A Novel Neurotoxoid Vaccine Prevents Mucosal Botulism
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免疫学杂志 2005年第4期
Abstract
The threat posed by botulism, classically a food- and waterborne disease with a high morbidity and mortality, has increased exponentially in an age of bioterrorism. Because botulinum neurotoxin (BoNT) could be easily disseminated by terrorists using an aerosol or could be used to contaminate the food or water supply, the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases has classified it as a category A agent. Although clearly the development of a safe and effective mucosal vaccine against this toxin should be a high priority, essentially no studies to date have assessed mucosal immune responses to this disease. To bridge this gap in our knowledge, we immunized mice weekly for 4 wk with nasal doses of BoNT type A toxoid and a mutant of cholera toxin termed E112K. We found elevated levels of BoNT-specific IgG Abs in plasma and of secretory IgA Abs in external secretions (nasal washes, saliva, and fecal extracts). When mice given nasal BoNT vaccine were challenged with 4 x 103 LD50 of BoNT type A (BoNT/A) via the i.p. route, complete protection was seen, while naive mice given the same dosage died within 2 h. To further confirm the efficacy of this nasal BoNT vaccine, an oral LD50 was determined. When mice were given an oral challenge of 5 μg (2 x oral LD50) of progenitor BoNT/A, all immunized mice survived beyond 5 days, while nonimmunized mice did not. The fecal extract samples from nasally vaccinated mice were found to contain neutralizing secretory IgA Abs. Taken together, these results show that nasal BoNT/A vaccine effectively prevents mucosal BoNT intoxication.
Introduction
It is now clear that parenteral immunization usually fails to elicit protective mucosal immune responses (1). Various alternate routes of Ag-vaccine delivery have been suggested by researchers, but none has shown more promise for the induction of Ag-specific Ab responses in the upper mucosal compartments (e.g., the respiratory tract) than nasal administration or in the gastrointestinal (GI)3 tract than oral administration (2, 3, 4, 5, 6, 7). Our own previous studies have shown that gut-associated lymphoreticular tissue-directed oral immunization effectively induces Ag-specific secretory IgA (S-IgA) Ab responses in the small intestinal lamina propria (iLP) and the submandibular glands, but not in the lung or the lamina propria of the nasal or reproductive tracts (5, 6, 7, 8, 9). In most current nasal immunization studies, the vaccine is instilled into each nostril (usually 5 μl/nostril), and normal inhalation results in the effective delivery of the vaccine, presumably into nasopharyngeal-associated lymphoreticular tissues (NALT). Such NALT-mediated nasal immunization has been shown to elicit significant immunity in the respiratory, GI, and reproductive tracts as well as in the nasal and oral cavities. These findings suggest that the nasal route of Ag delivery may possess potential advantages in the induction of mucosal immunity.
Native cholera toxin (CT) and mutants of CT (mCTs) are effective mucosal adjuvants and have been widely used for nasal immunization with protein Ags, bacterial components, viruses, or virus-related peptides for the induction of protective immunity associated with S-IgA and plasma IgG Ab responses (4, 10, 11, 12). Nasal immunization with the weakly immunogenic OVA along with mCT as adjuvant resulted in S-IgA anti-OVA Ab responses in various mucosal external secretions (4). Furthermore, mice nasally immunized with pneumococcal surface protein A Ag plus mCT revealed pneumococcal surface protein A-specific S-IgA Ab responses associated with effective protection against capsular serotype 3 Streptococcus pneumoniae A66 (10). These Ag-specific S-IgA Ab responses were associated with polarized Th2-type responses in cervical lymph nodes (4, 10). Nasal immunization with diphtheria toxoid plus mCT E112K has also been shown to induce protective immunity to the diphtheria exotoxin (11). Furthermore, our recent studies showed that young adult as well as aged mice given a nasal vaccine of tetanus toxoid along with a chimera of mCT-A E112K and heat labile toxin (LT) B subunit were protected when mice were challenged via the systemic route with tetanus toxin (12, 13).
