In this study, we have shown that severe combined immunodeficient/beige mice reconstituted with hyperimmune Balb/c lymphocytes can be used as a model to demonstrate adoptive and passive protection against plague infection. Reconstitution of severe combined immunodeficient/beige mice was successful in nine out of ten mice as demonstrated by spleen colonisation and sustained circulating immunoglobulin titres. Furthermore, an increase in antibody titre was evident after a booster immunisation of reconstituted mice. Presence of circulating antibody correlated with protection against a systemic plague challenge and indicated that in reconstituted mice adoptive transfer of a functional immune system had occurred. The severe combined immunodeficient/beige mouse was also used to demonstrate passive protection against inhaled and systemic plague infection. The reconstituted severe combined immunodeficient/beige mouse model demonstrating protective immunity against plague will be further developed to identify the immune cell subsets responsible for this protection.
Yersinia pestis is the causative agent of plague in mammals. Plague is endemic in certain areas of the world with South East Asia currently being the major focus . Plague is primarily a disease of rodents; however, humans are believed to be at risk of infection if they come into contact with an infected animal, either directly or through a bite from an infected flea vector . The disease can manifest itself in several ways depending on the route of entry of the organism into the host. Bubonic and septicaemic forms of plague result usually from the bite of an infected animal or flea and the other main form of the disease is pneumonic plague, a result of the inhalation of Y. pestis. [3, 4]. Whatever the route of infection, the disease has a high mortality rate (>50%) in untreated cases .
Currently, protective immunity to plague can be conferred by immunisation with killed whole cell vaccines, although these vaccines may not protect against all forms of the disease . Recently, a new improved subunit vaccine has been developed that generates mucosal and systemic immunity conferring protection against the pneumonic and bubonic forms of plague infection [7, 8]. The subunit vaccine comprises the F1- and V-antigens from Yersinia pestis administered after adsorption to alhydrogel. The roles of the antigens in plague infection are unclear; however, it is believed that the capsular F1-antigen has antiphagocytic activity, whilst the V-antigen exerts an anti-host effect through the modulation of tissue cytokine levels [9, 10].
An understanding of the mechanisms of protective immune responses afforded by vaccines is essential to allow the rational design of new and improved vaccines by identifying the key protective elements in immunity. These protective immune mechanisms can potentially be investigated using the severe combined immunodeficient/beige (SCID/Bge) mouse model. SCID/Bge mice carry two genetic defects that result in a lack of functional murine immune effector cells. The murine SCID mutation results in an absence of mature B- and T-lymphocytes and the beige mutation results in defective natural killer (NK) cells. As a result of these mutations, the SCID/Bge mouse lacks a functional immune system and is able to accept xenografts from a range of other species, including different strains of mouse . This process of immune system reconstitution can be achieved, for example, after the parenteral administration of a purified mixed lymphocyte population from a donor animal. Successful reconstitution of SCID/Bge mice leads to colonisation of lymphoid tissues with donor-derived immune cell subsets and establishes circulating donor immunoglobulins. This model offers a powerful tool for analysing the protective immune responses afforded by vaccines.
A problem associated with the SCID/Bge mouse is occasional reversion of the SCID phenotype in a small percentage of animals. These mice are termed ‘leaky’ and are normally removed from experiments; consequently, it is important that colonies of SCID/Bge mice are regularly screened for leakiness. The immunodeficient status of the SCID/Bge mouse means that specialised housing and procedural techniques are necessary in order to prevent infection from a possible environmental contaminant.
In this study, we have used the SCID/Bge mouse model to investigate the mechanisms of protective immunity to plague conferred by the subunit vaccine. Adoptive transfer of protective immunity to plague was attempted by reconstitution of SCID/Bge mice with hyperimmune donor Balb/c lymphocytes. Subsequently the immune response to boosting was monitored in these reconstituted mice. Additionally, the ability to protect against plague infection through passive transfer of antibody only, into naive SCID/Bge mice was investigated.
