Attenuated strains of S. enterica
provide a well-studied vehicle for oral presentation of heterologous antigens to stimulate mucosal, humoral, and cell-mediated immunity (for a review, see reference 20
). Attempts to deliver many bacterial and viral antigens (including antigens from Shigella
, E. coli
, Vibrio cholerae
, Francisella tularensis
, Clostridium tetani
, streptococci, dengue virus, hepatitis B virus, human papillomavirus, influenza virus, and measles virus) via attenuated Salmonella
vectors have been made (20
). An important advantage of live attenuated oral delivery systems for a rapid response for public health use and emergencies is the ability to circumvent the need for syringes and needles or for administration by a trained health care professional.
There is already significant evidence demonstrating that an anti-PA immune response can protect against aerosolized anthrax spore exposure. However, the existing PA-based injectable vaccine has been associated with adverse reactions, and the recommended immunization regimen requires six or more doses over a period of 18 months. Our goal has been to develop an oral vaccine for human use that can be rapidly distributed and self-administered in a few doses over a short time period and that protects against anthrax infection. Because of the impressive safety record and long protection induced by the licensed oral typhoid vaccine, we used S
. Typhi strain Ty21a as the delivery platform for PA. The plasmid constructs used included a codon-optimized synthetic B. anthracis
PA gene, which also lacked two proteolytic cleavage sites, thus providing enhanced resistance to proteolytic degradation of the PA protein produced (32
). Use of this enhanced PA gene resulted in expression of PA that was greater (10-fold) than that obtained with the wild-type PA gene. In vivo-inducible promoters, such as the htrA
), were employed to maximize PA production in the target host while low expression was maintained during broth growth, thus enhancing strain stability during vaccine manufacture. Genetic fusions of the synthetic PA gene to the C terminus of HlyA were generated to allow extracellular secretion of PA (Fig. ) (9
). The expression plasmids containing inducible promoters displayed greater stability than the constitutive lpp
promoter-based plasmids in Ty21a even after 50 generations of growth in the absence of antibiotic selection (Table ), a key quality for vaccine manufacture.
The AVA BioThrax vaccine contains approximately 20 μg of PA per dose (21
). Semiquantitative analysis of PA expression in the Salmonella
host showed that the amount of PA produced from an optimized PA expression plasmid system, such as the HtrA-PAop
system, was approximately 3 μg of PA per 1 × 109
cells. The current Ty21a human dose in the United States can be as high as 6 × 109
CFU, so a comparable human dose for Ty21a producing PA might be roughly equivalent to a dose of the licensed AVA BioThrax vaccine. Moreover, the use of strong in vivo-inducible promoters may result in the production of even higher levels of PA over time in the host. Western blot analysis of PA expression showed that PA was efficiently targeted to the extracellular space by use of a secretion signal, as almost all PA was detected in the supernatant fraction (Fig. ). In contrast, PA lacking the HlyA secretion signal was detected mainly in the pellet fraction.
serovar Typhi is a human-specific pathogen, and there are no animal models in which to evaluate orally administered, live, attenuated S
. Typhi vaccine candidates. However, i.p. immunization and challenge of mice has been employed as a preclinical model to demonstrate immune stimulation by S
. Typhi-vectored vaccine candidates. Thus, the immunogenicity and protective efficacy of the Salmonella
constructs were evaluated using the A/J mouse model, because anti-PA-based protective efficacy can be demonstrated with this strain (43
). In addition, as reported by one of us previously, the anthrax disease progression in this mouse strain challenged with aerosolized Sterne spores is similar to that seen for other species, such as rabbits and nonhuman primates, challenged with fully virulent B. anthracis
Mice were immunized three times, 2 weeks apart, with Ty21a expressing PA under control of the nirB
promoter. These vaccine constructs were able to induce high antibody titers (GMT, 30,000) in A/J mice (Fig. ). Other recent studies using live attenuated Salmonella
expressing PA have not generated such robust anti-PA responses (7
) unless the vaccine construct was introduced via the intravenous route (8
). Secretion of PA from the host bacterial cell enhances PA-specific serum IgG antibody production, as the vaccine constructs expressing nonsecreted PA induced a GMT of only 600. The lower titers elicited by the construct expressing PA under control of the nirB
promoter are due to yields of PA slightly lower than those obtained with the htrA
promoter (data not shown).