Clostridium botulinum is an anaerobic bacterium that produces a powerful exotoxin termed botulinum neurotoxin (BoNT), which induces flaccid paralysis (14, 15, 16, 17). The BoNTs, serologically divided into seven immunologically different types (A through G), are released from the bacterium as a single polypeptide chain (14, 15, 16, 17). Among these seven serotypes, types A, B, and E are typically associated with botulism in humans, and type C has been the most common cause of disease in domestic animals. However, both C and D induce botulism in cows (15, 16, 17, 18). As with foodborne botulism, type A is responsible for 60%, type B for 30%, and type E for 10% (18) of cases in humans. It should be emphasized that bioterrorists could potentially use any or all seven serotypes. The molecular mass of each exotoxin type is 150 kDa (14, 15, 16, 17). Each consists of two polypeptide chains, an L chain (50-kDa) and an H (100-kDa) chain. The main targets of BoNT are the peripheral cholinergic nerve endings and particularly the cholinergic neuromuscular junction. BoNT inhibits exocytosis, inducing the flaccid paralysis characteristic of botulism and eventually leading to death (19, 20, 21, 22, 23, 24, 25).
The bacterium C. botulinum and its derived BoNTs enter the body most commonly through the GI tract via tainted food. In the case of a bioterrorist event, the BoNT would most likely be disseminated by airborne or possibly waterborne routes. BoNTs are classified as one of the six highest risk agents for bioterrorism (category A agents) by the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases. Any chance of surviving this extremely potent and lethal toxin rests upon prolonged intensive care. The current pentavalent botulinum vaccine (serotypes A-E) is used for at-risk populations such as scientists researching BoNTs, health care providers dealing with their clinical manifestations, and the military (26). Furthermore, the current pentavalent vaccine is administered via the parenteral route and induces prolonged pain and swelling at the site of injection (26) (H. F. Staats, unpublished results) To minimize these side effects, several research groups have attempted to develop BoNT vaccines using recombinant C-terminal half of the H chain of BoNT peptide or synthetic epitopes (27, 28, 29, 30, 31, 32). Furthermore, a Venezuelan equine encephalomyelitis virus replication vector system provided effective protection against BoNT intoxication (33). However, given the nature of botulinum intoxication, it is essential to devise more effective mucosal vaccines to prevent the intoxication at both mucosal and systemic tissue compartments.
In this study, we have examined whether a nasal vaccine consisting of botulinum neurotoxoid type A (BoNToxoid/A) and mCT E112K as adjuvant would effectively induce protective immunity to BoNT/A challenge. Our results indicate that this nasal vaccine induced BoNT-specific Ab responses in both mucosal and systemic lymphoid tissues. Furthermore, the nasal vaccine prevented botulinum intoxication following either mucosal or parenteral challenge.
Materials and Methods
Mice
Young adult (6–8 wk old) C57BL/6 mice were purchased from the Frederick Cancer Research Facility (National Cancer Institute). Upon arrival, all mice were immediately transferred to microisolators and maintained in horizontal laminar flow cabinets and provided sterile food and water ad libitum. Experiments were performed using young adult C57BL/6 mice between 6 and 8 wk of age. The health of the mice was tested semiannually, and mice of all ages used in these experiments were free of bacterial and viral pathogens.
Preparation of BoNToxoid/A
The BoNT/A from C. botulinum type A 62 was used for toxoid preparation. The BoNT/A was purified using previously described methods (15). Briefly, purified BoNT/A was detoxified at 30°C by dialyzing against 0.2% Formalin in 0.1 M phosphate buffer (0.2 mg/ml, type A toxin containing 3.5, 106 LD50/ml). The detoxification proceeded at 30°C. The formalized toxin was sampled at intervals for mouse inoculation until it became completely nontoxic. At each interval, two mice were i.p. administered with 0.5 ml of the sample and then observed for 4 days. The formalized toxoid was shown to be nontoxic because mice neither died from intoxication nor showed any specific symptoms of intoxication such as muscle spasms, stiffening, or any other abnormal signs during the observation period. The toxoid was kept at 4°C until used (15, 34).
