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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

The subunit vaccine SV1 (20 μg F1 + 10 μg rV270) has been identified as the optimal formulation in mice, which provided a good protection against plague in mice, guinea pigs and rabbits. To compare SV1 and SV2 (200 μg F1 + 100 μg rV270) with live attenuated vaccine EV76, antibody responses, protective efficacy, cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-10 and IL-12) production, CD4/CD8 ratio and CD69+ T-cell activation marker were determined in sera of the immunized Chinese-origin rhesus macaques, Macaca mulatta. The immunized animals with SV1 or SV2 developed higher anti-rV270 IgG titre, while those immunized with EV76 elicited a negligible IgG to V antigen, indicating that subunit vaccine (SV) had an advantage over EV76 in terms of the indispensable role of anti-V antibody against Yersinia pestis. There was no significant antibody titre difference between SV1 and SV2, suggesting that the immune response may have been saturated at the dose level of SV1. There were no statistical changes for CD4/CD8 ratios, IL-4 and CD69 levels between the three-vaccine immunized groups. However, a significant higher level of IL-12 was observed in the EV76 immunized animals, indicating that EV76 had an advantage over SV in respect of cellular immunity. Complete protection was observed for the immunized animals with SV and EV76, revealing that SV has a similar protective efficacy with EV76 against 6 × 106 CFU of Y. pestis challenge by subcutaneous route in Chinese-origin rhesus macaques.


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 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 an awful infectious disease afflicting human populations, leading to millions of deaths. Plague has been classified as a re-emerging infectious disease recently by the World Health Organization [2] and has attracted a considerable attention because of its potential misuse as an agent of biological warfare or bioterrorism [3]. Although Y. pestis is the sole cause of plague, the disease may present clinically in three main forms, including the bubonic, septicemic and pneumonic plagues. Patients with bubonic plague can develop secondary pneumonic or septic infection. Pneumonic plague can then be spread from person-to-person via respiratory droplets generated from sneezing and coughing of the patients. Without timely and proper treatment, mortality is very high for plague [1].

To date, there is not an ideal plague vaccine for human use. Y. pestis killed whole cell (KWC) vaccines only have a short protection against bubonic plague and are needed for frequent boosting to maintain immunity [4]. Live attenuated vaccine EV76 was effective against bubonic and pneumonic plague, but it showed side effects of varying severity and has not been used in the Western world [4–6]. Recent studies are being focused on the development of acellular vaccines containing F1 and LcrV antigens [7]. F1 antigen is encoded by the caf1 operon, which is a capsule-like protein around the bacterium and has anti-phagocytic properties [8, 9]. LcrV antigen is a multifunctional virulence protein of the type III secretion system encoded on pCD1 plasmid, which affords both plague protection and immunosuppressive property [10]. The DNA vaccine based on Y. pestis F1 and LcrV antigens alone or in combination was efficacious against both bubonic and pneumonic plague [11–13]. However, DNA vaccines usually elicit lower and slower immune responses than conventional vaccines, and gene gun immunization that delivers DNA-coated particles into the dermis of the skin needs to be used for improving immune responses [12, 14]. In contrast, subunit vaccines have obvious advantages over the traditional vaccines (KWC vaccine and live attenuated vaccine) and DNA vaccines in terms of safety or efficacy. It has been demonstrated that F1 and LcrV antigens alone or in combination can protect mice against bubonic and pneumonic plague, but the mice vaccinated with F1 antigen alone fail to provide protection against F1-negative Y. pestis strains [15] and the vaccine based solely on LcrV cannot protect against some Y. pestis strains producing variants of LcrV [16]. Thus, to provide effective protection against plague, it is desirable that F1 and LcrV antigens should be administered together [17].

To develop a safe and effective plague subunit vaccine, we have extracted the highly purified natural F1 antigen from Y. pestis EV76 by a new purification strategy [18] and prepared a non-tagged rV270 protein containing amino acids 1–270 of LcrV from recombinant Escherichia coli BL21 cells [19]. The subunit vaccine comprising a dose level of 20 μg F1 and 10 μg rV270 (designated as SV1 in this study) has been identified as the optimal formulation in mice, which provided a good protective efficacy against Y. pestis challenge in mice, guinea pigs and rabbits [20]. It is known that different immunological responses are present in different kinds of animals [21, 22]. Therefore, plague subunit vaccines should be evaluated using different animal models before clinical trial in humans. In this study, a much higher dose level of SV2 (200 μg F1 + 100 μg rV270) was designed for observation of dose-dependent efficacy. Chinese-origin rhesus macaques (Macaca mulatta) were selected to compare immunological responses and protective efficacy of SV (SV1 and SV2) with those of live attenuated vaccine EV76. The aim of the present study is to find the difference between subunit vaccines and EV76 vaccine, helping to design the next-generation vaccines.

