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The Journal of Immunology, 2005, 175: 2510-2516.
Copyright © 2005 by The American Association of Immunologists

Plasmodium yoelii Can Ablate Vaccine-Induced Long-Term Protection in Mice1

Michelle N. Wykes, Yong-Hong Zhou, Xue Q. Liu and Michael F. Good2

Molecular Immunology Laboratory, Queensland Institute of Medical Research, Brisbane, Queensland, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Malaria is a serious cause of morbidity and mortality for people living in endemic areas, but unlike many other infections, individuals exposed to the parasite do not rapidly become resistant to subsequent infections. High titers of Ab against the 19-kDa C-terminal fragment of the merozoite surface protein-1 can mediate complete protection in model systems; however, previous studies had not determined whether this vaccine generated long-term protection. In this study, we report that functional memory cells generated by merozoite surface protein-1, per se, do not offer any protection. This is because the parasite induces deletion of vaccine-specific memory B cells as well as long-lived plasma cells including those specific for bystander immune responses. Our study demonstrates a novel mechanism by which Plasmodium ablates immunological memory of vaccines, which would leave the host immuno-compromised.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Malaria is a serious cause of morbidity and mortality in millions of individuals each year. People living in endemic areas build up partial immunity only after repeated attacks of malaria over several years (1, 2, 3). As such, it is clear that a vaccine is required, and a number of vaccine candidates are being developed. The 19-kDa C-terminal fragment of the merozoite surface protein-1 (MSP119)3 and the related molecule, MSP142 are vaccines that can protect monkeys and mice from infection (4, 5, 6, 7, 8, 9, 10, 11, 12, 13).

In humans, studies on natural immunity to MSP119 have found that despite multiple infections, <20% of children in Sierre Leone and only 60% of adult Gambians possess Abs to Plasmodium falciparum-MSP-119 (PfMSP-119) indicating that even lifelong exposure to this protein may be insufficient to induce an Ab response (14). The reason why 40% of adult donors from cross-sectional studies do not possess Abs to PfMSP-119 is not known as all donors were known to have been exposed to malaria over many years (15). Other studies on natural immunity to MSP119 found a scarcity of IgG anti-MSP119 responses in areas of hyperendemicity, both in terms of the number of responders and intensity of the response, while IgM responses were elevated (16, 17). Similarly, IgM responses to other Plasmodium Ags, including the circumsporozoite protein, have also been found to be elevated in areas of endemnicity (18, 19, 20, 21). IgM Ab is a sign of a primary Ab response, usually expected in previously unexposed individuals. Moreover, studies in the nonendemic area of Ndiop and the endemic area of Dielmo (in Senegal), found that few individuals had IgG3 anti-MSP119 Ab, even 3–4 mo after an epidemic of malaria when the infection had cleared (22). Although the parasite could have absorbed the Ab during infection, it was expected that infection would boost the number of memory and plasma cells and that the number of patients accumulating IgG3 anti-MSP119 would increase with time. Furthermore, there are anecdotal data that individuals lose immunity when they leave endemic areas (23). In any case, taken together, the data suggest that immunological memory of the parasite is not consistent in endemic areas (24).

Thus, the current study was initiated to assess whether MSP119 generated memory B cells and long-lived plasma cells (LLPC) in mice, and to determine whether these cells protected against lethal, Plasmodium yoelii YM infection. This study has shown formally for the first time that the vaccine generates memory B cells and LLPC. However, these functional memory B cells, per se, do not protect mice against infection. We then showed that Plasmodium ablates immunological memory of the vaccine and LLPC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

SPF, 6–8 wk old, BALB/c mice, and SCID mice on a BALB/c background were obtained from the Animal Research Centre (Perth, Western Australia). These studies have been reviewed and approved by The Queensland Institute of Medical Research Animal Ethics Committee.

Infectious challenge and monitoring parasitemia

Mice were always challenged with a lethal dose of (104) P. yoelii YM-infected RBC (pRBC) given by i.v. injection. To measure the level of infection in the blood (parasitemia), drops of blood were taken by tail snips and smeared on a glass slide. The slides were stained with a Giemsa stain, and the numbers of infected cells in at least 400 red cells or 20 fields were counted. To confirm sterile immunity (complete clearance of parasite) in immunized mice, 50 µl of blood was transferred from these mice to naive mice and parasitemia was monitored for 3 wk.

Immunization protocol for protection studies

BALB/c mice were immunized with 50 µg of MSP119 in Freund’s complete adjuvant per mouse, given s.c., followed by three additional doses of 20 µg of MSP119 in Freund’s incomplete adjuvant. The second dose was given s.c. and then the last two doses were given i.p., with at least 3-wk intervals between each dose. This protocol was shown previously to induce complete protection against a lethal infection (7, 8). To control for the effects of the adjuvant, parallel cohorts of mice were given PBS in adjuvant using the same four-dose protocol. The mice were rested for rested for 2–12 wk as stated in specific experiments.

Immunization protocol for apoptosis studies

BALB/c mice were immunized with three (instead of four) doses of MSP119 or with two doses of diptheria toxoid (DT) or keyhole limpet hemocyanin (KLH), (100 µg/mouse) as described above. To control for the effects of the adjuvant, mice were immunized with PBS first in Freund’s complete and then in Freund’s incomplete adjuvant as for the MSP119 groups, and rested for 10–16 wk.