Nasal immunization and sample collection
Mice were nasally immunized at weekly intervals for 4 wk with 20 μg of BoNToxoid/A and 5 μg of mCT E112K in PBS (4, 10, 11, 12, 13). Plasma and mucosal external secretions (nasal washes, saliva, and fecal extracts) were collected on day 28. Saliva was obtained from mice following i.p. injection with 100 μg of sterile pilocarpine (7). Fecal pellets (100 mg) were suspended into 1 ml of PBS containing 0.1% sodium azide and were then extracted by vortexing for 5 min. The samples were spun at 10,000 x g for 5 min, and the supernatants were collected as fecal extracts (5, 6, 7). The mice were sacrificed 7 days after the last immunization. The nasal washes were obtained by injecting 1 ml of PBS on three occasions into the posterior opening of the nasopharynx with a hypodermic needle (13).
Ab assays
Ab titers in plasma and mucosal secretions were determined by an ELISA (4, 5, 6, 7, 11, 12, 13). Briefly, Falcon microtest assay plates (BD Biosciences) were coated with an optimal concentration of purified BoNT/A (100 μl of 2 μg/ml) in PBS overnight at 4°C (35). Two-fold serial dilutions of samples were added after blocking with 1% BSA. To detect BoNT/A-specific Ab levels, HRP-conjugated, goat anti-mouse or , H chain-specific Abs were used (Southern Biotechnology Associates). For IgG Ab subclass determinations, biotinylated mAbs specific for IgG1 IgG2a, IgG2b, and IgG3 (BD Pharmingen) and peroxidase-conjugated goat anti-biotin Ab were used, as described elsewhere (11, 12, 13). Endpoint titers were expressed as the last dilution yielding an OD at 414 nm (OD414) of >0.1 U above negative control values after a 15-min incubation.
Enumeration of Ab-forming cells (AFCs)
The spleens were removed aseptically and single cell suspensions were prepared, as described elsewhere (4, 10, 13). For isolation of mononuclear cells from nasal passages (NPs) and iLP, a modified dissociation method was used. This method was based upon a previously described protocol using collagenase type IV (0.5 mg/ml; Sigma-Aldrich) enzymatic dissociation to obtain single cell preparations (4, 10, 13). Mononuclear cells were purified using a discontinuous Percoll gradient (Pharmacia Fine Chemicals). Mononuclear cells at the interface between the 40 and 75% layers were removed, washed, and resuspended in RPMI 1640 (Mediatech) supplemented with HEPES buffer (15 mM), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% FCS (complete medium). An ELISPOT assay was used to detect cells producing IgG and IgA Abs (5, 6, 7, 12, 13). Ninety-six-well nitrocellulose plates (Millititer HA; Millipore) were coated with 10 μg/ml BoNT/A for analysis of anti-BoNT/A-specific AFCs.
BoNT/A challenge
A 100-μl aliquot (2 μg/ml) of progenitor BoNT/A (2 x 107 i.p. LD50/mg; WAKO) diluted in 0.2% gelatin/PBS was given to each mouse via the i.p. route. For oral challenge, mice were starved for 12 h before oral delivery of BoNT/A and were then gastrically intubated with a 7.5% sodium bicarbonate isotonic solution 30 min before actual intubation of BoNT/A (5, 6, 7). The various doses (25 ng, 250 ng, and 2.5 μg/mouse) of BoNT/A were gastrically intubated into the duodenum of each mice (six mice/group) using a 22-gauge ball-tip intubation needle to establish the oral LD50 for BoNT/A (5, 6, 7). Individual mice were monitored daily for paralysis and death.
S-IgA Ab neutralization assay
Undiluted fecal extract samples (200 μl) from mice given either BoNT/A vaccine or a nonrelevant Ag (OVA) were incubated with 50 pg of progenitor BoNT/A (WAKO) in 0.2% gelatin/PBS for 30 min at room temperature (36). The solution mixture was then injected into mice via the i.p. route, and individual mice were monitored daily for paralysis and death.