Materials and methods

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

Antigens.  The native F1 antigen was prepared from Y. pestis EV76 by physical disruption, followed by a combination of ammonium sulphate fractionation and Sephacryl S-200HR column filtration chromatography [18]. The recombinant rV270 antigen was produced as a His-tag-fused protein by Escherichia coli transformed with recombinant plasmid pET28a-rV270; then cleaved with thrombin (Novagen, Merck KGaA, Darmstadt, Germany) and finally purified by Sephacryl S-200 HR column (GE Amersham Biosciences, Piscataway, NJ, USA) [19].

Animals.  Adult male and female Chinese-origin rhesus macaques were obtained from Laboratory Animal Research Center, Academy of Military Medical Science, China (licensed by Ministry of Health in General Logistics Department of Chinese People’s Liberation Army) and were raised in an air-conditioned laboratory with an ambient temperature of 21–25 °C, a relative humidity of 40–60% and a 12-h light/dark cycle. All the animals were 3–6 years old and weighed between 3 and 6 kg. Each animal was kept in a suspended stainless steel wire bottom cage and provided with a restricted diet of approximately 150 g of standard monkey keeping diet per day, and fresh fruit was given once daily to each of the animals, and water was available ad libitum during the entire course of this study. 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.

Animal immunizations.  The subunit vaccine F1 + rV270 comprised native F1 and rV270 antigens that were adsorbed to 25% (v/v) aluminium hydroxide adjuvant in PBS buffer. Adsorption of the proteins to the adjuvant was checked by subtracting protein in the supernatant from the total amount of proteins added. The live attenuated vaccine EV76 was obtained from the Lanzhou Institute of Biological Products (LIBP), China.

Fourteen Chinese-origin rhesus macaques were divided into four groups, including three experimental groups and one control group. Each one of three experimental groups contained four animals, and the alum-immunized control group had two animals (half male and half female). Three experimental groups of animals were intramuscularly injected in the forelimbs with the vaccines SV1 (20 μg of F1 and 10 μg of rV270 co-adsorbed to aluminium hydroxide adjuvant), SV2 (200 μg of F1 and 100 μg of rV270 co-adsorbed to aluminium hydroxide adjuvant) and EV76 [half of the human dose (8 × 108 cells)], respectively. Each animal of the control group was intramuscularly given 25% aluminium hydroxide adjuvant only. After the first immunization, on day 21, all the animals were boosted with an identical dose at the same injection sites.

Antigen-specific antibody assays.  Blood samples were collected from the forelimb veins of the immunized animals on week 2, 4 (1), 6 (3), 8 (5), 10 (7), 11 (9) or 12 (11) after primary immunization (the number in bracket was designated as time point post-secondary immunization). Sera were assayed for the presence of F1- and rV270-specific IgG by a modified ELISA [23]. Briefly, 96-well microtitre plates were coated with either F1 or rV270 antigens diluted to 500 ng/ml in 0.06 m sodium carbonate buffer (pH 9.6) and incubated for overnight at 4 °C. Non-specific binding was blocked with 0.1% casein in 0.01 m phosphate-buffered saline. Test sera were added to plates with serial dilution in 0.01 m PBS buffer containing 0.05% casein and incubated for 30 min at 37 °C. After five washes by 0.01 m PBS buffer containing 0.05% Tween-20 (pH 7.2), 100 μl of goat anti-monkey IgG labelled with horseradish peroxidase (Dakewe Biotech Co., Ltd, Shen Zhen, China) was added to each well and incubated for 20 min at 37 °C. The plates were washed three times in 0.01 m PBS buffer containing 0.05% Tween-20 (pH 7.2), and 100 μl of 0.01% peroxidase substrate 3, 3′, 5, 5′-tetramethylbenzidine (TMB) was added to each well. The reaction was stopped by the addition of 50 μl of 2.5 m H2SO4 per well, and then optical density (OD) was read at 450 nm with an ELISA plate reader (Bio-Rad, Hercules, CA, USA). The titre of specific antibody was estimated as the maximum dilution of serum giving an OD reading 0.2 units over background. Background values were obtained from serum samples collected from the non-immunized subjects. Antibody endpoint titre per immunization group is presented as the geometric mean endpoint titre to F1 or rV270 antigen.