Cell transfer studies

To define protection by memory B cells, lymphocytes were enriched from spleens of immunized or PBS-immunized mice (after >10 wk) by removal of stromal and APCs that adhere to plastic at 37°C within 30 min. The lymphocytes were then transfused i.v. into SCID or sublethally irradiated (550 cGy), naive, BALB/c mice to separate memory cells from serum Ab in immunized mice.

In vivo activation of memory responses

To measure memory Ab responses in vivo, immunized mice with memory cells or recipient mice with transfused memory cells were boosted i.v. with 10 µg of MSP119. The memory IgG, anti-MSP119 responses were measured in the serum 7 days after boosting with the vaccine by an Ag-specific ELISA or after 4 days by an Ag-specific ELISPOT assay of spleen and bone marrow (BM) cells. To measure activation of memory cells following infection, immunized and rested mice were challenged with 104 P. yoelii YM pRBC i.v., and parasitemias were monitored every 2 days.

Flow cytometric labeling

To identify memory B cells and plasma cells, spleen or BM cells were labeled to detect surface CD19 (clone 1D3-FITC) and CD27 (clone LG.3A10-biotin; memory B cells) or CD138 (clone 281-2-biotin; plasma cells) (BD Pharmingen) expression followed by Streptavidin-Quantum Red (Sigma-Aldrich). To identify cells that were apoptosing or activated, cells were labeled to detect cytoplasmic active caspase-3 (PE; early apoptosis marker) or Ki-67 Ag (PE; marker of proliferation indicating activation). The latter were purchased from BD Biosciences as kits. Nonspecific labeling was blocked by preincubation with purified rat Ig in 5% BSA/PBS. Data were acquired and analyzed using CellQuest software, and labeling with 7-aminoactinomycin D or propidium iodide (PI) excluded dead cells. Between 7.5 x 105 and 1.4 x 106 cells from each sample were analyzed so that at least 5 x 104 (average 9.1 x 104) CD19+/CD27+ memory B cells or CD138+ plasma cells were analyzed for expression of active caspase-3 and Ki-67. Labeling with isotype control Abs was used to set gates. The absolute number of apoptosing or proliferating cells were calculated by multiplying the percentage of caspase-3+ or Ki-67+ cells by the percentage of memory B cells or plasma cells in each sample and adjusting for either the total number of splenocytes or 107 BM cells. Samples were never fixed and left overnight as the flurochromes were affected by fixation.

Flow cytometric sorting

To determine whether apoptosing CD138+ cells were MSP119-specific, BM was taken from MSP119-immunized mice that had been rested for 13 wk and then infected i.v. with 104 P. yoelii YM pRBC. After 7 days, the BM cells from individual mice were labeled to detect CD138, annexin V, and PI (all from BD Biosciences) following preincubation with 5% rat serum in 5% BSA/PBS to block nonspecific labeling. Cells from each mouse that were CD138+/annexin V+/PI– or CD138+/annexin V–/PI– were sorted into subpopulations on a Dako Cytomation MoFlo cell sorter. Data were acquired and analyzed using Summit software. Four replicates of 1–2 x 105 sorted cells or total BM cells were then placed on ELISPOT plates previously coated with 10 µg/ml MSP119 to count numbers of vaccine-specific LLPC. It was not possible to isolate apoptosing CD138+/annexin V+/PI– cells from uninfected mice as an additional control, due to their extremely low frequency without infection.

ELISPOT assay (25, 26)

Multiscreen-HA plates were coated with 10 µg/ml MSP119, DT, or KLH. In most experiments, isolated spleen or BM cells were directly tested for Ab-secreting cells (ASC) using the published method (25, 26). However, for data in Fig. 4c, apoptosing CD138+ cells were sorted and then tested for MSP119 specificity. Four replicates were used for each BM or spleen sample with three to six mice per group.



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  		FIGURE 4. Analysis of MSP119-specific LLPC following infection. Cohorts of three mice (in two repeat experiments) were immunized with MSP119 or PBS in adjuvant and then rested for 13–16 wk. These mice were then either infected with P. yoelii YM or given MSP119 and, after a further 7 days, the cells of the BM were analyzed in vitro. a, Numbers of LLPC secreting IgG anti-MSP119 Ab in BM, before and after boosting with MSP119 or following infection. The PBS-immunized mice had no anti-MSP119-secreting cells indicating specificity of the assay. b, Numbers of CD138+ plasma cells in the BM expressing active caspase-3, a marker of early apoptosis. c, Numbers of anti-MSP119-specific LLPC in populations of early apoptosing (annexin V+/PI–) or viable (annexin V–/PI–) CD138+ cells in BM, compared with total BM cells. d, Apoptosis of CD138 plasma cells in the BM, 3 and 7 days following infection or being given MSP119.

 
Limiting dilution assay

Frequencies of KLH- or DT-specific memory B cells were assessed using an ELISPOT based limiting dilution assay (25, 26).

ELISA

Ab titers were determined as previously described using ELISA plates coated with 0.5 µg/ml MSP119 (27). Curves of absorbance against serum dilution were plotted and the reciprocal dilution of the end titer determined based on the absorbance of serum from naive mice.