2Materials and methods
SCID/Bge mice at 3–4 weeks of age, were supplied by CAMR, Porton Down, Salisbury, UK. The animals were housed and handled aseptically in Hepa filtered isolators and fed with sterile food and sterile water ad libitum. The absence of murine immunoglobulin (Ig) was screened by ELISA and mice having detectable murine Ig levels were excluded from experiments. Barrier-bred, female 6-week-old Balb/c mice free of mouse pathogens were obtained from Charles River Laboratories, Margate, Kent, UK.
All animal experimentation strictly adhered to the 1986 Scientific Procedures Act and to the Guidance on the Operation of the Animals (Scientific Procedures) Act, as promulgated by the ethics committee on animal experimentation within this research establishment.
2.2Immunisation of donor Balb/c mice
Donor Balb/c mice (n=25) were immunised intraperitoneally (i.p.) on days 0, 14 and 28 with the plague subunit vaccine comprising 10 μg each of F1- and V-antigen delivered after overnight adsorption to 25% (v/v) alhydrogel (Superfos Biosector, Denmark) at 4°C in 100 μl of phosphate-buffered saline (PBS) per animal.
2.3Preparation of hyperimmune lymphocytes and serum
Hyperimmune lymphocytes and serum were obtained on day 42 from Balb/c mice immunised as above. Mice were anaesthetised i.p.  before collection of blood samples by cardiac puncture from which sera was collected and pooled. Subsequently, anaesthetised animals were humanely killed by cervical dislocation and lymphocytes were isolated from spleen cell preparations after density centrifugation. This procedure has previously been shown to yield a pure (>90%) population of lymphocytes .
2.4Reconstitution and immunisation of SCID/Bge mice
SCID/Bge mice (n=10) were reconstituted i.p. with 1×107 hyperimmune lymphocytes isolated as detailed above (day 0). Reconstituted SCID/Bge mice were immunised 24 h later (day 1) with the plague subunit vaccine prepared and administered as detailed above. A second immunisation with the same subunit vaccine formulation was administered on day 111. Serial blood samples were obtained at intervals after reconstitution and circulating immunoglobulin levels were determined by ELISA.
2.5Passive transfer of hyperimmune serum
Serum was collected from hyperimmune Balb/c mice and untreated control Balb/c mice as detailed above. The serum was titred by ELISA for F1- and V-specific circulating IgG and IgG subclasses. Subsequently, a 500-μl volume of hyperimmune serum was administered i.p. to six SCID/Bge mice 20 h prior to a parenteral challenge of Y. pestis strain GB. A control group of six SCID/Bge mice received 500 μl of naive Balb/c serum. In a second experiment, 500 μl of hyperimmune serum was administered to 10 SCID/Bge mice 2 h prior to an aerosol challenge of Y. pestis strain GB.
Serum immunoglobulin levels were determined by ELISA as previously detailed . In brief, microtitre plates were coated with either F1- or V-antigens in PBS (5 μg ml−1) overnight at 4°C. Subsequently, the plates were blocked using 1% non fat dry milk powder in PBS+0.05% Tween 20 (2 h 37°C). Serum samples were double diluted on the plate and incubated for 1 h at 37°C. Binding of serum antibody was detected using a peroxidase conjugate against mouse polyvalent Ig (Sigma) used at a maximum dilution of 1:5000. Serum antibody titre was estimated as the maximum dilution of serum giving an absorbance at 405 nm (A405) reading 0.1 units over background and was presented as log10 reciprocal antibody titre per sample. The determination of IgG subclasses present and their titre was also determined for serum samples used in all passive transfer experiments. Peroxidase-labelled secondary antibodies against mouse, IgG, IgG1 and IgG2a (Harlan-Seralab) were each used at a maximum dilution of 1:4000. It has previously been reported that these secondary antibodies are specific and have an equivalent sensitivity of detection .