Serum cytokine analysis.  Sera were collected from the four groups of animals before immunization and on week 1, 2, 4 (1), 6 (3) and 8 (5) after primary immunization (the number in bracket was designated as time point post-secondary immunization). The levels of six cytokines IFN-γ, TNF-α, IL-2, IL-4, IL-10 and IL-12 were measured by using the monkey cytokine ELISA kits (U-CyTech biosciences, Utrecht, the Netherlands) according to the manufacturer’s instructions.

Flow cytometric analysis.  The monoclonal antibodies (anti-CD8-FITC, anti-CD4-PE, anti-CD3-percPcy5.5, anti-IgG2a-FITC κ isotype control and anti-IgG1-PE κ isotype control) used for flow cytometry in this study were purchased from BD Biosciences PharMingen (San Diego, CA, USA).

To investigate the changes of CD4/CD8 ratio and T-cell activation between preimmunization and post-immunization, peripheral whole blood was collected from each animal before immunization and at 7, 14, 21 and 28 (7) days after immunization (the number in bracket was designated as time point post-secondary immunization) and placed into a 15 -ml centrifuge tube containing heparin. The antibody combinations of CD3-percPcy5.5 + CD4-PE + CD8-FITC, CD3-percPcy5.5 + CD69-PE + CD20-FITC and IgG2a-FITC κ isotype + IgG1-PE κ isotype (isotypic control) were measured by flow cytometry in this study. Briefly, 100 μl of heparinized blood was placed in a polystyrene test tube that contained an antibody combination. The heparinized blood was reacted with each of the above-mentioned combinations of antibodies for 15 min at room temperature in the dark. After that, 2 ml of erythrocytolysin diluted with deionized water was added to the tube. The tube was sealed with parafilm and mixed gently by turning the tube upside down and then immediately incubated for 30 min at room temperature in the dark. The mixture was centrifuged at 177 g for 5 min, and the supernatant was discarded. The cells were finally resuspended in 700 μl of PBS buffer and then analysed by using Epics XL flow cytometer (Beckman Coulter Inc., Miami, FL, USA) with Expo 32 software for data storage and analysis.

Challenge with Y. pestis. Chinese-origin rhesus macaques were challenged with the virulent Y. pestis 141 strain in the Animal Biosafety Level 3 laboratory, which was isolated from Marmota himalayana in Qinghai-Tibet plateau and has a median lethal dose (MLD) of 5.6 colony-forming units (CFU) for BALB/c mice, 17.8 CFU for guinea pigs and New Zealand White rabbits by the subcutaneous route. All the immunized Chinese-origin rhesus macaques were challenged with 6 × 106 CFU by the subcutaneous route on week 10 after the primary immunization, and then, closely observed for 14 days. All the survival animals were killed humanely for a post-mortem examination. Livers, spleens, lungs, lymph nodes and blood from hearts of the challenged animals were removed to confirm if Y. pestis was presented in these organs.

Statistical analysis.  The differences of antibody titre, CD4/CD8 ratio and CD69 positive cell percentage among groups were compared by analysis of variance (anova) with sas 8.0 software (SAS, Raleigh, USA), and probability values of <0.05 were taken as significant.