Statistics
The data are expressed as the mean ± SEM, and mouse groups were compared with control mice using a Mann-Whitney U test with Statview II software (Abacus Concepts) designed for Macintosh computers. A p value of <0.05 or less was considered significant.
Results
Induction of BoNT-specific S-IgA Ab responses
To date, no studies have assessed BoNT-specific mucosal IgA Ab responses. To bridge this gap in our knowledge, we first examined BoNT/A-specific mucosal immune responses in mice given nasal BoNToxoid/A plus mCT E112K as mucosal adjuvant. Nasal washes, saliva, and fecal extracts were collected 1 wk after the last nasal immunization with BoNToxoid/A plus mCT E112K and were then subjected to BoNT/A-specific ELISA. Significant S-IgA Ab responses were induced in external secretions of mice given nasal BoNToxoid/A plus mCT E112K; however, mice given nasal BoNToxoid alone did not exhibit BoNT-specific S-IgA Ab responses (Fig. 1). Interestingly, higher levels of BoNT/A-specific S-IgA Abs were detected in fecal extracts than in other mucosal secretions such as saliva and nasal washes. In contrast, nasal washes contained significant levels of anti-BoNT/A-specific IgG Abs (reciprocal log2 titer 5). As one might expect, mice systemically immunized with BoNToxoid/A plus mCT E112K showed high titers of BoNT/A-specific plasma IgG (reciprocal log2 titer 22), but not S-IgA Abs (data not shown). These results are the first evidence that BoNT/A-specific S-IgA Ab responses can be induced by nasal immunization with BoNToxoid plus an appropriate mucosal adjuvant (e.g., mCT E112K).
FIGURE 1. Comparison of BoNT/A-specific IgA Ab responses in nasal washes, fecal extracts, and saliva of mice given nasal BoNT vaccine with () or without () mCT E112K. Each mouse group was nasally immunized once/wk for 4 consecutive weeks with 20 μg of BoNToxoid alone or in together with 5 μg of mCT E112K as mucosal adjuvant. Seven days after the last nasal immunization, S-IgA Ab levels in nasal washes, fecal extracts, and saliva were determined by a BoNT/A-specific ELISA. The values shown are the mean ± SEM for 12 mice in each experimental group. The asterisks indicate a significant difference between the mean of groups given BoNT/A vaccine in the presence or absence of adjuvant; *, p < 0.05.
BoNT/A-specific plasma Abs in mice given the nasal vaccine
Since it has been shown that nasal immunization induces Ag-specific immune responses in systemic as well as mucosal sites, we sought to assess BoNT/A-specific plasma Ab levels after nasal vaccine delivery and found that significant responses were induced (Fig. 2A). The level of BoNT/A-specific IgG Abs in mice given nasal vaccine (reciprocal log2 titer 23) was comparable to that of systemically immunized mice (reciproal log2 titer 22). Furthermore, mice given the nasal vaccine with mCT E112K as mucosal adjuvant also showed elevated BoNT/A-specific IgG Ab levels in plasma (Fig. 2A), while mice given nasal BoNToxoid alone exhibited only minimally detectable levels of BoNT/A-specific plasma IgG or IgA Ab responses (Fig. 2A). Furthermore, mice given nasal BoNToxoid/A plus mCT E112K showed significant levels of plasma IgG1, IgG2a, and IgG2b, but not IgG3 Ab responses (Fig. 2B). However, anti-BoNT/A IgG2a Ab levels were lower than IgG1 and IgG2b subclass Ab responses (Fig. 2B). These results clearly show that our mucosal BoNT vaccine evokes two layers of BoNT/A-specific immune responses: S-IgA Abs in mucosal external secretions, and IgG and IgA Abs in plasma.
