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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

In our previous studies, we found that plague vaccines can induce long-term antibody response, but no significant antibody boost was observed when the immunized mice were challenged with virulent Yersinia pestis. However, a booster vaccination of subunit vaccine on week 3 after primary immunization elicited a significantly higher antibody titre than a single dose, whereas no significant antibody titre difference was observed between a single dose and two doses of EV76 vaccination. To address these issues, in this study, we first investigated the kinetics of memory B cells and plasma cells in the mice immunized with EV76 or F1 protein by flow cytometry and then determined antibody titre in five groups of mice immunized with various vaccination strategy. The results showed that memory B cells dropped to a low level at day 56 after primary immunization. In contrast, plasma cells were maintained for more than 98 days. The group with primary immunization of EV76 and booster of F1 antigen developed a higher antibody titre than the group with immunization of F1 antigen and booster of EV76. This result supports a hypothesis that an excess of antigens can neutralize pre-existing antibodies, and then the redundant antigen induces antibody boost. Taken together, a boost of antibody titre after revaccination may be dependent on the existence of memory B cells and an excess of antigen vaccination. In addition, this study showed an ideal immunization strategy that involves first immunization with a live attenuated vaccine, such as EV76, and then with a subunit vaccine.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Plague is a zoonotic disease caused by the gram-negative bacterium Yersinia pestis, which is usually transmitted to humans from infected rodents via the bite of an infected flea [1]. Historically, plague was a lethal infectious disease that changed the path of human's civilization. Plague has been recently classified as a re-emerging disease by the World Health Organization [2] and has attracted considerable attention because of its potential misuse as an agent of biological warfare or bioterrorism [3]. To prevent this disease, both live attenuated vaccines (LAV) and killed whole-cell vaccines (KCV) against plague have been used in humans since the early part of the 20th century. However, KCV do not protect against pneumonic plague in addition to bubonic plague [4]. The LAV EV76 is effective against bubonic and pneumonic plagues, but it has side effects of varying severity and has not been used in the Western world at present [4-6]. The DNA and subunit vaccines based on Y. pestis F1 and LcrV antigens alone or in combination were efficacious against both bubonic and pneumonic plagues and had obvious advantages over traditional vaccines in terms of efficacy or safety and are being developed currently [4, 7-10].

In our previous work, to develop a safe and effective plague subunit vaccine, highly purified native F1 antigen from Y. pestis EV76 was extracted using a new purification strategy [11], and a non-tagged rV270 protein containing amino acids 1–270 of LcrV was prepared from recombinant Escherichia coli BL21 cells using thrombin digestion [12]. The subunit vaccine, which comprises a dose 20 μg F1 and 10 μg rV270 that were adsorbed to 25% (v/v) alhydrogel in PBS buffer (SV1), provides a good protective efficacy against Y. pestis challenge in mice, guinea pigs, rabbits [9] and rhesus macaques [10]. In addition, long-term protection and antibody response for LAV EV76 and the subunit vaccine F1 + rV270 were determined in mouse model. The complete protection against lethal subcutaneous challenge of Y. pestis could be achieved up to day 518 after primary immunization. Antibodies to F1 and rV270 were still detectable over a period of 518 days. Interestingly, after challenging with Y. pestis on day 56, 126 or 518 post-primary immunization, no significant anti-F1 antibody titre boost was observed in the group SV1 or the group EV76 [13]. This result seems not to be consistent with the conclusion that memory B cells can quickly produce more antibodies when they are exposed to the same antigen [14, 15].