Results

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

Antibody responses to F1 and rV270

The titres of F1-, V- or rV270-specific antibody in all immunized animals were determined on weeks 2, 4, 6, 8, 10, 11 and 12 after primary immunization. The antibody titres on week 11 and 12 were obtained from serum samples of survival animals post-challenge. The geometric mean of antibody titres and the standard error of the mean from each group of Chinese-origin rhesus macaques were calculated from the data obtained by ELISA. The antibody responses to F1, V or rV270 over a time course of 12 weeks in three immunized groups of animals were shown in Figs 1 and 2. The statistical analysis showed that there was no significant anti-F1 IgG titre difference among three groups of immunized animals during the course of immunization (= 0.20, = 0.8237). In contrast, the immunized Chinese-origin rhesus macaques with SV1 or SV2 subunit vaccine developed significantly higher anti-rV270 IgG titres than those immunized with EV76 elicited anti-V IgG titres during the observation course of 12 weeks (= 8.01, = 0.01), whereas there was no statistical difference between SV1 and SV2 (F = 0.1, P = 0.7643). No anti-rV270 IgG and anti-F1 IgG were detected in the two control animals that only received aluminium hydroxide adjuvant in PBS buffer. After challenging with Y. pestis on week 10, a significant anti-F1 antibody titre boost was observed in three groups of immunized animals during the time course of 14 days. An anti-rV270 IgG titre boost was also seen in two groups of subunit vaccines, but still no anti-rV270 IgG titre elevation was observed in the group EV76 within 2 weeks.

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Figure 1.  Development of IgG titres to F1 in Chinese-origin rhesus macaques immunized with different vaccines at different times post-immunization.

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Figure 2.  Development of IgG titres to rV270 or V antigen in Chinese-origin rhesus macaques immunized with different vaccines at different times post-immunization.

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Serum cytokine analysis

The levels of six serum cytokines IFN-γ, IL-12, IL-4, TNF-α, IL-2 and IL-10 were determined in all four groups of Chinese-origin rhesus macaques before and on week 1, 2, 4, 6 and 8 after primary immunization by using the monkey cytokine ELISA kits. The results showed non-detectable IFN-γ, TNF-α, IL-2 and IL-10 in the sera of all the tested animals before and after immunization. In contrast, IL-12 was detectable in the sera of all four groups of animals before and after immunization. Nevertheless, an elevated level of IL-12 was observed in the sera of EV76 group of animals after immunization (Fig. 3). Statistical analysis demonstrated that EV76 group of animals had significantly higher IL-12 level than other three groups of animals (= 4.84, = 0.0375), among which no significant differences were seen during the course of observation (= 0.28, = 0.6159). After immunization, IL-4 was detectable in the sera of SV1, SV2 and EV76 groups of animals, whereas non-detectable IL-4 was observed in control group. Statistical analysis revealed that the three tested groups of animals had significant higher IL-4 level than the control group (= 979.2, < 0.0001), and there was no significant IL-4 concentration difference among the three tested groups of animals (Fig. 4) (= 2.52, = 0.1354).

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Figure 3.  Expression levels of IL-12 were determined before immunization and on week 1, 2, 4, 6 and 8 after immunization with EV76, SV1, SV2 and adjuvant in Chinese-origin rhesus macaque sera.

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Figure 4.  Expression levels of IL-4 were determined before immunization and on week 1, 2, 4, 6 and 8 after immunization with EV76, SV1, SV2 and adjuvant in Chinese-origin rhesus macaque sera.

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Analysis of CD4/CD8 ratio and T-cell activation

To investigate the effect of SV1, SV2 or EV76 on T-cell activation, we determined the CD4/CD8 ratio and T-cell activation marker CD69 in peripheral blood of the immunized Chinese-origin rhesus macaques with SV1, SV2, EV76 and aluminium hydroxide at different time points. There was no significant difference for CD4/CD8 ratio between SV1, SV2, EV76 and aluminium hydroxide groups during the time course of 28 days (= 1.65, = 0.2302). Likewise, there was also no significant difference of CD4/CD8 ratios between the four treatment groups at any time points (P > 0.05). CD69 T lymphocytes representing the T-cell activation in peripheral blood were evaluated as a percentage. Statistical results demonstrated that there were no significant differences between SV1, SV2, EV76 and aluminium hydroxide groups during the time course of 28 days (= 1.08, = 0.3936). Likewise, there was also no significant difference of CD69 percentage between the four treatment groups at any time points (P > 0.05). However, levels of CD69 percentage in SV1, SV2 and EV76 groups were slightly elevated (although not statistically significant) after first and secondary immunization than those before first and secondary immunizations, respectively (Fig. 5).

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Figure 5.  T-cell activation marker CD69 was evaluated as a percentage in peripheral blood of each group of the immunized Chinese-origin rhesus macaques with SV1, SV2, EV76 and adjuvant before immunization and at days 7, 14, 28 after first immunization.

