This study demonstrated that oral booster doses of Y. pestis-derived antigens were at least as effective at eliciting protective antigen-specific antibody responses as needle-based s.c. doses of the same antigens. In addition, we found for the first time that a plant-based vaccine against the etiologic agent of plague successfully protected mice from lethal Y. pestis challenge. The levels of F1-V in chloroplasts—up to 14.8% of the TSP—enabled the delivery of sufficient amounts of vaccine antigens in intact plant cells.
We observed that oral boosts of transgenic plant material containing the plague fusion antigen F1-V without adjuvant performed as well as s.c. boosts containing chloroplast-derived enF1-V with adjuvant at eliciting a predominant IgG1 titer in the serum of vaccinates. This type of response is indicative of an ongoing TH2 response (78
) and, in s.c. vaccinated animals, is typical of AlH-adjuvanted vaccines (32
). The TH2 response, specifically mediated by serum levels of F1-V-specific IgG1, has been shown to protect against both s.c. (95
) and aerosolized (3
) Y. pestis
challenge. Our results corroborate these findings. Orally boosted mice had similar or, in some cases, higher levels of antigen-specific IgG1. Further, these responses were consistent across the various antigens tested, F1, V, and F1-V. The anti-F1 and anti-V IgG1 responses were not significantly different from one another (P
> 0.05), indicating that the individual components of the F1-V fusion protein had relatively equal immunogenicities.
The IgG1 levels were generally 2 to 3 logs higher than the corresponding IgG2a levels. Not surprisingly, animals with the highest IgG1 titers were more likely to survive challenge with live Y. pestis
= 0.71) (Table ). Overall, oral F1-V boosters yielded somewhat higher IgG1 titers, more survivors, and a longer MTD than s.c. F1-V boosters. Oral WT group control-boosted mice had the lowest IgG1 titers of the three vaccinated mice, the fewest survivors, and the shortest MTD. Collectively, these results may suggest that the route of immunization or the adjuvant plays a critical role in driving the formation of a protective response in which both IgG1 and IgG2a are present. It is important to note, however, that the oral boosts in our study did not contain adjuvant, nor was the acidic pH of the gut neutralized, which has been done in other oral vaccination schemes (50
Because of the severe pathogenicity of Y. pestis
, treatment of plague is a high priority. The use of antimicrobial agents began in 1938 (65
) and has led to a dramatic drop in human mortality. Today, the worldwide fatality rate attributable to plague has fallen to less than 8% (26
). Natural isolates of Y. pestis
are uniformly susceptible to all antimicrobial agents active against gram-negative bacteria (8
). However, a “natural” strain resistant to multiple antibiotics was isolated in 1995 in the Ambalavao district of Madagascar (27
). The possibility of the occurrence of such multidrug-resistant strains in the natural environment, the ease of generating such strains under laboratory conditions (39
), and the potential use of such strains for bioterrorist attack, together with the rapidity and high lethality of the disease (for a review, see reference 5
), indicate that it is necessary to search for alternatives to antibiotics.
Immunization is now one of the major approaches being pursued to deal with potential Y. pestis
infection. Use of the serum of vaccinated rabbits to cure animals infected with Y. pestis
was first attempted more than 100 years ago (97
). Since then, several antigens have been shown to be able to produce protective immunity. Among these antigens are the F1 capsular (58
) and LcrV (or V) antigens (51
), both of which also contain immunodominant epitopes (38
). Passive administration of antibodies against target antigens protects macrophages from Y. pestis
-induced cell death, promotes phagocytosis (13
), and protects animals against both bubonic plague and pneumonic plague (4
). However, therapy based on a single antibody against a single antigen or epitope will be ineffective in the case of infection with a virulent strain lacking the antigen or expressing a different serological variant of the antigen (6
Human plague vaccines based on either a live, attenuated strain or a killed, whole-cell preparation (for a review, see reference 5
) are no longer commercially available. Given the severity of the infection and the potential of the organism as a bioterrorist agent, we describe a subunit vaccine produced in transgenic tobacco chloroplasts. This vaccine offers the advantage of employing two defined antigens, F1 and V, that are able to elicit high-level protection. Subunit vaccines have been shown to be less reactogenic than the former whole-cell vaccines (6
There have been several reports of s.c., intranasal, and oral vaccination strategies protecting animals from Y. pestis
challenge (for a review, see reference 82
). Oral plague immunization studies of attenuated Salmonella
vaccines have shown that these vaccines are effective (28
). But there are clinical concerns for vaccination of special-needs populations, such as the young, the old, pregnant women, and immunocompromised individuals. Another practical concern is plasmid maintenance and stability. We believe that the use of orally delivered plant-based vaccines may obviate these concerns and that such vaccines may be less reactogenic in the general population.