Long-lived plasma cells and memory B cells are responsible for the long-term humoral immunity elicited by vaccination. The serum antibody level is maintained by long-lived plasma cells. Memory B cells are in charge of driving the rapid anamnestic antibody response that occurs after re-exposure to antigen [16-18]. Memory B cells also play a role in replenishing the pool of long-lived plasma cells to maintain long-term antibody level in the absence of pathogen [18, 19]. To explore the reason why no significant anti-F1 antibody titre boost was observed in the animals immunized with plague vaccines after challenging with a virulent Y. pestis strain, the kinetics of memory B cell and plasma cell response was investigated in the mice immunized with plague subunit vaccine F1 or the live attenuated vaccine EV76 in this study.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References
Vaccines and animals

The F1 antigen was adsorbed to 25% (v/v) aluminium hydroxide in PBS to give the subunit vaccine containing 20 μg of F1 antigen. The live attenuated vaccine EV76 was obtained from the Lanzhou Institute of Biological Products (LIBP), China. BALB/c mice were obtained from Laboratory Animal Research Center, Academy of Military Medical Science, China. All protocols were approved by Committee of the Welfare and Ethics of Laboratory Animals, Beijing Institute of Microbiology and Epidemiology. All animal experiments were conducted strictly in compliance with the Regulations of Good Laboratory Practice for non-clinical laboratory studies of drug issued by the National Scientific and Technologic Committee of People's Republic of China.

Antibodies and reagents

Alexa Fluor 488-conjugated anti-mouse CD38, isotype: rat IgG2a, κ, Percp-Cy™5.5-conjugated anti-mouse CD27 and isotype: Armenian Hamster IgG were purchased from BioLegend (San Diego, CA, USA). Phycoerythrin (PE)-conjugated rat anti-mouse IgD, isotype: rat IgG2a, κ, allophycocyanin (APC)-conjugated anti-mouse CD19 and isotype: rat (LEW) IgG2a, κ were obtained from BD Pharmingen™ (San Diego, CA, USA). Single-colour control antibodies: Alexa Fluor 488 anti-mouse CD19 was obtained from BioLegend; PE rat anti-mouse CD19 and PerCP-Cy™5.5 rat anti-mouse CD19 were purchased from BD Pharmingen.

Animal immunizations

For determination of memory B cells and plasma cells, three groups of mice were immunized with EV76 [1/10 of the human dose (8 × 108 cells)], F1-Alu and Alu by subcutaneous route, respectively, and then given a boost immunization 28 days after the primary immunization. To investigate whether revaccination can induce a boost of memory B cell population again, another two groups of mice were immunized with F1-Alu or Alu at day 0, 28 and 77 by subcutaneous route. For determination of anamnestic Ab responses after boost immunization, another five groups of female BALB/c mice were used. Group I was only given a primary immunization of 1 of 5 of the human dose of EV76; group II was given a primary and secondary immunization of 1 of 10 of the human dose of EV76; group III was first given a primary immunization of 1 of 10 of the human dose of EV76, and then 20 μg of F1 antigen; group IV was first given a primary immunization of 20 μg of F1 antigen, and then 1 of 10 of the human dose of EV76; group V (control group) was given two injections of aluminium hydroxide.

Preparation of cell suspensions

Mice were killed on day 4, 7, 28, 42, 56, 77 and 98 after immunization, spleens were removed, and single-cell suspensions were prepared as previously described [20]. Briefly, spleens were forced through 300 mesh copper nets and suspended in PBS, respectively. Bone marrow cells from the immunized mice were collected by flushing the femurs and tibias with PBS [21]. Single-cell suspensions were obtained by passing the cells through 300 mesh copper nets. The cells were collected by centrifugation at 300 g for 10 min and then lysed with ACK lysis buffer (Invitrogen, Carlsbad, CA, USA) for 15 min. 1 × 106 cells were collected in a tube by counting with a light microscope prior to antibody staining.

Flow cytometry analysis

B cell subsets were determined on day 4, 7, 14, 28, 42, 56, 77 or 98 after primary immunization with F1 subunit vaccine or EV76 using flow cytometry. The splenic and bone marrow cells were incubated with a mixture of APC-labelled anti-mouse CD19, PerCP-Cy5.5-labelled anti-mouse CD27, Alexa Fluor 488-labelled anti-mouse CD38 and PE-labelled anti-mouse IgD. Cell staining and flow cytometric analysis were executed as previously reported [22]. At least 105 gated events were acquired using a FACS Calibur flow cytometer (BD Biosciences, San Diego, CA, USA) and analysed using the winmdi software (version 2.8; The Scripps Research Institute, San Diego, CA, USA).

