limited to sequences from the organism itself.

Keywords: Streptococcus pyogenes, group A streptococcus, vaccine, memory B-cells, memory T-cells, long-lived plasma cells, adoptive transfer, T-cell proliferation assay

Introduction

Infection with Streptococcus pyogenes (group A streptococcus; GAS) causes many clinical manifestations including pharyngitis, impetigo, scarlet fever, invasive infections such as toxic shock syndrome and necrotizing fasciitis as well as the post-infectious sequelae of rheumatic fever (RF) and rheumatic heart disease (RHD). The latter are a major problem in developing countries and indigenous populations world-wide, particularly in indigenous Australians who have the highest reported disease incidence rate (1). There is strong evidence that RHD is autoimmune in etiology (2). Current control strategies to prevent streptococcal infection which would prevent RHD and other associated diseases, are proving ineffective and it is believed that development of a vaccine represents the best primary prevention solution. However, because RHD is autoimmune in etiology, it is important for safety concerns to use the minimal amount of GAS sequence required in the vaccine.

A number of potential GAS vaccine candidates have been identified and are at various phases of development as reviewed elsewhere (3); however, the M protein is a major candidate and antibody responses specific for it can protect against Streptococcus pyogenes (4). J8 is a minimal epitope derived in part from the conserved region of the M-protein (12 amino acids) and contained within a sequence of 16 amino acids from the yeast DNA binding protein, GCN4 (designed to maintain the α-helical coiling of the 12-mer insert (5). J8 conjugated to diphtheria toxoid (DT) is a leading vaccine candidate designed to protect against all strains. Studies investigating the mechanism of protection by J8-DT demonstrated that immunization or transfusion of J8-DT-specific antisera/antibodies protected mice against lethal GAS challenge (6). CD4+ T-cells were also shown to be important for protection since depletion of this subset prior to challenge resulted in reduced protection. The data suggested that CD4+ T-cells functioned as helper T-cells for the vaccine-induced B-cell response. Neither the duration of protection nor the factors controlling any memory/recall response were known. This was a significant issue since the vaccine contained minimal streptococcal sequence and specifically was designed not to contain any immunodominant T-cell epitopes derived from the M protein. T-cell help following vaccination came from stimulation by the diphtheria toxoid conjugate partner, not GAS sequences.

The persistence of long-term antibody titers for any vaccine is dependent on memory B-cells and long-lived plasma cells (LLPC). Memory B-cells differentiate rapidly (4–5 days) into antibody-secreting cells, which produce high affinity IgG antibody while a new primary immune response would take 10–14 days (7, 8). In contrast, LLPC survive in the bone-marrow in the absence of antigen for several years and continuously secrete antibodies (9–11), although titers diminish significantly over time (12). For many organisms a boost of antibody responses via a memory B-cell response may be critical for ongoing protection (13, 14).

Whether or not B-cells require T-cell help for a primary response depends on the type of antigen (15). The protein antigens possess the ability to recruit cognate CD4+ T-cell help through the TCR recognition of peptide-MHC class II complexes on the surface of APCs. On the contrary, the polysaccharides utilize multivalent membrane-Immunoglobulin dependent B-cell signalling (15). However, there is controversy as to whether memory B-cells specific for protein antigens require a memory T-cell response for optimal help (16, 17). Because the J8-DT vaccine was designed to contain a minimal B-cell epitope (defined by J8) but not a dominant T-cell epitope from GAS (to reduce the likelihood of any untoward autoimmune response) this issue is critical for success (18–20). While T-cell help following vaccination came from DT, there was great concern as to whether natural infection with GAS would boost the J8-specific antibody response. Any T-cell help for boosting would need to come from naive T-cells responding to GAS at the time of challenge.

The current study was therefore designed to assess whether immunization with J8-DT/alum would result in development of a long-lived protective immune response that could be boosted by exposure to limited numbers of GAS organisms, as might occur during natural exposure. We further dissected the role of T-cells in the memory responses and assessed whether and how these cells could protect against GAS infection. Our results demonstrate that the stimulation of a memory B-cell response requires T-cells but these need not be memory T-cells. The data suggest that vaccine design for other organisms could successfully utilise foreign T-cell epitopes chosen solely on their ability to induce a strong vaccine response.

Materials and Methods

Mice

Four to six week old BALB/c or SCID mice were obtained from the Animal Resource Centre, (ARC, Perth, Western Australia). All protocols were approved by the Institute’s ethics committee (Queensland Institute of Medical Research Animal Ethics Committee and Griffith University Animal Ethics Committee) in accordance with the National Health and Medical Research Council (NHMRC) of Australia guidelines.

Peptide synthesis

Peptides used in this study were synthesized in-house or synthesized commercially by Auspep Pty Ltd (Australia). Peptide J8 was conjugated to DT as described elsewhere (21). All peptides were stored lyophilised or in solution at −20°C.