One might expect that protection of the mucosal surface would be critical against aerosolized Y. pestis
. Mucosal protection is normally mediated by local production of secretory IgA (56
). Although IgA is the predominant humoral defense mechanism at mucosal surfaces, local IgM and IgG and, in the lower respiratory tract, serum IgG can also contribute significantly to immune defense (40
). Indeed, serum IgG1 has been shown to be the predominant isotype produced in response to recombinant plague vaccines and to be associated with protection against aerosolized Y. pestis
). The high IgG1 titers elicited in this study confirmed these findings and indicate that there was appropriate immune modulation by our plant-based vaccine.
The contribution of IgA to the protective response to plague is less clear. Previous oral plague vaccine strategies have been shown to induce low levels of serum IgA, although the expression was not examined for an association with parenteral protection (60
). In our study, we observed low but measurable levels of F1-V-specific IgA in the serum; however, this response correlated weakly with protection (r
= 0.26). More recently, an attenuated Salmonella
oral vaccine expressing both F1 and V antigens was shown to induce fecal IgA and serum IgG1/IgG2/IgG3 responses, as well as protection against both bubonic plague and pneumonic plague (96
). Again, no direct comparison of IgA with survival was made. Another recent study in which F1-V was mixed with the mucosal adjuvant Protollin and administered intranasally did show that there was a statistical association between lung IgA levels and protection (43
). This finding is complicated by the fact that there were also statistical associations between lung and serum IgG levels and protection. Collectively, these data suggest that an appropriate mucosal adjuvant may be required to induce secretory IgA. The ability to passively protect mice with IgA alone may need to be shown to firmly establish the contribution of IgA to overall protection from plague.
Other studies have suggested the importance of the route of immunization to the overall levels and types of antibody to F1 and V, as well as protection against subsequent Y. pestis
). It is becoming increasingly clear that mixing of vaccine routes can have a positive effect on both antibody production and protection. It has been previously reported that in F1-V vaccinations, heterologous prime-boost regimens are at least as effective as homologous boost regimens for inducing serum anti-F1-V IgG1 responses (29
). In this study, all vaccinated mice received an initial s.c. injection, which effectively induced a potent systemic humoral response (Fig. ). Oral delivery resulted in the highest levels of serum IgG1 and IgG2a of all groups but negligible titers of F1-V-specific IgA in the feces of animals. At first, this finding appears to be paradoxical. However, the route chosen for induction of peripheral immunity may delay or prevent the induction of mucosal immunity (10
). Indeed, s.c. immunizations followed by oral boosts have been shown to suppress local intestinal antigen-specific IgA responses (34
). The latter observation appears to support our finding that there was a limited IgA response following oral boosts of F1-V.
This report of a plant-based plague vaccine demonstrates for the first time that high-level protection of mice from the lethal pathogenic effects of aerosol challenge can be elicited by oral delivery. Virus-based plant expression systems have been used successfully to confer protection against Y. pestis
challenge in guinea pigs and macaques upon s.c. immunization with purified F1-V antigens (57
). There is one previous report of an s.c. prime-oral boost strategy, involving Agrobacterium
-transformed tomato nuclei, but no pathogen challenge was performed (1
). The prime-boost strategy that we employed was designed to maximize antigen delivery to the immune system while minimizing processing time and costs. The efficacy of this strategy also indicates that oral immunization boosts are at least as effective as s.c. boosts for eliciting systemic humoral responses. In addition, if oral boosts are found to consistently perform at least as well as s.c. boosts, then the use of needle-based vaccines and the substantial costs associated with their use may need to be reexamined. Chloroplast technology is therefore an ideal system for oral delivery of vaccines.