F1-specific antibody assays

Sera collected from the immunized and control mice were assayed for the presence of F1-specific IgG by a modified ELISA [7]. The titre of specific antibody was estimated as the maximum dilution of the serum with an OD value of 0.2 units over background. Background values were obtained from serum samples collected from the animals of the group V.

Statistics

The differences of memory B cell and plasma cell percentage among groups of mice were compared by analysis of variance (ANOVA) with sas 8.0 software (SAS, Raleigh, NC, USA). Differences in antibody titre among groups at different time points were analysed with the Student's t-test. A probability value of < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Kinetics of memory B cell responses

To investigate whether or not memory B cells arise after inoculation of plague vaccines, the frequencies of memory B cells were determined within the spleens and bone marrow using flow cytometry. The spleen and bone marrow cells were harvested from six mice immunized with EV76 or F1-subunit vaccine on day 4, 7, 14, 28, 42, 56, 77 or 98 after primary immunization, respectively. Following staining of spleen and bone marrow cells, the flow cytometric analysis was performed, and the results were shown in Fig. 1. The number of CD27+ memory B cells in the spleens or bone marrow increased initially on day 7, reached its peak in the spleens or bone marrow on day 42 or 56, respectively, after primary immunization, and then declined sharply to a low level in the spleens or bone marrow on day 56 or 77 after primary immunization. Statistical analysis showed that there was no significant memory B cell number difference between the F1 subunit vaccine and EV76 groups both in the spleens and in the bone marrow (> 0.05), but a significant difference in memory B cell population was observed between the spleens and bone marrow (< 0.05).

image

Figure 1. Memory B cells generated in the spleens and bone marrow of the BALB/c mice immunized with EV76 or F1 antigen after two doses of immunization on day 0 and 28. (S) represents ‘spleen’ and (B) represents ‘bone marrow’.

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Plasma cell responses

Following the staining of spleen and bone marrow cells, the distribution of plasma cells in the spleen and bone marrow was analysed using flow cytometry (Fig. 2). The number of CD38+ plasma cells in the spleens was elevated from day 14, reached peak on day 42 after primary immunization and then entered stationary phase on day 56 in both the mice immunized with EV76 and those immunized with F1 subunit vaccine. Nevertheless, only a small number of CD38+ plasma cells were observed in bone marrow of the mice immunized with EV76 or F1-subunit vaccine. Statistical analysis showed no significant difference in CD38+ plasma cell number in the spleens between the mice immunized with F1 subunit vaccine and EV76 (> 0.05).

image

Figure 2. Plasma cells generated in the spleens and bone marrow of the BALB/c mice immunized with EV76 or F1 antigen after two doses of immunization on day 0 and 28. (S) represents ‘spleen’ and (B) represents ‘bone marrow’.

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Memory B cell responses after boosting immunization

We have shown that memory B cells are maintained in the spleens and bone marrow for several weeks after two doses of immunization. However, it is not clear whether boosting immunization can induce a boost of memory B cell population once more after memory B cells declined to a low level. To address this issue, 66 female BALB/c mice were given 0.1 ml subunit vaccine containing 20 μg of F1 antigen on day 0, 28 and 77 after primary immunization, respectively. The spleen and bone marrow cells were harvested from the immunized mice on day 4, 7, 14, 28, 49, 63, 77, 98, 105, 119 and 132 after primary immunization, respectively. Following staining of spleen and bone marrow cells, the flow cytometric analysis was performed, and the results were shown in Fig. 3. The elevated level of CD27+ memory B cells in the spleens or bone marrow was observed after the third immunization.

image

Figure 3. Memory B cells generated in the spleens and bone marrow of the BALB/c mice immunized with F1 antigen after three doses of immunization on day 0, 28 and 77.