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Observation of protective efficacy

All the four groups of Chinese-origin rhesus macaques were challenged with 6 × 106 CFU Y. pestis strain 141 by the subcutaneous route on week 10 post-primary immunization, followed by observation for 14 days. Complete protection was observed for the three groups of animals immunized with SV1, SV2 and EV76 without the development of symptoms of plague during the 14-day post-challenge observation. In contrast, the two control animals succumbed to a same dose of Y. pestis 141 challenge, indicating that the adjuvant alone had no effect on protection or survival of the animals. The results of protective efficacy were presented in Table 1.

Table 1.   Protective efficacy of Chinese-origin rhesus macaques against challenge with 6 × 106 CFU of virulent Yersinia pestis strain 141 by the subcutaneous route.
Treatment groupsChallenge time (Weeks)Challenge dose (CFU)Survival/Total
SV1106 × 1063/3
SV2106 × 1064/4
EV76106 × 1064/4
Adjuvant106 × 1060/2

A post-mortem was carried out on two control animals, which succumbed to the challenge during the 14-day post-challenge observation period. Microbiological examination showed that Y. pestis were isolated from the livers, spleens, lungs, lymph nodes and blood from hearts of the two dead animals, indicating that the death of animals was caused by the systemic infection of Y. pestis. The survivors were killed humanely and autopsied for microbiological analysis on day 14 post-challenge, Y. pestis were not isolated from livers, spleens, lungs, kidneys, brains, lymph nodes and cardiac blood of all the killed animals, demonstrating that Y. pestis have been eliminated from the survival animals (Table 2).

Table 2.   Tissue pathogen load in Chinese-origin rhesus macaques following Yersinia pestis challenge.
GroupsLiversSpleensLungsKidneysCardiac bloodBrainsLymph nodes
  1. ‘+’ represents ‘Y. pestis load’.

  2. ‘−’ represents ‘no Y. pestis load’.

SV1
SV2
EV76
Adjuvant+++++++

Discussion

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

In our previous study, the subunit vaccine comprising a dose level of 20 μg F1 and 10 μg rV270 (SV1) has been identified as the optimal formulation in mice, which provided a good protection against Y. pestis challenge in mice, guinea pigs and rabbits [20]. In addition, this formulation provided the long-term protection and antibody response over a period of 518 days in mouse model [24]. In the present study, we compared the antibody responses among SV1, SV2 and EV76 in Chinese-origin rhesus macaques. There was no significant anti-F1 IgG titre difference among three groups of immunized animals after immunization. In contrast, the immunized Chinese-origin rhesus macaques with SV1 or SV2 subunit vaccine developed significantly higher anti-rV270 IgG titres than those immunized with EV76 vaccine elicited anti-V IgG titres. The results revealed that SV1 and SV2 had an advantage over EV76 in terms of the indispensable role of anti-V antibody against Y. pestis challenge [21, 25]. There was no significant anti-F1 or anti-rV270 antibody titre difference between SV1 and SV2, suggesting that the immune response may have been saturated at the dose level of 20 μg F1 and 10 μg rV270. This will be helpful in designing a clinical trial, because the intention is to elicit the greatest level of immune response while limiting the risk of potential toxicity and reactivity to the vaccinees.

After challenging with virulent Y. pestis strain 141, a significant anti-F1 antibody titre boost was observed in three groups of immunized Chinese-origin rhesus macaques, whereas an anti-rV270 IgG titre elevation was only observed in two groups of subunit vaccines. This result seems to be consistent with the conclusion that the memory B cells could quickly produce more antibodies when they expose to the same antigen once again [26–28]. However, no anti-V IgG titre boost was observed in the immunized animals with EV76 within 2 weeks. This might be attributed to the low anti-V IgG titre, because EV76 vaccine developed a higher anti-F1 IgG titre and an almost undetectable antibody titre to V antigen. On the other hand, it has been demonstrated that the immune responses afforded by a virulent infection are higher than those conferred by vaccination with different avirulent strains, which in turn is more permanent than that produced by plague subunit vaccines [22]. This conclusion may further explain why a significant antibody titre boost was observed in three groups of immunized Chinese-origin rhesus macaques after challenging with virulent Y. pestis strain 141. To our surprise, no significant anti-F1 or anti-rV270 antibody titre boost was observed in mice, guinea pigs and rabbits immunized with SV1 or EV76 after challenging with virulent Y. pestis strain 141 in our previous study [20]. These observations may be explained from the report that different immunological responses are present in different kinds of animals [22]. However, whether an infection in an immunized animal produces antibodies more rapidly and more effectively than the susceptible animal was not demonstrated [22]. Our results indicated that the immunized Chinese-origin rhesus macaques with SV or EV76 had an antibody elevation after infected with virulent strain Y. pestis 141, whereas no evident antibodies specific to F1 and V antigens was detectable in the control animals after challenging with virulent Y. pestis strain 141 during the time course of 14 days, demonstrating that immunized Chinese-origin rhesus macaques with SV or EV76 generated antibodies more rapidly than susceptible animals.

