Edited by: Si-Yang Huang, Yangzhou University, China
Reviewed by: Marisa Silvia Castro, Institute of Studies on Humoral Immunity (IDEHU), Argentina; Pedro Xavier-Elsas, Federal University of Rio de Janeiro, Brazil
This article was submitted to Infectious Diseases - Surveillance, Prevention and Treatment, a section of the journal Frontiers in Public Health
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Infection treatment vaccine (ITV) can lead to sterile protection against malaria infection in mice and humans. However, parasite breakthrough is frequently observed post-challenge. The mechanism of rapid decline in protection after the last immunization is unclear. Herein, C57BL/6 mice were immunized with 103, 105, or 107 ITV thice at 14-day intervals. Mice were challenged with 103 parasites at 1, 3, and 6 months after last immunization and the protection was checked using blood smear. The phenotypes of B cells were analyzed by flow cytometry. The levels of serum cytokines were quantified using cytometric bead array. The 103 ITV vaccination group exhibited 100% protection at 1 month after last immunization, and the 105 group showed sterile protection at 3 months after last immunization. However, the 107 group showed only partial protection. Further, the protection declined to 16.7% at 6 months after last immunization in 105 and 107 groups, whereas it maintained for more than 60% in 103 group. The number of memory B cells (MBC) decreased along with the decline in protection. However, programmed cell death protein 1 (PD-1) expressed on MBCs did not show significant variation among the three groups. Interestingly, CD19+CD1dhiCD5hi B cells, defined as B10 cells, exhibited negative regulation with respect to protection. The numbers of CD19+CD1dhiCD5hi B cells in the 103 group at 1 months and in the 105 group at 3 months post-immunization were the lowest compared to those in the other groups. Moreover, the serum levels of interleukin 10 (IL-10) in these two groups were also significantly lower than those in other groups. We conclude that higher immunization dose may not lead to better protection with the malaria vaccine as CD19+CD1dhiCD5hi B cells can downregulate ITV protection against malaria via IL-10 secretion. These results could facilitate the design of an effective long-lasting malaria vaccine with the aim of maintaining MBC function.
Malaria is still one of the three most important infectious diseases worldwide, resulting in 228 million clinical cases and 405,000 deaths in 2018, mostly in Africa in children under 5 years of age (
The blood stage of the malarial parasite life cycle is responsible for all the clinical symptoms of malaria (
A malaria infection-treatment-vaccine (ITV) as anti-malaria drug prophylaxis is effective against the blood stage of Plasmodium (
Over the past decade, a number of studies have demonstrated that regulatory B cells (Bregs) are crucial in the maintenance of immune tolerance and suppression of inflammation (
In this study, we mainly explored the role of MBCs and B10 cells in immune dose-mediated long-term protection decline of malaria blood stage ITV, and clarified the role of PD-1 in MBC and B10-related cytokines in this process. This study will thus provide a new research direction to explore the mechanism of decline in long-term protection of ITV, and provide a theoretical basis for improving the long-term protection of malaria vaccine as well as for the design and application of a more effective vaccine.
Female C57BL/6 mice and BALB/c mice were obtained from the Hunan Silaike Jingda Laboratory Animal Co. Ltd. All mice ranged in age from 6 to 8 weeks when the experiments were initiated. All mice were maintained in the experimental animal centers of Guilin Medical University. The lethal strain of
First, cryopreserved
Before challenge, the absence of blood stage parasite infection was confirmed by Giemsa staining of thin blood smears. Mice were challenged with 103 Py-iRBCs by intravenous (i.v.) injection at 1, 3, and 6 months (mo) after the last immunization. Blood stage infection was examined daily from 3 days post-challenge to the days until parasitemia disappeared or the mice died. Parasitemia was calculated as the percentage of iRBCs.
Spleens were collected at 1, 3, and 6 months after the last immunization, and splenocytes were prepared as described previously (
Initially, 106 cells were resuspended in 50 μL fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline (PBS) supplemented with 2% heat-inactivated fetal bovine serum (FBS); Gemini Bio-Products). Then, the mixture was incubated for 10–15 min at 4°C. Next, 50 μL of the 2X antibody cocktail was added to each tube (for the unstained sample, 50 μL of FACS buffer was added), vortexed gently or tapped to mix, and incubated at 4°C for 20–40 min in dark. Next, after washing with FACS buffer, 200 μL FACS buffer was added to resuspend the cells. The cells were analyzed using FACSCanto II instrument (BD Biosciences, San Jose, CA, USA), and the data were analyzed with FlowJo version 10 software.
The whole blood of mice was obtained by cardiopuncture before euthanization, and serum was harvested by centrifugation. The levels of the proinflammatory cytokines interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), IL-12p70, and the anti-inflammatory cytokine, IL-10, in serum samples were quantified using the cytometric bead array (CBA) Mouse Inflammation Kit (BD Biosciences), according to the manufacturer's instructions. Briefly, mouse inflammation standards were prepared with 2 mL of assay diluent, and by doubling the dilution to 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, and 1:1,024. Six mouse inflammation capture beads were mixed thoroughly. Next, 50 μL of mixed capture beads were incubated with the same volume of each mouse inflammation standard dilution or each sample. Then, 50 μL mouse inflammation PE detection reagent was added to all assay tubes and incubated for 2 h at room temperature, in dark. After washing with washing buffer, samples were analyzed on a FACSCanto II instrument (BD Biosciences), and the data were analyzed with FCAP Array version 3 software (
The data were analyzed using GraphPad Prism version 5 software. Non-parametric tests (Mann-Whitney test) and two-way analysis of variance (ANOVA) were used to compare groups, and
First, we investigated the protection against
Protection against 103
BALB/c | PBS | 0% (0/5) | 0% (0/5) | 0% (0/5) |
105 ITV | 100% (5/5) | 100% (5/5) | 80% (4/5) | |
C57BL/6 | PBS | 0% (0/5) | 0% (0/5) | 0% (0/5) |
105 ITV | 100% (5/5) | 40% (2/5) | 20% (1/5) |
To explore the protection resulting from immune doses, we immunized groups of C57BL/6 mice with 103, 105, and 107 ITV and challenged them with 103 parasite iRBCs by i.v. injection at 1, 3, and 6 months after the last immunization (
Protection assay of different immunization dose groups.