FIGURE 2. Comparison of BoNT/A-specific IgG, IgA, and IgG subclass Ab responses in plasma of mice given nasal BoNT vaccine alone () or together with mCT E112K as adjuvant (). Each mouse group was nasally immunized once/wk for 4 consecutive weeks with 20 μg of BoNToxoid alone or together with 5 μg of mCT E112K as mucosal adjuvant. Seven days after the last nasal immunization, IgG or IgA (A) and IgG subclass (B) Ab levels in plasma were determined by a BoNT/A-specific ELISA. The values shown are the mean ± SEM for 12 mice in each experimental group. The asterisks indicate a significant difference between the mean of groups given BoNT/A vaccine in the presence or absence of adjuvant; *, p < 0.01.
BoNT/A-specific AFCs in mice given the nasal vaccine
To confirm that BoNT/A-specific Ab responses were induced in both mucosal and systemic lymphoid tissues, mononuclear cells from NP, iLP, and spleen were taken from mice nasally immunized with BoNToxoid/A plus mCT E112K as mucosal adjuvant. These lymphoid cells were then subjected to a BoNT/A-specific ELISPOT assay to determine the numbers and isotypes of AFCs present. Mice given nasal vaccine exhibited high numbers of BoNT/A-specific AFCs in IgA effector sites such as NPs and iLP (Fig. 3). Increased numbers of BoNT/A-specific IgG AFCs were noted in NP and iLP of mice given mCT E112K as nasal adjuvant when compared with the group of mice given BoNToxoid/A alone. Furthermore, mice given BoNToxoid/A and mCT E112K as nasal adjuvant showed significant numbers of anti-BoNT IgA and IgG AFCs in spleen, while mice given BoNToxoid alone showed only minimal numbers of BoNT/A-specific IgA or IgG AFCs (Fig. 3). These results clearly show that BoNT/A-specific immunity mediated through the NALT immune system is robustly induced in both mucosal and systemic lymphoid compartments.
FIGURE 3. Analysis of BoNT/A-specific AFCs in mice given nasal BoNT vaccine with () or without () mCT E112K as adjuvant. Seven days after the last nasal immunization, mononuclear cells isolated from the NP, iLP, and spleen were examined using a BoNT/A-specific ELISPOT assay to determine the numbers of IgG and IgA AFCs. The results represent the mean values ± SEM for 15 mice in each experimental group. The asterisks indicate a significant difference between the mean of groups given BoNT/A vaccine in the presence or absence of adjuvant; *, p < 0.01.
Induction of protective systemic immunity by the nasal BoNT vaccine
Because nasal immunization with BoNToxoid/A plus mCT E112K as mucosal adjuvant induced both mucosal and systemic Ab responses, it was important to determine whether these Ag-specific Ab responses could protect mice from BoNT/A intoxication. To make this determination, groups of mice were immunized nasally with BoNToxoid/A plus mCT E112K or BoNToxoid without adjuvant at weekly intervals for 4 consecutive weeks. One week after the last immunization, the mice were then challenged i.p. with a lethal dose (0.2 μg/mouse: 4000 x i.p. LD50) of BoNT/A, which induces paralysis and death within 30 min. Mice given nasal BoNToxoid/A plus mCT E112K as mucosal adjuvant were completely protected for the first 24 h (Fig. 4), while mice given BoNToxoid alone were not protected from paralysis and died within 24 h of challenge (Fig. 4). These findings clearly suggest that BoNT/A-specific, plasma IgG Abs in mice induced by the BoNT/A nasal vaccine were protective.
FIGURE 4. Nasal immunization with BoNToxoid/A together with mCT E112K as mucosal adjuvant elicits protection against systemic challenge with BoNT/A. Mice were immunized nasally with either 15 μg of BoNToxoid/A and 5 μg of mCT E112K (?) or 20 μg of BoNToxoid without adjuvant () three times at weekly intervals. Mice were challenged with a lethal dose (4000 x i.p. LD50) of BoNT/A in 0.1 ml of PBS including 0.2% gelatin via the i.p. route. Each group consisted of six mice, and the data are representative of three separate experiments.