Immunization, sample collection and challenge

Cohorts of 20–30 BALB/c mice were subcutaneously immunized at the tail base with 30 µg of J8-DT or DT. The antigens prepared in PBS were adsorbed onto alum (Alhydrogel, Brenntag Biosector, Denmark) at room temperature for one hour with slow mixing before administering into mice. To control for the effect of adjuvant, parallel cohorts of mice were given alum in PBS. Mice were immunized by subcutaneous route on days 0, 21 and 28 according to an established protocol. This protocol, in the past, has offered high J8-specific protective antibody titers against GAS. To study the longevity and protective efficacy of immune response, the mice were rested for 14–16 weeks post-immunization. Serum samples were collected at various time-points from immunized and control cohorts, before and after rest, to determine the antigen specific IgG levels by ELISA. To quantify the number and location of antibody secreting cells (ASC), bone marrow and splenocytes of immunized and control mice were analysed by ELISPOT as depicted in figure 1. Assessment of protective efficacy of long-lived and memory B-cell responses was carried out by monitoring mice survival following a sub-lethal intraperitoneal challenge with M1 GAS or by determining the reduction in bacteremia following an intravenous low-dose infection with M1 GAS strain.

Figure 1. Antigen specific IgG isotypes and antibody secreting cells in the spleens and bone-marrow of BALB/c mice immunized with J8-DT or DT.

Figure 1

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(a) A schematic of experimental protocol employed to investigate the longevity of antibody responses induced by vaccination with J8-DT. Cohorts of BALB/c mice (n= 30/group) were immunized IP with J8-DT/alum or DT/alum on days 0, 21 and 28. One week after the last boost (day 35) J8-specific (b) and DT-specific (c) IgG subclass compositions were detected in the serum samples. Data representative of three or more independent experiments and results are shown as means ± standard errors of the means for 15–20 mice in each group. To enumerate the antigen-specific antibody secreting cells (ASC), spleens and bone marrow were analysed on day 35 post-immunization. The numbers of J8 and DT-specific antibody secreting cells that were resident in spleen (d) or had migrated to the bone marrow (e) were quantified by ELISPOT using 5 mice per group. Data representative of three or more independent experiments and results are shown as means ± standard errors of the means for at least 5 mice in each group. Significance determined by two-way analysis of variance (ANOVA) throughout the figure, where ***p<0.001.

Cell purification for in vivo and in vitro assays

Lymphocyte populations (B and T-cells) were purified from splenocytes for in vitro proliferation studies as well as in vivo transfer studies. Spleens from immunized and control mice were mashed and passed through a 0.70 μM cell strainer to obtain single cell suspensions. The cells were washed twice in PBS+1%BSA by centrifugation at 1300 rpm for 10 minutes. The resuspended cell pellet was treated with Gey’s solution for 2 minutes at room temperature in order to lyse red blood cells. The reaction was stopped with the addition of 10× the volume of PBS+1%BSA, and the cells were washed twice. B-cells and T-cells were obtained from the same spleen using positive selection strategy for B-cells. For positive selection of B-cells, splenocytes were incubated with CD19 microbeads (Miltenyi Biotech) at 4°C for 20 minutes. The cells were then washed twice with MACS buffer and run through two LS columns (Miltenyi Biotech). For in vitro assays, the B-cell-depleted splenocyte fraction (hereafter referred to as “T-cell enriched fraction”) was retained and used as a source of T-cells. For in vivo functional studies, after positively selecting B-cells the T-cells were purified by negative selection using Pan T-cell isolation kit (Miltenyi Biotech) as per manufacturer’s instructions. Following incubation with Biotin-Antibody cocktail, the cells were labelled with Anti-biotin micro-beads. The magnetically labelled non-T-cells were depleted by retaining them on the column and the resulting T-cell enriched effluent was collected. The isolated cells and the effluent were stained with appropriate antibodies for cell-surface markers for B and T-cells and analysed by FACS to determine cell purity. In general the purity level were >96% for B-cell and 92–95% for T-cells.

Adoptive transfer studies

To determine the efficacy of memory responses independent of circulating serum antibodies, splenocytes from BALB/c mice immunized with J8-DT/DT or PBS were adoptively transferred to recipient SCID mice. After isolation of splenocytes, RBC lysis was carried out using Gey’s solution as described above. Following two washes, the cells were counted, resuspended in 200 µl sterile PBS and transferred intravenously into SCID mice. Each mouse received one spleen equivalent.

To assess the role of different lymphocyte populations, post-immunization with J8-DT/DT or PBS the BALB/c mice were rested for 14–16 weeks. The B or T-cells populations from spleens of these mice were purified (prepared using the method described earlier) and utilised for adoptive transfer studies. For the mice receiving B or T-cells, each SCID mice received one mouse equivalent of splenic B or T-cells.

