Correlates of Immunity Elicited by Live Yersinia pestis Vaccine

  • Vivian L. Braciale
  • Michael Nash
  • Namita Sinha
  • Irina V. Zudina
  • Vladimir L. Motin
Part of the Infectious Disease book series (ID)

49.1 Introduction

Yersinia pestis is the causative agent of plague, one of the most severe bacterial infections in the history of mankind. Y. pestis has been squarely placed on the category A select agent list because of its potential to be used as an agent of biowarfare and bioterrorism (1). Most human plague cases usually present as one of three primary forms, i.e., bubonic, septicemic, or pneumonic, with the latter two having a high mortality rate (2). Currently, no plague vaccine exists in the United States, although until 1999, a formaldehyde-killed, whole-cell vaccine was available for military and laboratory personnel. This vaccine required a course of injections over a period of six months and was effective against bubonic plague. However, the protection was short-term and annual boosters were required; additionally, the incidence of side effects, such as malaise, headaches, elevated temperature, and lymphadenopathy, was high (in ̃10% of those immunized with vaccines); and the vaccine was expensive. Moreover, the protection of killed, whole-cell vaccine against the pneumonic form of plague was uncertain (3). A live-attenuated vaccine is available in the former Soviet Union (SU) countries. This vaccine Y. pestis E V, line NIIEG, represents a non-pigmented version of the virulent strain isolated in Madagascar. The vaccine strain is attenuated due to deletion of the 102-kb locus Pgm, which includes the hemin-storage region Hms and a cluster of genes encoding the siderophore-based yersinia bactin biosynthetic/transport systems (2, 4). A comparison of killed and live plague vaccines showed that the latter was more efficient when immunized animals were challenged with the fully virulent strain of Y. pestis by three routes of administration in three strains of mouse (5). The live plague vaccine has been adopted for human use for more than 70 years, and it is still the vaccine of choice in the former SU countries for those working around plague. The vaccine is considered to be effective against all forms of plague; however, the safety of this vaccine in humans is questionable, because it retains some virulence, and in most countries (including the United States) live vaccines such as this are not licensed (6, 7, 8).

Currently, the vaccine considered to present the best prospects for human use is a subunit vaccine consisting of either the mixture of LcrV and F1 antigens or their peptide fusion form F1-LcrV (3, 8). The LcrV protein (V antigen) of Y. pestis is a multifunctional protein involved in modification of innate immune responses to this pathogen, as well as in regulation of expression and translocation of Yop effectors of the type III secretion system (T3SS; refs. 9 and 10). The F1 antigen is a capsular subunit protein located on the surface of the Y. pestis cell, which is thought to have anti-phagocytic properties (11, 12).

In this study, we have compared both humoral and cellular immune responses to the formaldehyde-killed and live plague vaccines elicited in BALB/c mice. Since it is not possible to test the safety and efficacy of these vaccines in human volunteers, it was important to identify immune correlates of protection. We had the rare opportunity to characterize the Y. pestis -specific antibody and T-cell-mediated immune responses of individuals previously immunized with the live-attenuated vaccine strain EV and compare these responses to those elicited in BALB/c mice.

49.2 Results and Discussion

49.2.1 Experimental Outline for Immunization in the Murine Model

Y. pestis vaccine strain EV76 was used to make both killed and live plague vaccines for immunization of BALB/c mice and following in vitro assays. Killed vaccine was prepared by the same methods used in the past to produce a plague vaccine for human immunization (13). The procedure included the growth of Y. pestis on a complex solid medium for 72 hours at 37°C, followed by killing the cells with formaldehyde. The killed vaccine stocks were stored in bulk at 4°C with 0.5% of phenol as a preservative. Live vaccines were prepared freshly prior to each immunization and consisted of the cells grown either at 26 or 37°C in the Heart Infusion Broth for 30 hours with aeration. Final cell concentration was measured by spectropho-tometer at an optical density of 600 nm, followed by plating of the culture in 10-fold dilutions (colony-forming units).