Mucosal BoNT/A-specific S-IgA Abs prevent enteric botulism
Because our nasal BoNT/A vaccine induced BoNT/A-specific S-IgA Ab responses in mucosal sites, it was important to determine whether these mucosal S-IgA Abs also provided protection against mucosal BoNT/A intoxication. To do so, we initially established an oral challenge system using gastric intubation. Because nasal washes, but not fecal extracts, contained significant levels of BoNT/A-specific IgG Abs, it was logical to devise an oral challenge system to elucidate the role of BoNT-specific S-IgA Abs for mucosal intoxication. Mice were starved and pretreated orally with 7.5% sodium bicarbonate isotonic solution before oral challenge. When various doses (25 ng, 250 ng, and 2.5 μg/mouse) of BoNT/A were administered into the duodenum of each group of mice (six mice/group), three of six mice given the highest dose (2.5 μg/mouse) died within 24 h. All mice in the other groups survived until the end of the experiment (7 days) (Table I). Thus, we established that 2.5 μg was a LD50 for oral challenge. Based upon these results, mice given nasal vaccine consisting of BoNToxoid/A and mCT E112K were orally challenged with a dose of 2 x LD50 of BoNT/A 1 wk after the last immunization. All mice in this mucosally vaccinated group survived for 7 days after oral challenge with BoNT/A (Fig. 5). Conversely, naive mice and mice given BoNToxoid alone died within 24 h after challenge (Fig. 5). These results indicate that mucosal BoNT/A-specific S-IgA Ab responses induced by our nasal vaccine containing BoNToxoid/A and mCT E112K protect mice from mucosal BoNT/A intoxication.
Table I. Determination of the oral challenge dose for BoNT/A
FIGURE 5. Nasal immunization with BoNToxoid/A and mCT E112K as mucosal adjuvant prevents enteric intoxication. Mice were immunized nasally with 20 μg of BoNToxoid/A and 5 μg of mCT E112K (?) or 15 μg of BoNToxoid without adjuvant () three times at weekly intervals. Mice were challenged with a lethal dose (2 x oral LD50) of BoNT/A in 0.25 ml of PBS including 0.2% gelatin via the oral route. Each group consisted of six mice, and the data are representative of three separate experiments.
Mucosal BoNT/A-specific S-IgA Abs possess neutralization activity
To further establish a role for BoNT/A-specific S-IgA Abs, mice were challenged with BoNT/A pretreated with fecal extract samples. When mice were challenged with 1 x LD50 i.p. dose of BoNT/A that had been preincubated with a fecal extract sample, one-half survived for 3 days and one-third survived until the end of the experiment. In contrast, when mice were injected with BoNT/A and fecal extracts from mice nasally immunized with OVA plus mCT, three-quarters died of BoNT/A intoxication within 1 day (Fig. 6). These results indicate that BoNT/A-specific S-IgA Abs play a key role in the protection against BoNT/A intoxication.
FIGURE 6. BoNT/A-specific S-IgA Abs in fecal extracts of mice given nasal vaccine neutralize BoNT/A toxicity. Fecal extracts (200 μl) from mice given nasal BoNToxoid/A (?) or OVA () plus mCT E112K were incubated with 1 x i.p. LD50 of BoNT/A in 0.2 ml of PBS including 0.2% gelatin for 30 min at room temperature. Each mixture was then injected into mice via the i.p. route. Each group consisted of nine mice, and the data are representative of two separate experiments. The asterisks indicate a significant difference between the groups; *, p < 0.01.
Discussion
The current botulinum vaccines, both pentavalent (serotpyes A-E) and monovalent (serotype F), are used for at-risk populations such as scientists, health care providers, and the military (26, 37). Professional health care workers are well trained in avoiding mucosal BoNT intoxication while working with BoNTs. Thus, the current vaccines that induce only systemic immunity to BoNTs may be sufficient to protect these professional or military populations. However, natural botulism is disseminated by food, water, or air, and so a bioterrorist attack would most likely target mucosal surfaces. It is feared that the currently available botulinum vaccine will most likely not prevent such mucosal BoNT intoxication. Although passive immunization has been reported to prevent inhalational BoNT intoxication (38), it is not at all certain that such a systemic BoNT-specific Ab treatment would provide effective protection against food- or waterborne botulism. Furthermore, the mucosal tissue damage induced by BoNTs needs to be carefully evaluated. Our studies are aimed at developing a mucosal vaccine that can prevent mucosal BoNT intoxication as well as provide systemic immune protection comparable to that offered by the two current vaccines.