In vivo activation of memory response

To measure memory antibody responses in vivo, immunized mice with memory cells or recipient SCID mice with transfused memory cells were boosted i.v. with 10 µg of J8-DT/DT or 1000 CFU of GAS M1 strain. The antigen-specific memory IgG were measured in the serum of mice 5 or 7 days post-boost, with antigen or infection, by ELISA. To determine protective efficacy of long-lived and memory immune responses, the immunized-rested-boosted (I-R-B) BALB/c and recipient SCID mice post-boost were challenged with GAS M1 strain.

ELISPOT assay

Antigen specific ASC in the bone marrow (BM) and spleens of donor and recipient mice post-immunization, post rest, post-boost and post-challenge were quantified by ELISPOT. Multiscreen-HA plates were coated with 5 µg/ml of J8 or DT in carbonate coating buffer (pH 9.6). Isolated spleen or BM cells were directly tested for ASC using a published method (22, 23).

ELISA

At the indicated time-points, blood samples (10 µL) were collected from individual mice by tail snip. J8 and DT-specific IgG titers were determined as described elsewhere (24). NUNC Immunoplates (Flow laboratories) were coated with 100 µl of J8 or DT at 5 µg/ml concentration. The end-point titers were determined as the highest dilution of serum for which the OD was 3 SD above the mean OD of control wells containing serum from naïve mice. The IgG subclass composition of sera was analysed by ELISA using subclass-specific HRP-conjugated secondary antibodies (Zymed, Invitrogen).

In-vitro cell proliferation studies

To measure T-cell responses to antigens, an in vitro proliferation assay that utilises EDU (5-ethynyl-2’-deoxyuridine, a thymidine analogue) incorporation into the DNA of proliferating cells was used (25). Briefly, T-cell enriched lymphocyte fractions from J8-DT immunized mice were seeded into round bottomed, 96 well plates at 2.5×105 cells per well in complete IMDM media. The cells were then cultured in the presence or absence of various antigens including J8i, J8, DT and J8-DT (Table 1). T-cells stimulated with 2ug/well of Con A were used as positive controls. Following incubation at 37°/5% CO2 on day 3 (72 h post stimulation), 20 µM EDU was added to each well for 18–24 h. On day 4 (96 h post stimulation) the cells were harvested, washed and labelled with various cell surface markers for T-cells. Detection of EDU incorporated into the proliferating CD4+ T-cells was accomplished by using the “Click-IT EDU Alexa-Fluor 647 kit for Flow Cytometry” (Molecular Probes, USA) following manufacturer’s instructions. The cell population positive for CD3-AF-488 and CD4-PerCp-Cy5.5 were analysed for the percentage of proliferating CD4+T-cells.

Table 1.

Peptides used in the study to investigate in vitro and in vivo responses.

Name of peptide Peptide sequence
J8 QAEDKVKQSREAKKQVEKALKQLEDKVQ*
J8i SREAKKQVEKAL
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*

Sequence of chimeric peptide J8 derived from the M-protein of GAS. M-protein derived sequence is underlined and GCN4 derived sequence is italicised.

Flow cytometry labelling

Antibodies utilized to check the purity of isolated B and T-cells include CD19-PE, CD3-FITC, CD4-APC, CD8-perCP and their corresponding isotype controls. CD3-AF-488 and CD4-PerCp-5.5 were used in EDU assay. All the antibodies were sourced from BD Biosciences, US. Flow cytometry was conducted on a FACSCanto II (Becton Dickension), and data analysis achieved on FCS Express (De Novo Software).

Results

Primary immune responses to J8-DT

To assess the efficacy of J8-DT immunization in inducing long-lived immune responses, multiple cohorts of BALB/c mice were immunized with J8-DT, DT or PBS (all in alum) following a protocol that is established in our laboratory. Within 35 days of immunization, J8-DT/alum immunized mice developed significantly higher (p<0.001) titers of J8-specific IgG1 compared to the negligible titers in cohorts of mice immunized with DT/alum or PBS/alum (Figure 1a, b). As expected, the incorporation of alum with the vaccine induced Th2 responses resulting in a predominance of serum J8-specific IgG1 antibodies over IgG2b and 1 b. Similarly, very high (~106) DT-specific IgG1 titers were achieved in cohorts immunized with J8-DT or DT (Figure 1 c).

Enumeration of antigen-specific antibody secreting cells (ASC) in spleen and bone marrow, on day 37 post-immunization, found that following immunization, the majority were resident in the spleen and only a small fraction had migrated to the bone-marrow (BM) (Figure 1 a, d and e). The ASC in the spleen represent primary B-cells and long and short lived plasma cells whereas the majority of the ASC in the BM would be LLPCs. Mice immunized with J8-DT had similar number of J8 and DT-specific ASC in spleen and BM, while DT immunized mice showed significantly higher (p<0.001) numbers of DT-specific ASC both in spleen as well as in the BM compared to J8-specific ASC (Figure 1 d and e).