Evaluation of any plague vaccines first on rodents, and then on non-human primates, and finally on humans was generally considered as a standard process [29]. Several species of non-human primates including hamadryas baboons [30, 31], vervet monkeys, rhesus monkeys and African green monkeys have been used for evaluation of EV vaccines. It has been known that EV strain was shown to be virulent in African green monkeys and vervet monkeys, but not in rhesus monkeys and guinea pigs [32, 33]. Welkos et al. [34] reported that a pigmentation-deficient (Pgm−) strain of Y. pestis strain C092 was virulent in African green monkeys and outbred Swiss Webster mice by the aerosol route. The classical EV-type strains are known to possess Pgm− and virulence-associated characteristics in different laboratories [35]. In our previous study, we found that EV76 preserved in China was pathogenic for mice, but not for guinea pigs and rabbits [20]. In the present study, no local and systemic reactions associated with EV76 were observed in Chinese-origin rhesus macaques by intramuscular route, indicating that this species of non-human primate was not sensitive to EV76 vaccine. This result is in accordance with the report that EV strain showed some virulence by s.c. for African green monkey, but not for rhesus macaques [35]. In the current study, we also found a negligible IgG to V antigen in immunized Chinese-origin rhesus macaques with EV76. This observation is in agreement with our previous and other reports that animals given EV76 or KWC vaccine had a higher anti-F1 IgG titre and an almost undetectable titre to V antigen [6, 20, 36–38].

It has been reported that alum-adjuvanted subunit vaccines induce robust humoral immunity [37, 39], whereas live attenuated vaccine EV76 elicit both humoral response and cell-mediated immunity [6, 40, 41]. To investigate the difference of cytokine profile between subunit vaccines and live attenuated vaccine EV76, we determined six cytokines IFN-γ, TNF-α, IL-2, IL-12, IL-4 and IL-10 in the sera of the immunized Chinese-origin rhesus macaques by using the monkey cytokine ELISA kits. Cytokine IL-4 production retained to a detectable level in the sera of three groups of immunized animals, and no significant difference of IL-4 level was observed between subunit vaccine and live attenuated vaccine EV76. It has been demonstrated that IL-4 is an important activator of humoral immunity and implicate in both antimicrobial host defence and pathogenesis of diseases with an inflammatory component [42]. Therefore, IL-4 production is consistent with humoral responses elicited by subunit vaccines or live attenuated vaccine EV76. In addition, a significant higher level of IL-12 was observed in the sera of EV76 group of animals after immunization, compared with the subunit vaccine SV1 or SV2. This result indicated that live attenuated vaccine EV76 had an advantage over SV vaccines, because IL-12 has a central function in initiating cellular immune responses. Y. pestis is an intracellular pathogen, and cellular immunity can contribute to eliminate Y. pestis from infected animals [40, 43, 44]. However, other four cytokines IFN-γ, TNF-α, IL-2 and IL-10 were not detectable in the sera of immunized animals with both EV76 and subunit vaccines. These results may be explained from the previous report stating that IL-4 has a modest inhibitory effect on the IL-12-induced priming for IFN-γ production and very efficiently prevents the priming for IL-10 production [45]. In addition, IL-4 is also known to inhibit the production of TNF-α and IL-2 in activated CD4 T cells and mast cells [46, 47].