Our study provides the first evidence that nasal administration of BoNToxoid/A plus mCT E112K effectively induces BoNT/A-specific S-IgA Ab responses in addition to plasma IgG Abs. Thus, Ag-specific Ab responses were seen in plasma and mucosal secretions, while Ag-specific AFCs were detected in the NPs, the iLP, and the spleen, clearly showing that both mucosal and systemic immunity were induced in mice given the nasal BoNT vaccine. In this study, we have developed a unique oral BoNT/A challenge model that allows for investigation of protective mucosal S-IgA Ab responses. With this novel method, the levels of BoNT required for intoxication can be quantitated.
In showing that mice given a nasal BoNToxoid with mCT as mucosal adjuvant were protected from an oral challenge dose of 2 x LD50 BoNT/A, this study provided the first evidence that BoNT/A-specific S-IgA Abs play an important role in protecting against mucosally delivered BoNT/A. Furthermore, our study has also shown that BoNT/A-specific IgG Ab responses in plasma of mice given nasal vaccine also provide effective protection against i.p. BoNT/A intoxication. These results clearly demonstrate that this nasal BoNT/A vaccine establishes two layers of immune protection, one at the mucosal surfaces themselves and the second in the blood circulation via protective IgG Abs.
To assess the potential of mucosal vaccines, studies have relied on the oral delivery of mutant BoNT/B and on the nasal administration of the whole H chain component of BoNT/A. Although these reports showed successful induction of BoNT-specific Ab responses, they limited themselves to an examination of the IgG Ab isotype and did not investigate a role for S-IgA Ab responses in BoNT intoxication (39, 40). In contrast, our study has shown that BoNT/A-specific S-IgA Ab responses were induced in various mucosal secretions when mice were given a nasal BoNT/A vaccine. Indeed, like Ag-specific neutralizing plasma IgG Abs, BoNT/A-specific S-IgA Abs in fecal extract samples neutralized biologic activity and prevented BoNT/A intoxication. Furthermore, mice possessing high levels of BoNT/A-specific S-IgA Abs were completely protected against oral challenge with BoNT/A. These results indicate that intestinal mucosal BoNT/A-specific Abs, which are predominantly of the S-IgA isotype, play an important role in the prevention of GI tract botulism. To directly confirm the role of BoNT-specific S-IgA Abs in BoNT/A intoxication, we are currently testing the efficacy of nasal BoNT/A vaccine in both IgA-deficient and polymeric Ig receptor-deficient mice.
It is not enough, however, to consider the effects of BoNT on the GI tract. Because bioterrorists are more likely to disseminate BoNT by air than by food or water, it is of paramount importance to protect again nasal BoNT intoxication. For an example of terrorists’ predilection for an airborne method of delivery, one need go no farther than the failed attempt by the Japanese cult Aum Shinrikyo to disseminate BoNT in downtown Tokyo (37).
The immunopathologic threat posed by nasal BoNT intoxication may be of a different order than those posed by orally ingested BoNT, because the nasal cavity is directly connected with the CNS through the olfactory nerves and epithelium as well as the olfactory bulbs. These neuronal tissues express an abundance of different types of gangliosides, including GD1a and GT1b, which are potent binding receptors for the BoNT/B serotype (41). The other type of ganglioside-binding toxins, such as CT and heat LT of Escherichia coli, has been shown to cause significant neuronal tissue damage when administered via the nasal route (42). Such a prospect of neuronal tissue damage underlines once again how imperative it is to devise a novel mucosal vaccine for the prevention of nasal BoNT intoxication. Our current study has shown that nasal BoNToxoid/A plus mCT E112K induces significant levels of BoNT/A-specific S-IgA and IgG Ab responses in nasal washes, indicating that this nasal vaccine protocol provides potent protection against nasal BoNT intoxication as well as neuronal tissue damage. We are currently developing a nasal BoNT challenge system to examine the efficacy of this mucosal BoNT vaccine and to assess any potential BoNT toxicity for the olfactory tissues and the CNS.