Statistics

Error bars shown are means ± SEM. Values of p were calculated using the Mann-Whitney nonparametric t test, with a two-sided tail, based on pooled data from two to four replicate experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Protection by MSP119

To determine whether MSP119 generated memory responses, cohorts of six mice (in two repeat experiments) were immunized with either MSP119 (7, 8) or with PBS in adjuvant. After 2 wk, anti-MSP119 titers were assessed in these mice to confirm that vaccination with MSP119 had generated high titers of vaccine-specific Ab (Fig. 1a). Other cohorts of MSP119- or PBS-immunized mice were infected i.v. with 104 pRBC to assess protection by the vaccine. The progression of infection was monitored in the blood (parasitemia) by taking blood smears. Although all PBS-immunized mice died within 8 days, all MSP119-immunized mice survived for >48 days (when testing by blood-transfer showed complete clearance of the parasite; Fig. 1b). These studies clearly showed that vaccination with MSP119 conferred excellent protection against infection, 2 wk after the completion of immunization. The death of all PBS-immunized mice showed that the adjuvant had no effect on protection or survival of mice.



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  		FIGURE 1. Anti-MSP119 titers and protection from infection following MSP119 or PBS immunizations. Cohorts mice were immunized with MSP119 or PBS. After 2 wk, data show anti-MSP119 titers in serum (a) and parasitemias following challenge with a lethal dose of P. yoelii YM (b). Mean titers are indicated within the bars in the figure. The data represent one of two experiments with groups of six mice, which had similar results.

 
To study long-lived protection, all the other cohorts of immunized mice were rested for 10 wk, as vaccine-specific primary immune cells subside in 4–6 wk (27, 28). Anti-MSP119 titers were then measured in the serum of these mice and they had extremely high titers of anti-MSP119 Ab (Fig. 2a). Because the half-life of IgG Abs are generally only 7–23 days long (depending on the isotype), these high titers reflected accumulation of Ab produced by LLPC and possibly some residual Ab from the short-lived plasma cells that could not be differentiated. LLPC have been shown to survive and secrete Ab for at least 1 year (26, 29, 30). To measure immunity and protection in these mice, parallel cohorts of mice then either were given a small dose (10 µg) of MSP119 to recall memory responses or infected to measure protection. Boosting of these mice with MSP119 activated memory B cells and increased IgG vaccine-specific Ab titers within 7 days (Fig. 2b) compared with levels before boosting (Fig. 2a). Average IgG1 anti-MSP119 titers were 8 x 106 before boosting with MSP119 (Fig. 2a) and increased by ~20% following boosting (Fig. 2b). IgG2a, IgG2b, and IgG3 titers increased by ~60, 10, and 600%, respectively, following boosting (Fig. 2b). When parallel cohorts of these immunized and rested mice were infected (with no prior boosting), all the mice survived infection but did develop patent parasitemia for >22 days (Fig. 2c).



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  		FIGURE 2. Protection by MSP119-specific Ab and/or memory cells or anti-MSP119 serum. Cohorts of six mice were immunized with MSP119 or PBS in adjuvant and were then rested for 10 wk. a, Anti-MSP119 titers were measured in immunized mice after resting for 10 wk. b, Anti-MSP119 titers were measured in immunized mice that were rested for 10 wk, then boosted with MSP119 and serum was then tested after a further 7 days (titers seen include accumulated Ab from LLPC as well as residual Ab from the immunization protocol (a) and Ab from activated memory cells). c, Parasitemias in cohorts of immunized mice that were rested for 10 wk and then challenged with a lethal dose of P. yoelii YM. d, Anti-MSP119 titers in SCID mice given memory cells from mice immunized with either MSP119 or PBS, which were then boosted with MSP119. e, Parasitemias in SCID mice given memory cells from mice immunized with either MSP119 or PBS and then challenged with a lethal dose of P. yoelii YM. f, Parasitemia in naive mice given serum from either naive or anti-MSP119-immune mice that were challenged with a lethal dose of P. yoelii YM. These experiments are a continuation of experiments in Fig. 1. Mean titers are indicated within or over the bars in the figure.

 
To measure protection by memory cells, per se, spleen cells from parallel cohorts of immunized and rested mice (a source of memory cells) were transferred to SCID mice (one spleen equivalent per mouse) to eliminate accumulated, circulating Ab. We further defined memory B cells by the production of high titers of IgG Ab within 7 days of exposure to the immunogen or infection (31). A new immune response would take 9–14 days to produce significant quantities of IgG Ab in serum. As expected, when recipient mice were boosted with MSP119, very high titers of memory Ab, specific for the vaccine were produced (Fig. 2d). However, when memory cells from an identical group of immunized and rested mice were transferred to SCID mice, which were then infected, the protection was equivalent to that mediated by cells from PBS-immunized mice (Fig. 2e). Although the memory cells were capable of producing Ab in uninfected mice (Fig. 2d), these cells did not even delay onset of parasitemia in parallel-infected mice.

Because mice with memory cells and accumulated Ab had survived a lethal infection (Fig. 2c), naive mice were given hyperimmune anti-MSP119 serum to determine the role of Ab in clearing infection independent of memory B cells. All mice cleared parasitemia (Fig. 2f), further indicating that while Ab accumulated from LLPC could protect mice, memory cells did not contribute to control of the parasite.