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Anamnestic Ab responses after boost immunization

The above results indicated that memory B cells elicited by plague vaccines are short lived, which is likely to explain why no significant anti-F1 antibody titre boost was observed after the immunized animals were challenged with Y. pestis on day 56, 126 or 518 post-primary immunization [13]. However, the results cannot explain why boost immunization with a subunit vaccine elicits a significantly higher Ab titre than a single dose of immunization, whereas no significant Ab titre difference was observed between a single dose and two doses of EV76 vaccination [9]. We have once suggested a hypothesis that circulating antibodies may combine with the F1 capsule antigen exposed to newly invasive Y. pestis to prevent the live bacteria from eliciting the immune response [13]. To prove the hypothesis, in this study, five groups of ten mice were immunized with different immune strategy, and then the Ab titres were determined at different time points. Group I was only given a primary immunization of 1 of 5 of the human dose of EV76; group II was given a primary and secondary immunization of 1 of 10 of the human dose of EV76; group III was first given a primary immunization of 1 of 10 of the human dose of EV76, and then 20 μg of F1 antigen; group IV was first given a primary immunization of 20 μg of F1 antigen, and then 1 of 10 of the human dose of EV76; group V was only given a primary injection of aluminium hydroxide, from which serum background values were obtained. There was no statistical Ab titre difference (> 0.05) between the mice given a single dose of EV76 vaccine and those receiving two doses at all time points. By comparison, the group III developed a statistically higher antibody titre than the group IV at all time points (< 0.05) (Fig. 4).

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Figure 4. Development of IgG titres to F1 antigen in the BALB/c mice immunized with different strategy. Four experimental groups EV76 (1/5), EV76 (1/10 × 2), EV76 + F1 and F1 + EV76 were boosted on day 28 after the primary immunization.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

In our previous work, the antibody response for EV76 and the subunit vaccine F1 + rV270 could be maintained for up to 518 days. However, no significant anti-F1 antibody titre boost was observed after the immunized mice were challenged with virulent Y. pestis 141 on day 56, 126 or 518 post-primary immunization [13]. Memory B cells could quickly produce more antibodies when they are exposed to the same antigen [7, 9, 13], and antibody level in serum is maintained by long-lived plasma cells. In addition, memory B cells also play a role in replenishing the pool of long-lived plasma cells to maintain long-term antibody level in the absence of pathogen. Both long-lived plasma cells and memory B cells are responsible for the long-term humoral immunity elicited by vaccination [18, 19]. Based on this conclusion, we presumed that EV76 or subunit vaccine F1 + rV270 did not elicit or induced short-lived memory B cells, and long-lived plasma cells may be responsible for the long-term humoral immunity. To verify this presumption, the kinetics of memory B cell and plasma cell response were investigated in the mice immunized with plague subunit vaccine F1 or EV76 in the present study.

B lymphocytes are phenotypically and functionally heterogeneous. The surface molecule CD19 has been demonstrated to be specifically expressed on all B-lineage cells [23]. CD38 is highly expressed on plasma cells and plasmablasts, whereas CD27 is characteristically present on memory B cells. Membrane IgD presents a restricted expression pattern, which allows for differentiation between switched and unswitched memory B cells. Combining these surface markers with a multicolour flow cytometric analysis allows for determining memory B cells and plasma cells [24, 25]. In the present study, the frequencies of memory B cells and plasma cells were determined according to expression profiles of cell surface molecules reported in a previous report [25]. As shown in Fig. 1, flow cytometry analysis showed increases in the number of memory B cells in the spleens and bone marrow 1 week after the primary immunization, but memory B cells were short lived and their frequencies decreased rapidly to a low level in the spleens and bone marrow by 6 and 8 weeks, respectively. Because the number of memory B cells in the mice immunized with plague vaccines has declined to a low level on day 56 after primary immunization, no significant anti-F1 antibody titre boost after the challenge with virulent Y. pestis is understandable. Moreover, the number of memory B cells in the spleens was significantly higher than that in the bone marrow. This result is consistent with a previous study that the majority of memory B cells are present in the spleen [26]. As shown in Fig. 2, the number of plasma cells in the mouse spleens, not in bone marrow, increased 7 days after the immunization with plague vaccines, entered stationary phase starting on day 56 and persisted for 98 days (observation end time) after primary immunization. These results indicated that the majority of plasma cells are spleen resident and long lived, compared with memory B cells. Plasma cells are detectable for more than 98 days following a primary infection, which might be responsible for the long-term maintenance of Ab.