Long-term immune responses in mice immunized with J8-DT

The above cohorts of immunized mice were then rested for approximately 14–16 weeks (~110 days) to allow all primary immune cells and short-lived plasma cells to subside resulting in only memory B-cells (MBC) and long lived plasma cells (LLPCs) surviving (Figure 1 a). These LLPCs maintain a basal level of antibodies in the serum. A comparison of J8-specific serum IgG titers in J8-DT immunized mice between day 35 and day 110 post-immunization found an approximately 16-fold reduction in IgG1 titers (Figures 1 b and 2 a). However these titers were still significantly higher (p<0.001) compared to the levels in DT or PBS immunized cohorts (Figure 2 a). DT-specific IgG titers in J8-DT or DT immunized cohorts were also maintained in the serum post 110 days of immunization (Figure 2 b). IgG1 still remained the predominant isotype post rest (Figure 2 a and b).

Figure 2. Antigen specific IgG isotypes and antibody secreting cells in the bone-marrow and spleens of BALB/c mice post 110 days of immunization.

Figure 2

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Post-immunization with J8-DT/DT or PBS (all in alum), the mice (n=25/group) were rested for 14–16 weeks. After 110 days of immunization the J8-specific (a) and DT-specific (b) serum IgG isotype titers were detected. Data representative of three or more independent experiments and results are shown as means ± standard errors of the means for 15–20 mice in each group. The numbers of antigen-specific LLPCs in the bone marrow (c) and ASCs in the spleen (d) were detected by ELISPOT using 5 mice per group. Data representative of two or more independent experiments and results are shown as means ± standard errors of the means for at least 5 mice in each group. Significance determined by two-way analysis of variance (ANOVA) where ***p<0.001.

Next, ELISPOT assays were used to quantify J8 and DT-specific ASC (LLPC) in the bone-marrow and spleens of J8-DT, DT or PBS-immunized-rested mice. It was apparent that at 110 days post-immunization, only few ASC were residing in the spleen (Figure 2 c) whereas the majority of LLPC had migrated to the bone marrow (Figure 2 d). The spleens from mice immunized with J8-DT or DT had typically around or fewer than 100 LLPC/spleen specific for DT or J8 respectively (Figure 2 c). J8-DT immunized mice had similar numbers of J8 and DT-specific LLPC in the bone marrow (~2000 ASC per 107 BM cells) whereas DT immunized mice had a significantly higher (p<0.01) number of DT-specific LLPC (~2000 LLPC per 107 BM cells) compared to J8-specific LLPC (~ 80 LLPC per 107 BM cells) (Figure 2 d).

J8-DT immunized mice have a memory response to J8-DT

To determine if J8-DT immunization generated memory B-cells that were specific for the vaccine, mice were immunized with the vaccine and rested for 14–16 weeks. To recall memory responses, half (n=10/group) of the mice from each cohort of immunized-rested mice were boosted (I-R-B) with a small dose (10 µg) of antigen (J8-DT or DT) whereas the remaining mice (n=10/group) were left non-boosted (I-R). Following a boost with 10 µg of antigen, we observed an 8-fold rise in J8-specific antibody titers by day 7 compared to titers on day 4 (p<0.01) (Figure 3 a). To confirm that this rise in J8-specific IgG titers was due to activation of memory B-cells and not a new primary immune response, which typically takes 9–14 days to generate, we also included control naive groups that were immunized with J8-DT/alum via a subcutaneous route or J8-DT alone via an intravenous route. These control cohorts did not show any detectable J8-specific IgG until day 14 post-immunization, thereby confirming that the rise in antibody levels in immunized-rested and boosted (I-R-B) mice is not a primary immune response. Further, the antibody levels in the immunized-rested (I-R) cohort (without boosting) did not show any change, further supporting that it was the activation of memory B-cells that resulted in the rapid rise in antibody levels. There was no noticeable rise in DT-specific IgG titers following boosting (Figure 3 b).

Figure 3. In-vivo activation of memory responses.

Figure 3

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Following a period of 110 days post-immunization with J8-DT/DT or PBS, the rested mice were either boosted (I-R-B) with homologous antigen or left non-boosted (I-R). Serum samples were collected at various time-points (day 1, 4, 7 and 14) post-boost and IgG levels were determined. A follow-up of J8 (a) and DT (b) specific IgG levels at various time-points is shown. The control naïve mice (n=10 each) were either given J8-DT alone (via IV route) or J8-DT/alum (via subcutaneous route). Data representative of two or more independent experiments and results are shown as means ± standard errors of the means for 10 mice in each group. Significance determined by two-way analysis of variance (ANOVA) where **p<0.01.