Apoptosis of memory B cells

To determine why MSP119-specific memory B cells were unable to confer any protection, cohorts of three mice (in three repeat experiments) were immunized with MSP119 or PBS and after 10–12 wk, when primary immune cells had subsided, spleen cells containing memory cells were transferred to sublethally irradiated naive mice. Recipients then either were infected or given MSP119 to activate MSP119-specific memory cells. After 4 days, when parasitemia was still subpatent (below detectable levels), an ELISPOT assay was used to enumerate the number of MSP119-specific memory cells that had differentiated into MSP119-specific ASC. These studies found a very significant reduction in the numbers of MSP119-specific ASC in the spleen of infected mice compared with mice boosted with MSP119 (p < 0.004; Fig. 3a). Similarly, infected mice had significantly fewer numbers of MSP119-specific ASC in their BM compared with mice boosted with MSP119 (p < 0.03; Fig. 3b). These experiments showed that memory B cells were either lost or not activated following infection. The absence of any MSP119-specific ASC in spleens of PBS-immunized mice showed that new ASC were not generated within 4 days of vaccine-boosting or infection (Fig. 3, a and b).



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  		FIGURE 3. Analysis of memory B cells following infection. Cohorts of three mice (in three repeat experiments) were immunized with MSP119 or PBS in adjuvant and then rested for 10 wk. The spleen cells from these mice were then transferred to irradiated mice, which were then either infected with P. yoelii YM or given MSP119 to activate memory B cells. The spleen and BM cells from the recipient mice were then studied in vitro after 4 days. a and b, The numbers of memory B cells that had differentiated into anti-MSP119-specific ASC and were resident in spleen (a) or had migrated to BM (b) were quantified by ELISPOT assays. c and d, Numbers of CD19+/CD27+ cells per spleen that expressed markers of proliferation (Ki67+) indicating activation (c) or early apoptosis (d; active caspase-3+).

 
To directly analyze these memory B cells following boosting with the vaccine or infection, we used flow cytometry. The expression of CD27 (in combination with CD19) is an accepted marker of memory B cells in humans (32, 33). In contrast, in mice, CD27 is acquired by Ag-primed B cells at a centroblast stage of development and is expressed on a lower frequency of mouse memory B cells (34). Thus, we hypothesized that a proportion of MSP119-specific memory B cells would also express CD27 and CD19. As such, we labeled spleen cells from mice in experiments related to Fig. 3, a and b, to determine whether CD19+/CD27+ cells were expressing markers of early apoptosis (active caspase-3) or proliferation (Ki-67), which indicated activation. We found that 6-fold more CD19+/CD27+ cells per spleen showed proliferation following vaccine-boosting compared with infection (p < 0.015; Fig. 3c). In contrast, 6-fold more CD19+/CD27+ cells per spleen underwent apoptosis following infection compared with vaccine-boosting. These observations indicated that CD19+/CD27+ (centroblasts and memory) B cells may be deleted by apoptosis following infection (p < 0.002; Fig. 3d). As such, data showing a significant loss of MSP119-specific ASC following infection, combined with the reduction in numbers of activated CD27+ B cells and increased numbers of apoptosing CD27+ B cells following infection, strongly suggested that the parasite was causing a loss of vaccine-specific memory B cells by inducing their apoptosis.

Apoptosis of LLPC

All plasma cells (short lived and LLPC) express CD138+, but after 10 wk, only LLPC survive in significant numbers in the BM (26). To determine whether Plasmodium affected LLPC, cohorts of three mice (in two repeat experiments) were immunized with either MSP119 or PBS in adjuvant to generate plasma cells and rested for 13 to 16 wk, after which only LLPC would survive. One half of each cohort of mice was infected, and the other half was boosted with MSP119. After 7 days, when parasitemia was still subpatent (below detectable levels) because of circulating Ab, numbers of MSP119-specific LLPC were quantified in the BM by an ELISPOT assay. Although the numbers of LLPC following boosting with MSP119 were equivalent to numbers before boosting, infection consistently reduced the numbers of MSP119-specific LLPC by >50% (p < 0.02; Fig. 4a). The absence of MSP119-specific LLPC in PBS-immunized mice confirmed that no new vaccine-specific plasma cells had migrated to the BM within 7 days of infection or boosting with the vaccine (Fig. 4a).

To determine whether LLPC were deleted by apoptosis, BM cells from the above cohorts of mice were labeled to determine whether CD138+ plasma cells were expressing markers of early apoptosis (active caspase-3). Significant apoptosis of CD138+ plasma cells was noted following infection of both MSP119-immunized (p < 0.002) and PBS-immunized (p < 0.01) mice compared with mice that were boosted with the vaccine (Fig. 4b). Moreover, because a small proportion of pre-B cells express CD138, we sorted apoptosing CD138+ cells from the BM of infected mice to show that they were vaccine-specific LLPC. For this, we sorted apoptosing (CD138+/annexin V+/PI–) or viable (CD138+/annexin V–/PI–) cells from the BM of MSP119-immunized and rested mice that had then been infected, and tested their MSP119 specificity by an ELISPOT assay. The apoptosing CD138+ cells had nearly 200-fold more MSP19-specific cells than the viable CD138+ cells confirming the loss of vaccine-specific LLPC during a malaria infection (p < 0.0001; Fig. 4c). We could not examine apoptosing (CD138+/annexin V+/PI–) cells in the MSP119-boosted group as their numbers were too low for sorting. Clearly the apoptosing CD138+ cells could not have been Ag-specific pre-B cells that do not secrete Ab. The apoptosis of plasma cells became evident between days 3 and 7 following infection (Fig. 4d).

Effects of Plasmodium on bystander immune responses

Because we had noted apoptosis of CD138+ cells in PBS-immunized mice (Fig. 4b) that did not have vaccine-specific cells, we then investigated whether LLPC specific for other immunogens were also apoptosing during infection. Cohorts of five mice (in two repeat experiments) were immunized with DT or KLH to generate memory B cells and LLPC, specific for these immunogens. These mice were then rested for 17 and 13 wk, respectively, so that primary cells could die and only memory cells and LLPC survived. One half of these cohorts of mice were infected and all mice were studied after 4 days.