A modified ELISA was used to analyse antibody secretion by spleen cells as follows: sterile microtitre plates were coated with either F1- or V-antigens in PBS (5 μg ml−1) overnight at 4°C, under aseptic conditions. Subsequently the plates were blocked with DMEM plus 20% (v/v) foetal calf serum (FCS) (1 h 37°C), prior to aliquoting 100 μl of the individual spleen cell suspensions. The cells were double diluted on the plate and then allowed to incubate for 48 h (5% CO2 37°C). The plates were subsequently washed in PBS (×3), PBS+0.2% Tween 20 prior to the addition of the peroxidase-labelled secondary antibody conjugate. Incubation was continued for 20 h (+4°C) prior to automated washing (×4) in PBS+0.2% Tween 20 and addition of a soluble peroxidase substrate, 2,2-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). The amount of antibody secreted by well was quantified by measuring A405 and the log10 reciprocal antibody titre was derived.
2.7Challenge with Y. pestis
SCID/Bge mice were challenged under the Advisory Committee for Dangerous Pathogens (ACDP) Category 3 containment conditions. Aliquots of Y. pestis GB strain containing approximately 103 colony forming units (cfu) were injected subcutaneously in a 0.1-ml volume, into reconstituted SCID/Bge mice and control unreconstituted SCID/Bge mice.
To investigate passive protection against a systemic plague infection, SCID/Bge mice received either hyperimmune serum or control naive mouse serum as detailed above and were challenged with approximately 105 cfu of Y. pestis GB. The challenge inoculum was prepared at 28°C as previously described .
Strain GB was isolated from a fatal human case of plague and has an MLD of <5 cfu in SCID/Bge mice by the subcutaneous route (unpublished data). This strain was derived from strain MP6 and is fully virulent in Balb/c mice with an MLD of 1 cfu via the subcutaneous route and 900 cfu via the respiratory route .
A second group of SCID/Bge mice received hyperimmune serum prior to a challenge with an aerosol of Y. pestis GB. The inoculum for aerosol challenge was prepared as previously described . Aerosol particles were generated using a Collison spray and conditioned in a modified Henderson aerosol apparatus. SCID/Bge mice were placed in a head-only exposure chamber and exposed for 10 min to a 105 cfu dose of Y. pestis. The aerosol stream was maintained at 55% relative humidity (±5%) and 19°C (±1°C). During each exposure period, a sample of Y. pestis was taken directly from the sampling port, using an all-glass impinger (AGI-30) and plated onto Congo Red agar to quantify the available dose of Y. pestis delivered.
Challenged mice were observed over a period of 14 days. Humane end points were strictly observed during this time so that no animal became distressed. At the end of the observation period, survivors were humanely culled and samples of blood, spleen and liver were streaked over the surface of Congo Red agar plates. The plates were incubated at 28°C for 48 h and then observed for the growth of Y. pestis.
3.1Assay of immunoglobulins in reconstituted SCID/Bge mouse sera
Initial experiments investigated the reconstitution of SCID/Bge mice with hyperimmune murine lymphocytes by determining the antibody response to both V-antigen and F1-antigen in these mice at various time points after reconstitution. Results presented in Table 1 indicate the log10 reciprocal serum antibody titres at four time intervals after reconstitution. It can be seen that an antibody titre to both the F1- and V-antigens was demonstrated in 9 out of 10 reconstituted SCID/Bge mice at 56 days after reconstitution. These 9 mice were considered to be successfully reconstituted with donor lymphocytes. A second immunisation of the same formulation subunit vaccine was given on day 111 after reconstitution and it can be seen that on day 123, antigen-specific serum Ig levels were recorded at increased levels in the remaining reconstituted mice. In the single non-responding SCID/Bge mouse (results not shown) a second immunisation did not result in detection of a specific antibody response. It can also be seen from the results in Table 1 that the antibody titre to V-antigen was higher than that to F1-antigen and this is typical of the immune response to the plague subunit vaccine seen in previous investigations [7, 8].
Table 1. Antibody titres to F1- and V-antigens in SCID/Bge mice at various time points after reconstitutiona
Days after reconstitution
Mean log10 antibody titre±S.E.M.
n, number of SCID/Bge mice.
aReconstitution was attempted for 10 SCID/Bge mice and mean antibody titres were determined for SCID/Bge mice that were successfully reconstituted.
bReconstituted SCID/Bge mice received a boost immunisation with the subunit vaccine for plague on day 111.