As shown in Fig. 1, the number of memory B cells in the spleens and bone marrow started to increase 7 days after the primary immunization, but it declined to a low level 77 days after the primary immunization. It is not clear whether revaccination can induce a boost of memory B cell population again. To address this issue, the mice were first given two doses of immunization with the subunit vaccine F1 on day 0 and 28 and then boosted with the subunit vaccine F1 on day 77 after the primary immunization. The kinetics of memory B cells in the spleens and bone marrow was determined at different time points. The results (shown in Fig. 3) showed the increases in the number of memory B cells in the spleens and bone marrow from day 7 to 63 after two doses of immunization with the subunit F1 vaccine. These data further corroborated the results from the previous part of the current research (shown in Fig. 1), whereas a boost of memory B cells was observed once more after the third immunization in the present study. This result indicated that when memory B cells declined to a low level, a revaccination can elicit a memory B cell number boost again. On the other hand, this result also meant that the increase in memory B cell was specifically caused by the immunization with plague subunit F1 vaccine.

The current data may explain our previous results [13] that no significant anti-F1 antibody titre boost was observed after the mice immunized with EV76 or subunit vaccine F1 + rV270 were challenged with virulent Y. pestis 141 on day 56, 126 or 518 post-primary immunization. However, they cannot explain another result, in which the mice receiving two doses of subunit vaccine elicit a significantly higher Ab titre than those given a single dose of immunization, whereas no significant Ab titre difference was observed between a single dose and two doses of EV76 vaccination [9]. We have once suggested a hypothesis [13] that circulating antibodies may combine with the F1 capsule antigen exposed to newly invasive Y. pestis to prevent the live bacteria from eliciting rapid Ab production upon revaccination. To further prove this hypothesis, five groups of mice were immunized with different immune strategy as described in the methods. There was no statistical Ab titre difference (> 0.05) between the mice given a single dose of EV76 vaccine (group I) and those receiving two doses (group II) at all time points, which reaffirm our previous study [9]. In contrast, there was no Ab difference between the group III (first given EV76 vaccine and then F1 antigen) and the group IV (first given F1 antigen and then EV76 vaccine) after the primary immunization, but the group III elicited a significantly higher Ab titre than the group IV after the secondary immunization. These results may be explained that the Ab induced by primary immunization in the group IV can combine with the F1 capsule antigen exposed to newly invasive Y. pestis to prevent the live bacteria from eliciting rapid Ab production, whereas the pre-existing antibody in the group III cannot neutralize a large quantity of newly added F1 antigen completely, and the redundant F1 antigen can stimulate memory B cells to produce Ab rapidly. Taken together, eliciting an antibody titre boost after the secondary immunization may be dependent on the existence of memory B cells and an excess of antigen vaccination. Therefore, to elicit higher antibody production by plague vaccines, secondary immunization should be performed within 2–8 weeks after primary immunization. In addition, the present study showed an ideal immunization strategy that involves first immunization with EV76 and then with a subunit vaccine.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Financial support for this study came from the National Natural Science Foundation of China (contract no. 81171529) and the National High Technology Research and Development Program of China (863 programme) (contract no. 2012AA02A403).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References