J8-specific memory B-cells are functional against a GAS infection

To measure recall of memory responses by GAS, splenocytes from parallel cohorts of mice (immunized with J8-DT, DT or PBS and rested for 14–16 weeks) were transferred to naive SCID mice and either boosted with the vaccine (10 µg of J8-DT) or given a low dose infection (1000 CFU of GAS) (Figure 4 a). The J8-DT immunized mice demonstrated a significant increase (p<0.001) in J8-specific IgG following boost with antigen or infection by day 5 post-boost compared to their non-boosted counterparts. By day 7, there was a further 10 to 14-fold increase (p<0.001) in J8-specific IgG titers following antigen and infection boost respectively, which reflected activation of memory B-cells (Figure 4 b). Further, transfused memory B-cells did not differentiate to ASC in the absence of antigen boost or infection. The absence of significant antibody titers in non-boosted control groups excluded the possibility that short or long-lived plasma cells were responsible for the increase (Figure 4 b). These results thus confirmed that antibodies were derived from activated memory B-cells and not from plasma cells.

Figure 4. Analysis of IgG levels and antibody secreting cells in SCID mice post adoptive transfer of splenocytes.

Figure 4

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(a) A schematic of experimental protocol employed to investigate the protective efficacy of J8-DT- induced memory responses. To study memory response independent of circulating antibodies and LLPCs, the splenocytes from immunized-rested mice (n=15 mice per group) were transferred to naïve SCID mice (n=15 mice per group). The recipient SCID mice were then boosted intravenously either with antigen (n=5 per group) or GAS M1 infection (n=5 per group). Mean J8-specific IgG titers on day 5 and 7 post-boost (b) and mean J8-specific ASC in spleen (c) and bone-marrow (d) at day 7 are shown. The mice with no boost (n=5 per group) served as controls. Data representative of three or more independent experiments and results are shown as means ± standard errors of the means for at least 5 mice in each group. Significance determined by two-way analysis of variance (ANOVA) where ***p<0.001. Mann-Whitney test was used to compare similar groups from day 5 and 7 (***p<0.001).

We then quantified the numbers of J8-specific memory B-cells in the spleen and bone marrow of SCID mice, on day 7 post stimulation, using an ELISPOT assay (Figure 4 c and d). The SCID mice transfused with J8-DT splenocytes whether boosted with antigen (>3000 MBC/spleen) or infection (>4000 MBC/spleen) had significantly higher (p<0.01) numbers of J8-specific MBC in their spleens compared to basal numbers in DT (<100) or PBS (<20) control mice (Figure 4 c). As expected, the number of ASC that had migrated to the bone marrow, at that stage, was negligible (Figure 4 d). This confirmed that the rise in serum IgG levels was due to activation of memory B-cells that resulted in their differentiation into antibody secreting plasmablasts and that the LLPCs were not a source of these antibodies.

J8-specific memory B-cells protect against a GAS infection

To measure protection against GAS by memory B-cells per se, cohorts of mice were immunized with J8-DT, DT or PBS to generate MBC and rested for 14–16 weeks prior to challenge. SCID mice were then transfused with splenocytes from these immunized mice and either boosted with antigen or 1000 CFU of GAS. A parallel cohort was also left as a non-boosted control. On day 8 post transfusion, all mice were challenged with a GAS infection (5×104 cfu/mouse) and bacteraemia was monitored from day 1 (Figure 4 a). All SCID mice that received J8-DT splenocytes (whether boosted with vaccine or low dose infection) and challenged with GAS infection controlled disease by day 8–12 (Figure 5 a, b, c). In contrast, all control SCID mice given splenocytes from DT (Figure 5 d, e, f) or PBS-immunized mice (Figure 5 g, h) developed high bacteremia and had to be sacrificed after approximately 20 days. SCID mice given J8-DT splenocytes cleared the infection more rapidly if given an antigen boost or infection prior to challenge, correlating with the activation of MBC prior to challenge. These mice developed peak bacteremia by day 3 and infection cleared by day 8. Recipient cohorts that did not receive any prior boosting before GAS challenge had negligible J8-specific IgG titers and a slightly more prolonged peak bacteremia with a very rapid clearance by day 12 (Figure 5 a). Finally, SCID mice recipients of DT splenocytes had a progressive increase in bacteremia starting from day 1 post-challenge irrespective of the level of DT specific IgG, highlighting the reduction in bacterial counts seen in J8-DT immunized cohort was due to J8-specific antibodies (Figure 5 d, e and f). SCID mice given PBS splenocytes whether non-boosted or boosted with antigen or infection were unable to control the infection (Figure 5 g, h).

Figure 5. Antigen-specific memory IgG levels and GAS bio-burden in SCID mice following a challenge with M1 GAS.