Analysis of antibody secretion by spleen cells from three reconstituted SCID/Bge mice prior to the boost immunisation on day 111 demonstrated a low level of antibody titre to both the F1- and V-antigens indicating the colonisation of spleen tissues with immune antibody secreting cells (results not shown).
3.2Assay of hyperimmune serum used for passive transfer
The profile of immunoglobulin subclasses present in the hyperimmune sera used in passive transfer experiments was determined and is represented in Figs. 1–2. It can be seen that the predominant immunoglobulin subtype for both sera samples against both F1- and V-antigens is IgG1, with lower levels of IgG2a being recorded. This is indicative of a T-cell immune response of a predominantly Th2 type. Again it can be seen that the antibody titre to the V-antigen is greater than to the F1-antigen
3.3Protection against challenge
Those SCID/Bge mice that demonstrated an antibody titre to F1- and V-antigens in Table 1 were protected against a systemic challenge dose of 140 cfu Y. pestis (approximately 30 MLDs) Control unreconstituted mice succumbed to this challenge with a mean time to death of 4 days. Furthermore, the single mouse that did not show evidence of reconstitution did not survive the challenge. The spleens, livers and blood from a sample of survivors (n=4) collected after the 14-day observation challenge macroscopically appeared normal and Y. pestis was not recovered from these tissues indicating that surviving mice had cleared the challenge organism.
The protective effect of passive transfer of hyperimmune serum against plague challenge can be seen in Figs. 3–4. Protection was achieved for 5 of 6 and 7 of 10 SCID/Bge mice against injected challenge and aerosol challenge, respectively. Y. pestis was not recovered from sections of spleen, blood, liver and additionally lung samples from aerosol challenged mice, in a sample of surviving mice after the 14-day observation period. This again indicated that clearance of the challenge organism had occurred.
An understanding of the underlying mechanisms of protective immunity mediated by a vaccine is essential in the rational design of new and improved vaccines. The work reported here demonstrated that reconstitution of SCID/Bge mice with hyperimmune Balb/c lymphocytes followed by immunisation with the subunit vaccine for plague, allowed antigen-specific antibody to be detected for up to 4 months after reconstitution. It was apparent in these experiments that 9 out of 10 SCID/Bge mice were successfully reconstituted. No antibody response was detected in the single remaining SCID/Bge mouse, even after a booster immunisation. This finding that some SCID/Bge mice do not show evidence of reconstitution concurs with observations by other investigators .
Antibody levels in reconstituted mice were shown to increase after a second boost immunisation of reconstituted SCID/Bge mice with the subunit vaccine for plague. These results, together with the evidence of antibody secreting cells in spleen preparations from reconstituted mice, suggested that donor hyperimmune Balb/c lymphocytes are colonising recipient SCID/Bge mice lymphoid tissues, leading to successful and evidently long-term reconstitution of functional immune lymphocytes.
The protective effect which this adoptive transfer of immune lymphocytes conferred against a systemic plague challenge was clearly demonstrated here. A sample of surviving mice showed evidence of complete clearance of challenge organisms at the end of the 14-day observation period. It is evident in these adoptive transfer experiments that the SCID/Bge mouse offers a valuable model to investigate the protective effect of a subunit vaccine for plague.
The SCID/Bge mouse also offers a good model to investigate passive transfer of immunity because the mouse has no circulating immunoglobulins allowing easier interpretation of any protective effects. Balb/c mice show a predominantly Th2-type immune response to the vaccine and this response is typical of immunisation with protein antigens in alhydrogel . It has been shown in this study that passive transfer of hyperimmune serum leads to a high degree of protection against injected and inhaled challenge. This protection suggests that host resistance to plague is principally mediated by antibody. It is also evident that this passive protection afforded by antibody alone is not complete, against either systemic or inhaled challenge and suggests that an additional component of the immune system is required for complete protection. For this reason, the SCID/Bge mouse model used in this study to investigate the protection afforded by the subunit vaccine for plague will be further developed to examine selective reconstitution with immune cell subsets. This will allow an assessment of the role of different immune cell subset populations in immune protection against plague infection.