Two subsets of T cells, namely T helper (Th)/T inducer (Ti) and T cytotoxic (Tc)/T suppressor (Ts) lymphocytes, are known to be involved in the regulatory function of the immune system. These two subsets are different from one another in terms of their surface characteristics and functions. Th/Ti and Tc/Ts cells express surface markers designated CD4 and CD8, respectively. In terms of their functions, the Th/Ti cell subset is involved in induction of the immune response, whereas the Ts cell subset is involved in the suppression of the immune response. A decrease in the CD4/CD8 ratio is often seen in both primary and secondary immune deficiencies or is indicative of impairment the cell-mediated immunity [48]. In this study, we determined CD4/CD8 ratios in the peripheral blood of immunized Chinese-origin rhesus macaques with SV1, SV2, EV76 and aluminium hydroxide. Consequently, no significant changes in CD4/CD8 ratios were observed between subunit vaccines and live attenuated vaccine EV76. It has been reported that the normal range of CD4/CD8 ratio was 0.33–3.57 with the mean of 1.27 [49]. Based on the normal range, it was found that the CD4/CD8 ratios of all tested animals were within the normal range of variation. These results indicated that Chinese-origin rhesus macaques keep in a state of balance of the immune system after they immunized with subunit vaccines (SV1, SV2) or live attenuated vaccine EV76.

Another goal of the current study was to evaluate T-cell activation in response to EV76, SV1 and SV2 vaccines in peripheral blood of the Chinese-origin rhesus macaques. Recently, the early activation antigen CD69 has generated interest as a possible marker for use in flow cytometry-based assays for cellular activation. The fact that CD69 is rapidly expressed upon T-cell activation and allows easy detection by flow cytometry suggests its possible utility as a marker for the rapid assessment of T-cell activation [50]. In this study, the early T-cell activation marker CD69 was expressed as a percentage of the total of CD3-positive cells. Our results indicated that there was no statistical difference of CD69 percentage between subunit vaccines (SV1, SV2) and live attenuated vaccine EV76 at any time points during the time course of 28 days. However, levels of CD69 percentage in SV1, SV2 and EV76 groups were slightly elevated after first and secondary immunization (although not statistically significant) than those before first and secondary immunizations, respectively. This finding seems to reveal that CD69 molecules are unstable in vivo, and perhaps a significant difference may be found within several hours after immunization.

Because protection against lethal challenge of new candidate plague vaccines cannot be ethically tested in humans, it is essential that an in vitro surrogate marker that can reliably predict the protective efficacy and as much testing as possible with animals be developed. In this study, SV1, SV2 and EV76 vaccines were evaluated for protective efficacy using Chinese-origin rhesus macaques. Complete protection against subcutaneous Y. pestis challenge was observed for the animals survived after immunization, whereas two animals in control group developed clinical signs of disease and succumbed to death 3–5 days after challenge. Unfortunately, one of four animals in SV1 group was dead during the course of transportation from Beijing to Qinghai, which may be caused by high altitude reaction. Therefore, only three animals in SV1 group were challenged finally. Post-mortem analysis of Y. pestis load in different organs of the animals that survived the challenge showed that Y. pestis have been eliminated from the survival animals, whereas Y. pestis have been isolated from organs of the two control animals that died of challenge. These observations demonstrated that subunit vaccines (SV1 and SV2) and live attenuated vaccine EV76 have a good protective efficacy against 6 × 106 CFU Y. pestis by subcutaneous route in Chinese-origin rhesus macaques. The current results are consistent with other previous reports that the immunized cynomolgus macaques [51, 52] and mice [7, 53] with subunit vaccines can completely eliminate challenge organism from their different organs. However, our previous results showed that Y. pestis can be eliminated from the most immunized animals, including mice, guinea pigs and rabbits, with subunit vaccines or EV76, but bacteria were recovered from a liver of one mouse survivor immunized with subunit vaccine [20]. Similar results were also found in the immunized guinea pigs with subunit vaccine [54] and immunized mice with KWC vaccine [36]. Taken together, live attenuated vaccine may have an advantage over subunit vaccine or KWC in clearing bacteria from animal bodies.

Although our challenge experiment with virulent Y. pestis strain 141 was limited to two control animals, we observed an acute and severe clinical illness in Chinese-origin rhesus macaques. Pathological analysis of the two control animals after challenged with Y. pestis strain 141 was shown to be typical of bubonic-septicemic plague (data not shown). It was revealed that Chinese-origin rhesus macaques seem to be a suitable animal model for the evaluation of plague vaccines.

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 Key Program for Infectious Diseases of China (2009ZX10004-4001). Ms. Jin Wang, Xiangxiu Qin and Lili Zhang are appreciated for their assistance in performing ELISA.

References

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