Groups of female BALB/c mice were immunized at the age of 6 to 8 weeks either with killed or live vaccines (Figure 49.1). Killed vaccine at a dose of 2 × 108 formaldehyde-treated cells was administered either with or without Inject Alum adjuvant (Pierce, Rockford, IL) via the subcutaneous (s.c.) route. Live vaccines, grown at two temperatures, were inoculated at a dose of 2 × 105 by the same s.c. route. Mice were boosted twice at intervals of 14 days by killed vaccine and once with live vaccines on day 28 after primary immunization. Blood samples were collected at days 0, 29, 35 and 43, followed by a terminal bleed and spleen harvest on day 57.
Figure 49.1.

Immunization and bleed schedule.

49.2.2 Murine Humoral Responses

Several major antigens of Y. pestis were cloned and expressed in E. coli as N -terminal fusions with a polyhistidine Tag, followed by affinity purification using an Ni 2+− charged resin. To test the antibody response in mice immunized with killed and live vaccine, we used the T3SS effectors YopM and YopE, known protective antigens LcrV and F1, and plague plasminogen activator Pla. The quality of purification of Y. pestis antigens and the results of Western blots using sera collected on day 43 are depicted in Figure 49.2.We found that the sera of mice immunized with live vaccines prepared from cells grown at 26 or 37°C reacted with LcrV and capsular antigen F1 (Figure 49.2B). Since the expression of both LcrV and F1 antigens is practically nonexistent in Y. pestis cultured at 26°C, the identical picture of Western blots from sera obtained after immunization with live vaccines prepared from cells grown at two different temperatures was the indicator that live vaccine organisms replicated in vivo. In contrast, antisera from mice immunized with killed vaccine, with or without adjuvant, contained antibodies to F1 but lacked immunoglobulin specific to LcrV (Figure 49.2C). This would be expected since F1 but not LcrV is a constituent of the input-killed Y. pestis prior to inactivation with formaldehyde. Since the killed vaccine could not replicate in the mouse, there was no LcrV production in vivo. These results were confirmed in enzyme-linked immunosor-bent assays (ELISA; Table 49.1).
Figure 49.2.

Antigenic specificity of immune antisera. (A) The silver-stained PAGE gel of purified recombinant Y. pestis antigens: 1- YopM, 2- YopE, 3- LcrV, 4- F1, 5- Pla. (B, C) Results of representative Western blots using antisera from mice immunized with live and killed vaccines, respectively.

Table 49.1.

ELISA antibody titers to F1 and LcrV antigens in vaccinated mice

Anti-F1, IgG

 

Vaccine

Week 4

Week 5

Week 6

Terminal

 

Mock

<1/100

<1/100

<1/100

<1/100

 

Killed

>1/12,500

>1/12,500

>1/12,500

>1/12,500

 

Killed+ Alum

>1/12,500

>1/12,500

>1/12,500

>1/12,500

 

Live, 26°C

>1/12,500

>1/12,500

>1/12,500

>1/12,500

 

Live, 37°C

>1/12,500

>1/12,500

>1/12,500

>1/12,500

 

Anti-LcrV, IgG

 

Vaccine

Week 4

Week 5

Week 6

Terminal

 

Mock

<1/100

<1/100

<1/100

<1/100

 

Killed

<1/100

<1/100

<1/100

<1/100

 

Killed + Alum

<1/100

<1/100

<1/100

<1/100

 

Live, 26°C

<1/100

>1/1,000

>1/1,000

<1/1,000

 