Figure 5

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SCID mice (n=12 per group) were transfused with J8-DT splenocytes (figure 5 a, b, c) /DT splenocytes (figure 5 d, e, f) or /PBS splenocytes (figure 5 g and h) from immunized-rested BALB/c mice. They were then boosted with either antigen or infection on day 1 post transfusion. A parallel cohort did not receive any boosting (AT-Ch) prior to challenge. Seven days post-boost (day 0 for challenge, on the graph), mice were challenged with a low dose (5×104 CFU/mouse) of GAS infection. The J8 and DT-specific serum IgG titers were monitored for up to day 18 post-challenge and are shown on right Y-axis. The CFU counts were also monitored at indicated time-points and are shown as solid lines on left Y-axis. Data representative of three or more independent experiments and results are shown as means ± standard errors of the means for 10–12 mice in each group.

At 20 days post-challenge, the numbers of J8-specific ASC were enumerated in these mice. SCID mice given J8-DT splenocytes had approximately 4000 J8-specific ASC per spleen with (antigen or infection) or without boosts and challenge with GAS compared to <100 ASC/spleen in mice given splenocytes from DT or PBS-immunized mice (Figure 6 a). These numbers were comparable to the numbers of MBC/ spleen seen in SCID mice given J8-DT splenocytes and vaccine and/or infection to quantify MBC (Figure 4 c). These studies show that GAS infection does not compromise the MBC responses as noted for other infections (26). Further, while ASC numbers in recipient mice were negligible in the absence of vaccine and/or infection (Figure 4 c), GAS challenge alone activates MBC to secrete antibody (Figure 6 a). Finally, small numbers of ASC had also migrated to the bone-marrow (Figure 6 b).

Figure 6. Persistence of antibody secreting cells in the recipient SCID mice post GAS challenge.

Figure 6

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The SCID mice recipients of J8-DT/DT or PBS splenocytes were boosted with antigen or infection. One cohort was left without any boost. On day 7 post-boost, the mice (n=12 per group) were challenged with a low dose GAS infection. The number of J8-specific antibody secreting cells in the spleen (a) and in the bone marrow (b) of SCID mice (n=7/group) on day 20 post infection are shown. Data representative of three or more independent experiments and results are shown as means ± standard errors of the means for 7 mice in each group.

The J8-DT induced memory B-cell response is T-cell dependent

To define the role of J8-DT-specific memory B and T-cells, independently, in protection against GAS, these cell populations were purified from splenocytes of mice immunized with J8-DT, DT or PBS that had then been rested for 14–16 weeks. Assessment of in vitro proliferative capacities of the enriched T-cells was carried out using a flow cytometry based EDU uptake assay where EDU added to the cell cultures is incorporated by cells replicating DNA. The enriched T-cells from J8-DT immunized and rested mice were stimulated in vitro with antigens including J8i (the minimal 12-mer B-cell epitope from within J8), J8, J8-DT or DT and their proliferation studied. The cells were labelled to detect EDU uptake into CD3+CD4+ T-cells by flow cytometry (figure 7 a–d). The CD4+ T-cells from J8-DT-immunized-rested mice responded to J8-DT (p<0.038) and DT (p<0.0001), but not to J8 (p>0.05) or J8i (p>0.05) compared to unstimulated T-cells (Figure 7 e). A recall proliferative T-cell response was observed to rM1 (data not shown) which may be attributed to cross reactivity between DT and the M-protein of GAS. This observation suggested that J8-DT specific memory T-cells are functional and can be recalled upon stimulation with J8-DT or DT.

Figure 7. In vitro proliferation of memory T-cells and in vivo protection assay with memory B-cells, T-cells and splenocytes.

Figure 7

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To measure activation of CD4+ T-cells in response to various antigens, EDU assay was carried out on T-cell enriched fraction from J8-DT immunized-rested mice. Following antigenic stimulation for 72 h the cells were incubated with optimized concentration of EDU. The percentages of CD3+CD4+ T-cells proliferating in the unstimulated cell population (a) or stimulated with J8 (b), DT (c) and J8-DT (d) are shown. Con A was used as a positive control. The numbers of EDU positive cells/well were determined after 16–24 h of incubation with EDU and are shown on y-axis (e). To assess the protective efficacy of lymphocyte subsets, SCID mice recipient of purified B or T-cells or total splenocytes (n=10 per group) from J8-DT/ DT or PBS immunized mice were challenged with M1 GAS strain. Protection induced in recipient SCID mice following GAS challenge is shown (f). Log-rank analysis was carried out to compare the survival curves in figure f. Significance is represented as * (where * is p<0.05 and **p<0.01). (Figure a–e) Data representative of two or more independent experiments and results are shown as means ± standard errors of the means for at least 5 mice in each group. Significance determined by two-way analysis of variance (ANOVA) where **p<0.01, ***p<0.001.