These studies found significantly more apoptosis of CD138+ cells in the BM of mice immunized with DT (p < 0.03) or KLH (data not shown) following infection compared with uninfected control mice (Fig. 5a). ELISPOT assays confirmed that infected mice had ~50% fewer DT-specific (Fig. 5b) and KLH-specific (data not shown) LLPC than uninfected control mice in the BM (p < 0.003) and ~30% fewer LLPC in the spleen (p < 0.035). By comparison, apoptosis of CD19+/CD27+ cells (Fig. 5c) and frequencies of DT-specific (Fig. 5d) and KLH-specific memory B cells (data not shown) per spleen were equivalent between infected and uninfected mice. We thus concluded that following infection, significant numbers of LLPC of various specificities were deleted, but non-Plasmodium-specific memory cells were unaffected.



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  		FIGURE 5. Effects of Plasmodium on long-term non-Plasmodium immune responses. Cohorts of five mice (in two repeat experiments) with memory B cells and LLPC specific for DT were either infected with P. yoelii YM or uninfected and studied after 4 days. a, Numbers of CD138+ plasma cells in BM that expressed active caspase-3, a marker of early apoptosis. b, Numbers of DT-specific LLPC in the spleen and BM. c, Numbers of CD19+/CD27+ B cells that expressed active caspase-3. d, Frequency of DT-specific memory B cells (MBC) per spleen.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study shows a novel mechanism by which Plasmodium evades immunological memory of vaccines. We initially established that the vaccine MSP119 could generate memory B cells and LLPC. Studies on protection then found that while these cell were functional, memory B cells per se did not offer any protection. We thus studied memory B cells directly and have evidence that these cells apoptose following infection. Finally, we found that LLPC of various specificities, including those specific for diphtheria toxoid, were also deleted following infection.

Our studies had observed that following infection, 14-fold fewer MSP119-specific memory B cells differentiated into ASC, compared with boosting with the vaccine. We also found 6-fold more apoptosis of CD19+/CD27+ B cells (which represent a proportion of memory B cells) and 6-fold lesser activation of these cells. The significant reduction in numbers of vaccine-specific ASC suggested that either memory B cells received direct apoptotic signals during activation or that these cells were not activated. Furthermore, another study has also found that numbers of apoptotic B cells are significantly increased during malaria (35) also indicating that Plasmodium affects B cell survival. There are suggestions that the disulphide bonds in MSP119 can impede processing of the Ag as MSP-119 in whole parasite has to compete with other parasite Ags for presentation (36). Although problems with processing of MSP119 may explain why fewer memory B cells differentiated into ASC, it does not explain why these cells underwent apoptosis. Another possibility is that the survival of CD4+ T cells may be compromised during infection (37, 38, 39), and this affects the survival or activation of memory B cells. Memory B cells require CD4+ T cells for their survival (40) and activation (28). As a final point, a very recent study of memory B cells in endemic areas found that exposure to malaria did not result in the establishment of stable populations of circulating Ag-specific memory B cells (41). In our studies, we have only investigated the deletion of MSP119-specific memory B cells in mice but there is evidence that IgM responses to other Plasmodium Ags are elevated in areas of endemnicity (18, 19, 20, 21), suggesting a possible problem with memory responses to the parasite. We thus speculate that repeated infection of humans could delete circulating parasite-specific memory B cells but further studies are required.

The current study also found that LLPC, specific for the vaccine as well as other immunogens, underwent apoptosis following infection. The apoptosis of LLPC in the BM suggests that deletion is initiated by inflammatory mediators that are released in response to infection, as these plasma cells are generally not known to interact with other cell types. The apoptosis of these LLPC in the BM was not likely to be a result of changes in homeostasis (42), as few parasite-specific plasma cells would develop in the spleen and migrate to the BM within in 7 days of infection. In fact none were noted following infection of PBS-immunized mice (Fig. 4a). Most new plasma cells would still predominate in the spleen (Fig. 3, d and e). Nonetheless, if an acute malaria infection does cause such severe homeostatic changes that result in the apoptosis of LLPC in the BM, these observations have significant implications for general immunity of individuals with clinical malaria.

Although our studies suggest that long-term protection by vaccines that mediate protection via Abs may not be viable, other studies to date on immunological memory following malaria have not seen significant problems. Those studies have focused on memory responses to the whole parasite, in many cases using an infection and cure regime to generate immunological memory (43, 44). In this case, the parasite would generate memory T cells and B cells and it is not easy to differentiate the contribution of each cell type in long-term protection. These protocols could also generate LLPC, which could secrete Ab for years to maintain protective Ab levels. Thus, we examined the function of memory cells independent of circulating Ab by transferring memory B cells into naive mice. Moreover, we used MSP119, which has clearly been shown to mediate protection via Ab alone, independent of T cell mediated protection (7). As such, any protection would be mediated by Ab produced by memory B cells. Furthermore, we also assessed whether memory B cells could produce Ab rapidly enough to offer protection and found that this was not the case.

Our study used Freund’s complete adjuvant, as MSP119 had previously been shown with this adjuvant to mediate complete protection and we wanted to determine whether memory responses generated with this adjuvant would also mediate protection. We found that all PBS (in adjuvant)-immunized mice died, indicating that the adjuvant alone had no effect on protection or survival of mice. Furthermore, the memory responses were not protective. As such, the adjuvant selected probably determines the quality and quantity of the immune response but otherwise does not directly affect protection.