Live, 37°C

<1/100

>1/1,000

>1/1,000

<1/1,000

 
Results for pooled bleeds showed that both live and killed vaccines elicited F1-specific antibodies. Antisera from immune mice bled on days 29, 35, and 43 showed titers greater than 12,500. The titers to F1 were high at four weeks and remained high regardless of the immunization protocol. Mice immunized with killed vaccine, however, did not develop antibodies specific for V antigen. Mice receiving live-attenuated vaccine showed low titers to LcrV at 4 weeks; these levels peaked at 6 weeks and began to decline at terminal bleed. Thus, the kinetics of antibody development to F1 differed from that to V antigen. Determination of IgG isotype usage to F1 antigen did not reveal a principal difference between groups of mice immunized with live or killed vaccines (Figure 49.3A). Similarly, there was little difference in IgG subtype pattern to LcrV in mice immunized with live vaccines grown at two different temperatures, although anti-LcrV titers seemed to be higher when Y. pestis EV76 cells were cultured at 37°C (Figure 49.3B).
Figure 49.3.

IgG isotypes determined on day 43. (A) Immunoglobulin subtypes to capsular antigen F1 for all groups of mice immunized either with killed or live vaccines; (B) Immunoglobulin subtypes to LcrV for the groups of mice immunized with live vaccine cells grown either at 26 or 37°C.

49.2.3 Murine T-cell-mediated Responses

In order to examine the murine T-cell-mediated responses, spleens were removed at the time of terminal bleed and splenocytes harvested for in vitro T-cell studies. A portion of the splenocytes were frozen and stored in liquid nitrogen for future studies. Another portion was stimulated with heat-killed (HK) Y. pestis in vitro. Samples from these cultures were tested for 3H-thymidine incorporation on days 3 and 7 of culture. The remaining cells from this in vitro stimulation were frozen and stored in liquid nitrogen for future studies. Following initial studies using spleens from individual mice from each group, the spleens were pooled for each experimental group. Preliminary data indicated that the immune splenocytes are stimulated to proliferate in the presence of HK Y. pestis (data not shown). As illustrated in Figure 49.4, immune splenocytes, but not naïve splenocytes, produced IFN-γ when stimulated with HK Y. pestis. Figure 49.5 shows the results of testing culture supernatants for IL-17, IL-3, IL-6, GM-CSF, IL-10, IL-5, and IFN-γ. IL-4, and IL-2 were below detection possibly due to consumption of these cytokines in the 3-day cultures. The most telling difference between the cytokines produced by splenocytes from mice given killed vs. live vaccine lay in the lack of IFN-γ, as was the case for the former, but not the latter. When the splenocytes were from mice vaccinated with live vaccine, and stimulated with HK versus live Y. pestis, differences were seen for the cytokines and chemokines produced. Stimulation with live cells of Y. pestis resulted in significantly higher production levels of KC, CM-CSF, IL-3 and IL-6, in comparison with the stimulation by HK cells (Figure 49.6). Also, stimulation of splenocytes with capsular protein F1 from mice immunized with killed Y. pestis resulted in an antigen-specific T-cell proliferative response (Figure 49.7).
Figure 49.4.

IFN-γ production by murine splenocytes stimulated in vitro with HK Y. pestis. Splenocytes from naïve mice, or mice immunized with live Y. pestis were stimulated in vitro with medium, 10 μg/ml PHA, or killed Y. pestis. Culture supernatants were harvested on days 3 and 6 and tested for IFN-γ

Figure 49.5.

Y. pestis elicited Th1/Th2 cytokines. Splenocytes from mice immunized with killed (top panel) or live (bottom panel) Y. pestis were stimulated in vitro with medium, 10 μg/ml PHA, or HK Y. pestis. Culture supernatants were harvested on days 3 and 6 and tested for IL-17, IL-3, IL-6, GM-CSF, IL-10, IL-5, and IFN-γ (See Color Plates).

Figure 49.6.

Cytokine and chemokine responses elicited by in vitro stimulation of immune T cells with live or HK Y. pestis. Splenocytes from mice immunized with live Y. pestis were stimulated in vitro with HK or live Y. pestis. Culture supernatants were harvested on days 5 or 6 and tested for RANTES, MIP-1α, KC, G-CSF, IL-17, IL-3, IL-6, GM-CSF, IL-10, IL-5, and IFN-γ

Figure 49.7.