Further, to investigate the role of purified B and T-cells in protection against GAS, in vivo protection assays were conducted. For in vivo studies, naïve SCID mice received purified B or T-cell from J8-DT, DT or PBS immunized-rested mice. In addition, one cohort that received total splenocytes from J8-DT/DT/PBS -immunized-rested mice was also included as a control (Figure 7 f). Following a sub lethal M1 GAS challenge, we observed 83% survival (p<0.01) in J8-DT splenocyte-recipient mice compared to 16% in J8-DT B-cell or T-cell recipients by day 18, suggesting the requirement of both B and T-cells to mount a protective immune response (Figure 7 f).

Naïve T-cells can help J8-DT memory B-cells to mount a protective immune response

We next analysed the T-cell dependency of memory B-cell responses. Adoptive transfer experiments were conducted where naïve SCID mice were transfused with various combinations of J8-DT memory as well as naïve B or T-cells as depicted in Figure 8. Twenty-four hours following adoptive transfer, the mice were infected intravenously with 50,000 CFU of M1 GAS. As readouts of protection, bacterial bio-burden was followed in blood samples collected at various time-points. Our data demonstrated that the cohort recipient of J8-DT memory B and naïve T-cells were protected significantly better (p<0.01) compared to the cohort receiving naïve B/memory T-cells (Figure 9 a). These mice also fared significantly better than mice receiving memory B-cells alone or memory T-cell alone (Figures 9 c). The cohort recipients of memory splenocytes were able to clear bacterial burden very efficiently, similar to the mice receiving memory B/memory T-cells (Figure 9 b and d). Furthermore, these cohorts demonstrated an early decline in bacterial burden that led to faster clearance (figure 9 b and d) compared to the recipients of J8-DT memory B and naïve T-cells (Figure 9 a). As expected, the cohort recipient of naïve B/naïve T-cells or naïve splenocytes did not show any reduction in bio-burden until day 12 (Figures 9 b and d). These data further demonstrated the need for both B and T-cells in J8-DT mediated protection.

Figure 8.

Figure 8

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A schematic of experimental protocol employed to investigate the synergistic effect of J8-DT- B and T-cells in protection against GAS.

Figure 9. Adoptive transfer and assessment of protective efficacy of J8-DT memory and naïve B and/or T-cells.

Figure 9

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To investigate synergistic effect of memory and naïve B and T-cells in protection against GAS, SCID mice (n=5 per group) were transfused with combinations of B and T-cells from J8-DT-immunized-rested or naïve BALB/c mice. As illustrated in schematic figure 10, cohorts of mice were transfused with J8-DT-B-cells/naive T-cells or naive B/naive T-cells (a), J8-DT-B-cells/J8-DT-T-cells or naive B/naive T-cells (b), J8-DT-B-cells or J8-DT-T-cells (c) and J8-DT splenocytes or naive splenocytes (d). Twenty-four h post-adoptive transfer, the mice were given an IV infection with 50,000 CFU of GAS M1 strain. The bacterial bio-burden in the blood (left Y-axis) and serum J8-specific IgG titers (right Y-axis) were monitored at indicated time-points as shown. Data representative of three or more independent experiments and results are shown as means ± standard errors of the means for at least 5 mice in each group. Significance determined by two-way analysis of variance (ANOVA) where *p<0.05, **p<0.01, ***p<0.001.

Discussion

While antibodies have been implicated in protection mediated by J8-DT/alum (6, 21), here we show that exposure to GAS can stimulate memory B-cells to respond rapidly and that this response can protect mice from the extant infection. For several vaccines there is good evidence that immunity can persist after immunization when antigen-specific antibodies are no longer detected (27–30). It was not known whether this applied to GAS, although previous work showing that the degree of protection was proportional to the serum J8-specific antibody titer (6, 21, 31) suggested that antibody would need to be present in high titer at the time of challenge for protection. We were particularly keen to know what titer of antibody might correlate with protection. By adoptively transferring memory cells into naïve immunodeficient mice we have been able to show that the titer at the time of challenge is in fact not critical; rather it is the presence of a memory B-cell (MBC) population per se that is important. Memory T-cells alone offered no protection. It is known that MBCs continuously re- circulate though the body thus allowing them to encounter and react to antigens at tissue sites (32). Following appropriate stimulation, MBCs undergo rapid proliferation, culminating in differentiation into plasma cells and in the secretion of high affinity IgG. The quick rise in antibody levels that we observed coming after 5–7 days of stimulation confirms that they are coming from MBCs, as a new immune response would take 9–14 days to develop. It seems that this quick response is critical.