Finally, in the context of human disease, our observations have two very significant implications for human vaccines: 1) while an immunized individual may control an initial infection due to accumulated Ab, the deletion of memory B cells and LLPC would leave the host susceptible to reinfection; 2) the deletion of bystander LLPC renders the host susceptible to other infections. Of course, not all cells may be deleted before new protective immune responses are generated and the infectious process could produce new memory B cells and LLPC. Nonetheless, while there are numerous valid arguments indicating that memory B cells persist in humans in the absence of Ag (24), there is no explanation as to why there is a scarcity of IgG responses against Plasmodium in areas of hyperendemicity (16), or why a significant proportion of west Africans do not possess IgG Abs to PfMSP-119 although they were exposed to malaria (15). As such, at this point, the apoptosis of IgG memory B cells and LLPC following infection is a logical explanation.


   Acknowledgments
 
We sincerely thank Dr. A. Saul for generously providing MSP119, to make this study possible. We also thank Drs. M. Y. Sangster, R. Thomas, G. Hill, J. Sprent, and E. Riley for their critical reading or discussion of this manuscript. We also thank Drs. L. Miller and A. Krettli for their intellectual input into the design of the immunization experiments.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Financial support was provided by the United Nations Development Program/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases, and National Health and Medical Research Council of Australia. Back

2 Address correspondence and reprint requests to Dr. Michael F. Good, Queensland Institute of Medical Research, The Bancroft Centre, 300 Herston Road, Brisbane, Queensland, Australia 4029. E-mail address: michaelG{at}qimr.edu.au Back

3 Abbreviations used in this paper: MSP119, merozoite surface protein-1; BM, bone marrow; ASC, Ab-secreting cell; LLPC, long-lived plasma cell; PfMSP-119, Plasmodium falciparum-MSP-119; pRBC, P. yoelii YM-infected RBC; DT, diptheria toxoid; KLH, keyhole limpet hemocyanin; PI, propidium iodide. Back