Capsular protein F1 stimulation of Y. pestis immune splenocytes. Splenocytes from mice immunized with killed Y. pestis were stimulated in vitro with medium, killed Y. pestis, 1 μg/ml ConA, or F1. Cultures were assayed on days 3 for 3H-thymidine incorporation.

49.2.4 Human Humoral Responses

Blood samples were obtained from two volunteers who had previously been immunized with live plague vaccine Y. pestis EV, line NIIEG. Donor A1 was immunized once approximately one month prior to the beginning of experiments. A pre-immune serum from Donor A1 was available. Then, Donor A1 was bled monthly for 8 months post-immunization. Testing of antisera Donor A1 in Western blots and ELISA for reactivity to purified Y. pestis antigens YopM, YopE, LcrV, F1, and Pla produced negative results. Donor A2 had multiple immunizations with the live plague vaccine with the last inoculation occurring at least 5 years prior to the current testing, therefore, pre-immune serum from this donor was not available. Despite a prolonged period of time that passed from the last immunization, Donor A2 had anti-F1 immunoglobulin in the blood, as detected via Western blot (Figure 49.8, lane 4). Also, we observed a weak positive signal of Donor A2's antiserum with Pla antigen (Figure 49.8, lane 5). However, this reaction was likely non-specific, since Pla protein displayed a similar signal when the sera of three non-immune donors participating in this study was used as a control (data not shown). Determination of IgG isotype usage to F1 antigen revealed that there was no a dominant subtype in Donor A2's antiserum (Figure 49.9). Thus, we observed a long-lasting circulation of anti-F1 antibodies in the blood of the person immunized with live vaccine, and this fact is in good agreement with previously published findings on human vaccination with Y. pestis EV, line NIIEG (14). Specific antibodies to LcrV were not detected in Donor A2's serum. This was not a surprising outcome, taking into account our observation that antibody titers to LcrV in mice immunized with live plague vaccine reached their maximum at 5 to 6 weeks, but then started to decline by week eight post-immunization (Table 49.1).
Figure 49.8.

Antigenic specificity of immune antisera from Donor A2. The left panel shows the silver-stained electrophoretic gel of purified Y. pestis antigens (lanes 1.YopM, 2. YopE, 3. LcrV, 4. F1, 5. Pla and 6. whole bacterial lysate of Y. pestis EV76 grown at 37°C. The right panel shows results of Western blot using antisera from Donor A2, who had been vaccinated with live attenuated Y. pestis.

Figure 49.9.

IgG isotypes to capsular antigen F1 in the serum of Donor A2.

49.2.5 Human T-cell-mediated Responses

Despite the lack of identifiable antibodies to several purified proteins of Y. pestis in the serum of Donor A1, we have found antigen-specific T-cell recall responses in this donor. The T-cell-mediated responses of Donor A2 have been tested as well. For examination of human T-cell-mediated responses, peripheral blood mononuclear cells (PBMCs) were isolated from blood collected from Donors A1 and A2 by centrifugation over Lymphocyte Separation medium. A portion of PBMCs were frozen and stored in liquid nitrogen for future studies. A portion of PBMCs were stimulated with HK Y. pestis grown at 37 or 26°C. These cultures were tested at days 3 and 7 for 3H-thymidine incorporation. The results of the stimulation have shown that both cells grown at 37 and 26°C provided a strong proliferative response, particularly on day seven (Figure 49.10). The remaining stimulated cells were frozen for future studies. T-cell-depleted PBMCs were transformed with EBV to generate autologous antigen-presenting cells for use in in vitro re-stimulation to propagate antigen-specific T-cell lines. PBMCs from non-immune (B2) and immune (A1 or A2) donors were stimulated with Y. pestis for proliferation assays, as well as for cytokine production. The PBMCs from B2 did not proliferate in the presence of Y. pestis, while PBMCs from A1 did (Figure 49.11). Figure 49.12 shows the results of testing culture supernatants of in vitro stimulation of human PBMC from immune Donor A2 with PHA, HK Y. pestis grown at 37°C and purified capsular antigen F1 for the number of cytokine and chemokine responses.
Figure 49.10.