Control | 0% (>60%) | 0% (>60%) | 0% (>60%) |
103 | 100% (–) | 60% (0.50%) | 66.7% (1.00%) |
105 | 60% (0.5%) | 100% (–) | 16.7% (0.90%) |
107 | 20% (1.86%) | 70% (2.50%) | 16.7% (2.90%) |
Mice in the control group showed detectable blood stage parasitemia 4 days after challenge and died at 19–21 days post-challenge. However, in immunized groups, the infection started from 3 to 7 days post-challenge and lasted with low-dose parasitemia only for 3–6 days (
In other words, malaria blood stage ITV long-term protection of the 107 group was generally worse than that of the 103 and 105 groups, and the long-term protection of ITV decreased with the prolongation of immunization time.
To characterize the protection resulting from immunization dose, we first observed the number of MBC variations in spleen. The results showed that the number of CD19+CD27mid MBCs in the three experimental groups decreased gradually over time (
The number of memory B cells and PD-1+ memory B cells in the spleens of the ITV-immunized mice.
In order to explore whether the immune dose could affect the increase in B10 cell abundance, splenocytes were collected and the number of CD19+CD1dhiCD5hi B cells was counted by flow cytometry. The results showed that at 1 month, the number of B10 cells of ITV-immunized mice in 103 and 105 groups was significantly lower than that in the control group (103:
The results showed that the immunized group with the best protective effect expressed the lowest number of B10 cells. This suggests that the expansion of B10 cells in ITV-immunized mice may result in reduced long-term protective efficacy of ITV.
Considering the important role of cytokines in the immunosuppressive effects of B10 cells (
At 1 and 3 months, the amount of IL-10 in the 103 and 105 groups was the lowest, respectively. Serum samples were collected from immunized mice at the indicated time points after the final immunization and the concentrations of cytokines were measured serially by a cytometric bead array (CBA). Two individual experiments were performed. The data are presented as the mean ± SD. Data were compared with the two-way analysis of variance (ANOVA). *
In addition, the level of IFN-γ was also inversely proportional to the level of IL-10 (
Taken together, the amount of IL-10 in mouse serum was positively correlated with the number of B10 cells, whereas the amount of IL-6 and IFN-γ was negatively correlated with the amount of IL-10. Thus, these data suggest that increased level of IL-10 may lead to decline in protection. The role of IL-6 and IFN-γ in this process needs to be verified by further experiments.
All clinical symptoms of malaria mainly occur due to the blood phase of the malaria parasite life cycle (
Herein, we explored the influence of immune dose on Plasmodium ITV protection longevity. C57BL/6 mice are more sensitive to
Studies have found that splenic MBCs played the most important role in the immune protection induced by Plasmodium blood stage ITV vaccine. The number of malaria parasite-specific MBCs is proportional to the immune protection of the host, and decrease of the number of specific MBCs leads to decrease in immune protection (
B10 cells, a small subset of CD19+CD24hiCD38hi B cells as well as CD19+CD1dhiCD5hi B cells in mice, are an immunosuppressive B cell type that stop the expansion of pathogenic, proinflammatory lymphocytes and play a significant role in suppressing autoimmune responses and preventing autoimmunity through the secretion of IL-10, IL-35, and transforming growth factor β (TGF-β) (
A previous study indicated that the main mechanism of the inhibitory action of B10 cells is IL-10 production (
In conclusion, we demonstrated that the immunization dose could mediate the long-term protection of Plasmodium blood stage ITV in C57BL/6 mice, and that a higher immunization dose could not produce a better long-term protective effect. The long-term protection of ITV was shown to be related to the increase in the number of CD19+CD1dhiCD5hi B (B10) cells, which may negatively regulate the long-term protective effect of malaria ITV by secreting IL-10. In addition, the decrease in long-term protection of ITV may be related to the decline of MBCs in the spleen of mice with an increase in immunization time. Our study thus elucidates the mechanism of high immunization doses inhibiting long-term immunity protection in a model of long-term protection decline of malaria blood stage ITV mediated by the immunization dose due to the regulatory role of B10 cells, thus providing a theoretical basis for the design of malaria vaccine.
All datasets generated for this study are included in the article/supplementary material.
This animal study was reviewed and approved by Animal Ethics Committee of the Guilin Medical University Institute of Medical Research.
HG, JP, and XL performed this study. HG analyzed the data. LJ collected samples. GM provided technical support. XP designed this work, supervised this study, and final revised the manuscript. HG drafted the work. JP, XL, LJ, and GM revised the work. All of the authors listed have approved the final version for published and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.