Received for publication March 24, 2005. Accepted for publication May 18, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Barragan, A., P. G. Kremsner, W. Weiss, M. Wahlgren, J. Carlson. 1998. Age-related buildup of humoral immunity against epitopes for rosette formation and agglutination in African areas of malaria endemicity. Infect. Immun. 66: 4783-4787. [Abstract/Free Full Text]
  2. Bull, P. C., B. S. Lowe, M. Kortok, C. S. Molyneux, C. I. Newbold, K. Marsh. 1998. Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria. Nat. Med. 4: 358-360. [Medline]
  3. Soe, S., A. Khin Saw, A. Htay, W. Nay, A. Tin, S. Than, C. Roussilhon, J. L. Perignon, P. Druilhe. 2001. Premunition against Plasmodium falciparum in a malaria hyperendemic village in Myanmar. Trans. R. Soc. Trop. Med. Hyg. 95: 81-84. [Medline]
  4. Daly, T. M., C. A. Long. 1995. Humoral response to a carboxyl-terminal region of the merozoite surface protein-1 plays a predominant role in controlling blood-stage infection in rodent malaria. J. Immunol. 155: 236-243. [Abstract]
  5. Blackman, M. J., A. A. Holder. 1992. Secondary processing of the Plasmodium falciparum merozoite surface protein-1 (MSP1) by a calcium-dependent membrane-bound serine protease: shedding of MSP133 as a noncovalently associated complex with other fragments of the MSP1. Mol. Biochem. Parasitol. 50: 307-315. [Medline]
  6. Good, M. F., D. C. Kaslow, L. H. Miller. 1998. Pathways and strategies for developing a malaria blood-stage vaccine. Annu. Rev. Immunol. 16: 57-87. [Medline]
  7. Hirunpetcharat, C., J. H. Tian, D. C. Kaslow, N. van Rooijen, S. Kumar, J. A. Berzofsky, L. H. Miller, M. F. Good. 1997. Complete protective immunity induced in mice by immunization with the 19-kilodalton carboxyl-terminal fragment of the merozoite surface protein-1 (MSP119) of Plasmodium yoelii expressed in Saccharomyces cerevisiae: correlation of protection with antigen-specific antibody titer, but not with effector CD4+ T cells. J. Immunol. 159: 3400-3411. [Abstract]
  8. Hirunpetcharat, C., P. Vukovic, X. Q. Liu, D. C. Kaslow, L. H. Miller, M. F. Good. 1999. Absolute requirement for an active immune response involving B cells and Th cells in immunity to Plasmodium yoelii passively acquired with antibodies to the 19-kDa carboxyl-terminal fragment of merozoite surface protein-1. J. Immunol. 162: 7309-7314. [Abstract/Free Full Text]
  9. Kumar, S., A. Yadava, D. B. Keister, J. H. Tian, M. Ohl, K. A. Perdue-Greenfield, L. H. Miller, D. C. Kaslow. 1995. Immunogenicity and in vivo efficacy of recombinant Plasmodium falciparum merozoite surface protein-1 in Aotus monkeys. Mol. Med. 1: 325-332. [Medline]
  10. Hui, G. S., C. Nikaido, C. Hashiro, D. C. Kaslow, W. E. Collins. 1996. Dominance of conserved B-cell epitopes of the Plasmodium falciparum merozoite surface protein, MSP1, in blood-stage infections of naive Aotus monkeys. Infect. Immun. 64: 1502-1509. [Abstract]
  11. Kumar, S., W. Collins, A. Egan, A. Yadava, O. Garraud, M. J. Blackman, J. A. Guevara Patino, C. Diggs, D. C. Kaslow. 2000. Immunogenicity and efficacy in aotus monkeys of four recombinant Plasmodium falciparum vaccines in multiple adjuvant formulations based on the 19-kilodalton C terminus of merozoite surface protein 1. Infect. Immun. 68: 2215-2223. [Abstract/Free Full Text]
  12. Kumar, S., F. Villinger, M. Oakley, J. C. Aguiar, T. R. Jones, R. C. Hedstrom, K. Gowda, J. Chute, A. Stowers, D. C. Kaslow, et al 2002. A DNA vaccine encoding the 42 kDa C-terminus of merozoite surface protein 1 of Plasmodium falciparum induces antibody, interferon-{gamma} and cytotoxic T cell responses in rhesus monkeys: immuno-stimulatory effects of granulocyte macrophage-colony stimulating factor. Immunol. Lett. 81: 13-24. [Medline]
  13. Singh, S., M. C. Kennedy, C. A. Long, A. J. Saul, L. H. Miller, A. W. Stowers, S. Kumar, F. Villinger, M. Oakley, J. C. Aguiar, et al 2003. Biochemical and immunological characterization of bacterially expressed and refolded Plasmodium falciparum 42-kilodalton C-terminal merozoite surface protein 1. Infect. Immun. 71: 6766-6774. [Abstract/Free Full Text]
  14. Egan, A. F., J. Morris, G. Barnish, S. Allen, B. M. Greenwood, D. C. Kaslow, A. A. Holder, E. M. Riley, J. A. Chappel, P. A. Burghaus, et al 1996. Clinical immunity to Plasmodium falciparum malaria is associated with serum antibodies to the 19-kDa C-terminal fragment of the merozoite surface antigen, PfMSP-1. J. Infect. Dis. 173: 765-769. [Medline]
  15. Egan, A. F., J. A. Chappel, P. A. Burghaus, J. S. Morris, J. S. McBride, A. A. Holder, D. C. Kaslow, E. M. Riley. 1995. Serum antibodies from malaria-exposed people recognize conserved epitopes formed by the two epidermal growth factor motifs of MSP119, the carboxy-terminal fragment of the major merozoite surface protein of Plasmodium falciparum. Infect. Immun. 63: 456-466. [Abstract]
  16. Nguer, C. M., T. O. Diallo, A. Diouf, A. Tall, A. Dieye, R. Perraut, O. Garraud. 1997. Plasmodium falciparum- and merozoite surface protein 1-specific antibody isotype balance in immune Senegalese adults. Infect. Immun. 65: 4873-4876. [Abstract]
  17. Nambei, W. S., M. Goumbala, A. Spiegel, A. Dieye, R. Perraut, O. Garraud. 1998. Imbalanced distribution of IgM and IgG antibodies against Plasmodium falciparum antigens and merozoite surface protein-1 (MSP1) in pregnancy. Immunol. Lett. 61: 197-199. [Medline]
  18. Boudin, C., B. Chumpitazi, M. Dziegiel, F. Peyron, S. Picot, B. Hogh, P. Ambroise-Thomas. 1993. Possible role of specific immunoglobulin M antibodies to Plasmodium falciparum antigens in immunoprotection of humans living in a hyperendemic area, Burkina Faso. J. Clin. Microbiol. 31: 636-641. [Abstract/Free Full Text]
  19. Achidi, E. A., H. Perlmann, L. S. Salimonu, P. Perlmann, O. Walker, M. C. Asuzu. 1995. A longitudinal study of seroreactivities to Plasmodium falciparum antigens in Nigerian infants during their first year of life. Acta Trop. 59: 173-183. [Medline]