PBMC stimulation with killed Y. pestis. PBMCs of Donor A1 were stimulated with medium, 10 μg/ml PHA, or HK Y. pestis grown at 26 or 37°C. Quadruplicate cultures were pulsed with tritiated thymidine on days 3 and 7.

Figure 49.11.

Antigen-specific stimulation of human PBMC. PBMCs from non-vaccinated Donor B2, and from Donor A1, vaccinated once with live attenuated Y. pestis were stimulated with PHA or HK Y. pestis. 3H-TdR incorporation was determined in day 6 cultures.

Figure 49.12.

Cytokine and chemokine responses elicited by in vitro stimulation of human PBMC from immune Donor A2 with PHA, HK Y. pestis grown at 37°C and purified capsular antigen F1. Culture supernatants were harvested on day 3 and tested for IL-10, IFN-γ, IL-4, TNF-α, IL-5, IL-7, IL-12, IL-13 and IL-17 (See Color Plates)

Finally, we have compared cytokine and chemokine responses of PBMCs from both immune donors A1 and A2 with those from two naïve donors (B4 and B5) after stimulation with HK Y. pestis cells (Figure 49.13). The results shown in Figures 49.11 to 49.13 clearly demonstrated plague cellular immunity in vaccinated individuals.
Figure 49.13.

Cytokine and chemokine responses elicited by in vitro stimulation of human PBMC from immune Donors A1 and A2 as well as from naïve Donors B4 and B5 with heat-killed Y. pestis. Culture supernatants were harvested on day 3 and tested for IL-8, IL-10, IFN-γ, IL-1β, GM-CSF, MCP-1, MIP-1β (See Color Plates)

49.3 Conclusions

Thus far, our findings support the premise that killed plague vaccine does not elicit antibodies to LcrV, and we would expect to find a lack of T-cell-mediated response to this protein after immunization with this vaccine. Using the live vaccine in the mouse, we have been able to elicit antibodies to LcrV and expect to find LcrV-specific T cells derived from these mice. We have yet to find Y. pestis -specific antibodies to LcrV in the human sera; however, our experiments in mice suggested that the time of circulation of anti-LcrV antibody at detectable levels might be short. In contrast, anti-F1 antibodies could be detected in human serum for years post-vaccination. The F1 antigen-specific T-cell recall responses could be detected in both humans and mice that were followed after all types of immunizations with plague vaccines. Overall, T-cell stimulation studies of PBMCs from human donors immunized once or multiple times with live plague vaccine indicated that there were Y. pestis -specific memory T cells in the blood of the vaccinated individuals. Our data are in good agreement with recently published observations on long-lasting T-cell responses in veterans of the Gulf conflict (1990–1991) immunized with killed plague vaccine (15).

Notes

Acknowledgments

We thank Dr. Valentina A. Feodorova for helpful discussions. This work was supported by grant U54 AI0577156 to NIH/NIAID Western Regional Center of Excellence for Biodefense and Emerging Infectious Diseases.

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Vivian L. Braciale
    • 1
  • Michael Nash
    • 1
  • Namita Sinha
    • 1
  • Irina V. Zudina
    • 2
  • Vladimir L. Motin
    • 3
  1. 1.Department of Microbiology and ImmunologyUniversity of Texas Medical BranchGalvestonUSA
  2. 2.Department of PathologyUniversity of Texas Medical BranchGalvestonUSA
  3. 3.Departments of Pathology/Microbiology and ImmunologyUniversity of Texas Medical BranchGalvestonUSA

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