Several studies using PA-based vaccines in rabbits, guinea pigs, and rhesus macaques have shown that there is not a positive correlation between the amount of total circulating PA-specific IgG and protection against B. anthracis
infection. However, a direct correlation was found between the titers of LeTx-neutralizing antibodies and protection against challenge in rabbits (30
). High titers (16,241) of toxin-neutralizing antibodies were induced by Ty21a-vectored derivatives secreting PA under control of the htrA
promoter and administered i.p. (Table ). In our model system, i.n. administration of similar vaccine candidates elicited significantly lower toxin-neutralizing titers (2,412) than i.p. administration. Interestingly, use of the Ty21a derivative producing PA intracellularly (HtrA-PAop
) resulted in very low titers of toxin-neutralizing antibodies. These observations demonstrate the importance of PA secretion by the bacterial vector, suggesting that the immune system cells can more easily access and process the secreted antigen. We appreciate the fact that the modified PA protein (without a furin cleavage site and with a noncleaved secretion signal at the C terminus) might affect the overall structure of PA and could result in elimination of important neutralizing epitopes. However, our constructs stimulated high serum anti-PA antibody titers (Fig. ) and high LeTx-neutralizing titers (Table ) in vaccinated mice.
Protective immunity induced by the Salmonella-
vectored PA vaccines was evaluated by i.n. challenging immunized mice with a lethal dose (>20 LD50
) of aerosolized spores of nonencapsulated, toxin-producing B. anthracis
Sterne strain 7702. Two different vaccine constructs producing secreted PA were tested in this study, and they generated a wide range of toxin-neutralizing antibody titers (1,702 to 16,241). Despite the wide range of toxin-neutralizing titers, all vaccine constructs producing secreted PA (regardless of i.n. or i.p. administration) provided complete protection against a lethal spore challenge. In contrast, only 40% of mice immunized with Ty21a producing PA intracellularly were protected, and this is a reflection of the low titer of functional antibodies produced in these animals (Table ). As expected, control mice immunized with the Ty21a vector alone were not protected, and all of them died within 5 days following challenge, a profile identical to that of naïve mice challenged in the same manner (23
). Two independent mouse immunogenicity (Fig. ) and challenge (Fig. ) studies were performed, and they yielded virtually identical results. LeTx-neutralizing titers, as shown in Fig. and Table , were determined only in a single mouse immunization study.
The data demonstrate that S. enterica
serovar Typhi strain Ty21a can serve as a suitable host for efficient production, secretion, and delivery of anthrax PA. Use of Ty21a exploits the extensive safety record of this existing licensed live attenuated oral typhoid vaccine, which has been administered to over 200 million individuals over 25 years with no documented cases of reversion to virulence or the occurrence of significant adverse reactions, such as reactive arthritis. In addition, field trials of Ty21a in developing countries have demonstrated generation of immunity lasting more than 7 years with 70 to 80% efficacy, depending on the formulation (19
). Recent collaborative studies with Aridis Pharmaceuticals LLC (San Jose, CA) have resulted in the manufacture of temperature-stabilized dried preparations of Ty21a that have a projected shelf life of 5 to 10 years at 4°C and ≥1 year at room temperature. Thus, this vaccine has potential for inexpensive production, a long shelf life, and distribution without refrigeration, valuable qualities for a biodefense vaccine.
The mouse has served as a suitable preliminary model for evaluating the immunogenicity and protective efficacy of the vaccine constructs. Two previous studies using attenuated Salmonella
to deliver PA in mice have relied on S. enterica
serovar Typhimurium as a delivery platform for PA (8
). However, a Salmonella
-based oral vaccine against anthrax infection in humans will most likely depend on Salmonella
serovar Typhi as a delivery vector. One previous study showing expression of PA in S
. Typhi reported only moderate immune responses against PA in vaccinated mice and provided no data on protective efficacy in this animal model (7
). Here we show that the safe, licensed typhoid vaccine strain S
. Typhi Ty21a can efficiently deliver optimized anthrax PA to induce a robust protective immune response against a lethal aerosolized anthrax spore challenge in vaccinated mice. The ultimate goal of these studies, a temperature-stable, oral human vaccine against anthrax infection that can be self-administered in a few doses over a 1-week period, now awaits further animal (rabbits) and phase I human testing.