  20. Nagendran, K., R. Ramasamy. 1996. Isotypes of naturally acquired antibodies to a repetitive and non-repetitive epitope on Plasmodium falciparum surface proteins in an endemic area of Sri Lanka. Indian J. Med. Res. 103: 142-145. [Medline]
  21. Brabin, B. J., L. Brabin, G. Crane, K. P. Forsyth, M. P. Alpers, H. J. van der Kaay. 1989. Two populations of women with high and low spleen rates living in the same area of Madang, Papua New Guinea, demonstrate different immune responses to malaria. Trans. R. Soc. Trop. Med. Hyg. 83: 577-583. [Medline]
  22. Diallo, T. O., A. Spiegel, A. Diouf, L. Lochouarn, D. C. Kaslow, A. Tall, R. Perraut, O. Garraud. 2002. Short report: differential evolution of immunoglobulin G1/G3 antibody responses to Plasmodium falciparum MSP119 over time in malaria-immune adult Senegalese patients. Am. J. Trop. Med. Hyg. 66: 137-139. [Abstract]
  23. Jelinek, T., C. Schulte, R. Behrens, M. P. Grobusch, J. P. Coulaud, Z. Bisoffi, A. Matteelli, J. Clerinx, M. Corachan, S. Puente, et al 2002. Imported falciparum malaria in Europe: sentinel surveillance data from the European network on surveillance of imported infectious diseases. Clin. Infect. Dis. 34: 572-576. [Medline]
  24. Struik, S. S., E. M. Riley. 2004. Does malaria suffer from lack of memory?. Immunol. Rev. 201: 268-290. [Medline]
  25. Slifka, M., R. Ahmed. 1996. Limiting dilution analysis of virus-specific memory B cells by an ELISPOT assay. J. Immunol. Methods 199: 27-35. [Medline]
  26. Slifka, M. K., M. Matloubian, R. Ahmed. 1995. Bone marrow is a major site of long-term antibody production after acute viral infection. J. Virol. 69: 1895-1902. [Abstract]
  27. van Essen, D., H. Kikutani, D. Gray. 1995. CD40 ligand-transduced co-stimulation of T cells in the development of helper function. Nature 378: 620-623. [Medline]
  28. Gray, D., P. Dullforce, S. Jainandunsing. 1994. Memory B cell development but not germinal center formation is impaired by in vivo blockade of CD40-CD40 ligand interaction. J. Exp. Med. 180: 141-155. [Abstract/Free Full Text]
  29. Slifka, M. K., R. Antia, J. K. Whitmire, R. Ahmed. 1998. Humoral immunity due to long-lived plasma cells. Immunity 8: 363-372. [Medline]
  30. Slifka, M. K., R. Ahmed. 1998. Long-lived plasma cells: a mechanism for maintaining persistent antibody production. Curr. Opin. Immunol. 10: 252-258. [Medline]
  31. Ochsenbein, A. F., D. D. Pinschewer, S. Sierro, E. Horvath, H. Hengartner, R. M. Zinkernagel. 2000. Protective long-term antibody memory by antigen-driven and T help-dependent differentiation of long-lived memory B cells to short-lived plasma cells independent of secondary lymphoid organs. Proc. Natl. Acad. Sci. USA 97: 13263-13268. [Abstract/Free Full Text]
  32. Klein, U., K. Rajewsky, R. Kuppers. 1998. Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells. J. Exp. Med. 188: 1679-1689. [Abstract/Free Full Text]
  33. Tangye, S. G., Y. J. Liu, G. Aversa, J. H. Phillips, J. E. de Vries. 1998. Identification of functional human splenic memory B cells by expression of CD148 and CD27. J. Exp. Med. 188: 1691-1703. [Abstract/Free Full Text]
  34. Xiao, Y., J. Hendriks, P. Langerak, H. Jacobs, J. Borst. 2004. CD27 is acquired by primed B cells at the centroblast stage and promotes germinal center formation. J. Immunol. 172: 7432-7441. [Abstract/Free Full Text]
  35. Helmby, H., G. Jonsson, M. Troye-Blomberg. 2000. Cellular changes and apoptosis in the spleens and peripheral blood of mice infected with blood-stage Plasmodium chabaudi chabaudi AS. Infect. Immun. 68: 1485-1490. [Abstract/Free Full Text]
  36. Hensmann, M., C. Li, C. Moss, V. Lindo, F. Greer, C. Watts, S. A. Ogun, A. A. Holder, J. Langhorne. 2004. Disulfide bonds in merozoite surface protein 1 of the malaria parasite impede efficient antigen processing and affect the in vivo antibody response. Eur. J. Immunol. 34: 639-648. [Medline]
  37. Hirunpetcharat, C., M. F. Good. 1998. Deletion of Plasmodium berghei-specific CD4+ T cells adoptively transferred into recipient mice after challenge with homologous parasite. Proc. Natl. Acad. Sci. USA 95: 1715-1720. [Abstract/Free Full Text]
  38. Wipasa, J., H. Xu, A. Stowers, M. F. Good. 2001. Apoptotic deletion of Th cells specific for the 19-kDa carboxyl-terminal fragment of merozoite surface protein 1 during malaria infection. J. Immunol. 167: 3903-3909. [Abstract/Free Full Text]
  39. Xu, H., J. Wipasa, H. Yan, M. Zeng, M. O. Makobongo, F. D. Finkelman, A. Kelso, M. F. Good. 2002. The mechanism and significance of deletion of parasite-specific CD4+ T cells in malaria infection. J. Exp. Med. 195: 881-892. [Abstract/Free Full Text]
  40. Bernasconi, N. L., E. Traggiai, A. Lanzavecchia. 2002. Maintenance of serological memory by polyclonal activation of human memory B cells. Science 298: 2199-2202. [Abstract/Free Full Text]
  41. Dorfman, J. R., P. Bejon, F. M. Ndungu, J. Langhorne, M. M. Kortok, B. S. Lowe, T. W. Mwangi, T. N. Williams, K. Marsh. 2005. B cell memory to 3 Plasmodium falciparum blood-stage antigens in a malaria-endemic area. J. Infect. Dis. 191: 1623-1630. [Medline]
  42. Sze, D. M., K. M. Toellner, C. Garcia de Vinuesa, D. R. Taylor, I. C. MacLennan. 2000. Intrinsic constraint on plasmablast growth and extrinsic limits of plasma cell survival. J. Exp. Med. 192: 813-821. [Abstract/Free Full Text]
  43. Carter, R., R. W. Gwadz, I. Green. 1979. Plasmodium gallinaceum: transmission-blocking immunity in chickens. II. The effect of antigamete antibodies in vitro and in vivo and their elaboration during infection. Exp. Parasitol. 47: 194-208. [Medline]
  44. Gwadz, R. W., L. C. Koontz. 1984. Plasmodium knowlesi: persistence of transmission blocking immunity in monkeys immunized with gamete antigens. Infect. Immun. 44: 137-140. [Abstract/Free Full Text]

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