Long-lived plasma cells (LLPC) are also important for on-going protection. It is known that LLPCs can survive and continue to secrete antibodies for extended periods of time (> 1 year) (10). The majority of these non-dividing cells are present in the bone marrow and their main function is to continuously secrete large amounts of specific antibody. Thus, we observed that high levels of J8-specific IgG were present in the serum of mice immunized 110 days previously. At 110 days post-immunization, the majority of the ASC are detected in the bone marrow confirming that the source of the antibodies detected in serum were LLPCs. However, titers measured by ELISA had declined significantly over that time. For example, on day 110 post-immunization a 16-fold drop in J8-specific IgG titers was noticed. Thus, the vaccine-specific primary immune response had subsided and the major fraction of antibodies detected in the serum thereafter were coming from LLPC at that time.

Having shown that both vaccine antigen and GAS could boost a MBC response, we then analysed the role of T-cells in this activation. Some earlier studies suggested that MBCs require CD4+ T-cells for their survival (17, 33) and activation (34, 35). Thus, using a vesicular stomatitis virus (VSV) infection model Ochsenbein et.al. (17) have demonstrated that differentiation of MBCs into short lived antibody-secreting cells always depends on primed specific T-cell help. Similarly in another study a polyclonal stimulus by bystander T-cell help was shown to be critical for proliferation and differentiation of human MBCs (33). However, there are also data showing that boosting of a B-cell response does not require memory T-cell help (16, 36, 37). Our data serve to dissect this issue. We have demonstrated that MBCs with the help of T-cells generate an antibody response that protects mice from a GAS challenge. Furthermore, our data also demonstrate that the T-cell help for J8-DT-specific immunity can come from naive T-cells. Thus, naïve T-cells were capable of stimulating J8-DT MBCs resulting in a protective immune response post-exposure to either immunogen or the pathogen. This highlights the usefulness of the J8-DT vaccine that contains a minimal B-cell epitope along with T-cell epitopes that do not belong to the pathogen, but to diphtheria toxoid. It has recently been shown that memory T-cells that express B-cell follicle homing molecule, CXCR5, are capable of providing accelerated help to B-cells (38). We observed that naïve as well as memory T-cells when transferred with MBCs, were both capable of reducing GAS bio-burden. However, there was a difference in the kinetics of bacterial clearance between the two groups with the naive T-cell recipient cohort demonstrating a delayed decline in bacterial burden compared to the cohort receiving memory T-cells. This outcome is likely to be due to the delayed proliferation of naïve CD4+ T-cells resulting in delayed T-cell help to memory B-cells.

Apoptosis of MBCs following infection with Plasmodium (26) and Trypanosoma brucei (39) had been implicated in the abolition of long-term protection in mice. Our studies demonstrated that vaccination with J8-DT resulted in a memory B-cell response that was capable of responding to a low inoculum of GAS. Of interest was the observation that this memory response was also protective without any prior boosting. The demonstration that the memory B-cell response can reduce bacteremia following a high dose challenge was further reassuring.

In vitro proliferation assays showed that J8 did not contain a T-cell epitope from GAS since there was no recall proliferative response to either J8i (a minimal B cell epitope from within J8) or to J8. J8-DT was designed to contain the absolute minimum component of GAS (12 amino acids from the M-protein) and previous studies in different mouse strains (21, 24) and in humans (40) have also shown that it does not contain a dominant GAS-specific T-cell epitope. In the in vivo protection studies T-cells alone did not offer any protection, further suggesting their role is primarily in the regulation of vaccine-induced B-cell immunity, as cognate CD4+ T-helper cells. There was thus concern that GAS infection would not boost a vaccine induced memory response. In showing here that memory T-cells are not required for boosting an antibody response we have allayed many concerns that this vaccine would not induce enduring protection. Furthermore, based on these data there may be reason to reconsider vaccine design for many challenging organisms. For example, in malaria, the leading but suboptimal vaccine candidate, RTS,S, was designed to include T-cell epitopes from the coat protein of the parasite to augment antibody responses to the B-cell epitope (41). Those T-cell epitopes are limited and polymorphic in the organism (42) and as such it may have been better to use more potent, non-parasite-derived, T-cell epitopes in the vaccine.

In conclusion, we have demonstrated that J8-DT induced B-cell responses are long lasting and result in the development of antigen-specific memory B-cells and LLPCs. Activation of memory B-cells with antigenic stimulus or GAS infection results in a quick and effective antibody response that is capable of clearing systemic GAS and protecting mice. Most importantly our data demonstrate that activation of MBCs does not require memory T-cells. These findings not only have significant importance regarding the efficacy of the J8-DT vaccine but they hold significance for vaccine design against other pathogens.

Acknowledgements

We thank Mr Graham Magor for technical assistance in animal experiments and Ms Karen Phillipps for help with flow cytometry experiments.

Funding: This work was supported by the National Institutes of Health (NIH, Grant number 5U01AI060579-05)), USA and the National Heart Foundation of Australia (grant numbers G08 B3838 and G09 B4282).

Footnotes

Competing Interests: The authors have declared that no competing interests exist.

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