Although the nasal vaccine regimen helped induce two layers of Ag-specific responses in mucosal and systemic lymphoid tissues, it succeeded in doing so only with the aid of a mucosal adjuvant. Concern has been expressed that the potent toxicity of toxin-based mucosal adjuvants such as CT and LT may induce CNS damage (42). To circumvent this problem, both current and our previous studies have examined whether nontoxic mCTs as well as cytokines and chemokines provide mucosal adjuvanticity. These studies have clearly shown that nasal application of cytokines or chemokines as mucosal adjuvants together with selected Ags induces Ag-specific Ab responses in both systemic and mucosal compartments (5, 10, 11, 12, 43, 44, 45, 46, 47). Although the toxicity of these cytokines and chemokines needs to be elucidated, our most recent studies show that nasal mCT E112K does not elicit nerve growth factor-1 induction indicative of neuronal tissue damage in the CNS of nonhuman primates (48). Thus, we feel confident that our current approach may yield a nasal vaccine able to generate effective immunity against botulism.
Ag-specific plasma IgG subclass Ab responses are excellent indicators of Th1- or Th2-type cytokine responses by CD4+ T cells. Our results showed significantly increased levels of IgG1, IgG2a, and IgG2b Ab responses in plasma of mice given nasal BoNToxoid/A plus mCT E112K when compared with those mice given nasal toxoid only. Among these elevated IgG subclass Abs, levels of anti-BoNT/A IgG1 and IgG2b Abs were higher than the IgG2a subclass Ab response. A similar pattern of Ag-specific IgG subclass responses has been seen in mice given native CT or mCT as nasal adjuvants, which are known to induce Th2-type cytokine responses (3, 4). Thus, we anticipate that BoNT/A-specific Ab responses induced by nasal BoNToxoid plus mCT E112K are mediated through Th2-type cytokine responses. To our knowledge, no studies have reported potential roles of Th1- and Th2-type responses by BoNT-specific CD4+ T cells. To this end, our studies are the first to suggest that BoNT/A-specific and dominant Th2-type responses are associated with protective immunity against botulism. More precise and direct studies of BoNT vaccine-induced Th1 and Th2 cytokine responses are currently underway in our laboratory.
In summary, our studies have now provided the first evidence that nasal immunization with BoNToxoid/A plus mCT E112K induces BoNT/A-specific S-IgA Ab responses in mucosal secretions as well as plasma IgG Ab responses. Furthermore, mice given nasal BoNT/A vaccine were protected from both parenteral and oral challenge with BoNT/A. These results directly demonstrate that mucosal immunization is able to provide two layers of immunity, a first line of defense at mucosal surfaces and a second line via systemic blood circulation. The precise roles of BoNT-specific S-IgA Abs will require further investigation; however, our findings in this study show that this BoNT-based mucosal vaccine is an effective and perhaps essential strategy for the protection of the population against botulism used as a weapon of bioterrorists.
Acknowledgments
We thank Dr. Kimberly K. McGhee for editorial advice and Sheila D. Turner for the preparation of the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This research was supported by National Institutes of Health Grants DE 12242, AI 43197, AI 18958, AI 35932, AI56286, DC 04976, and DK 44240, as well as grants-in-aid from CREST of Japan Science and Technology Corporation, the Ministry of Education, Science and Sports, and the Ministry of Health and Labor, Japan.
2 Address correspondence and reprint requests to Dr. Kohtaro Fujihashi, Department of Pediatric Dentistry, Immunobiology Vaccine Center, University of Alabama at Birmingham, 845 19th Street South, BBRB Room 761, Birmingham, AL 35294-2170. E-mail address: kohtarof@uab.edu
3 Abbreviations used in this paper: GI, gastrointestinal; AFC, Ab-forming cell; BoNT, botulinum neurotoxin; BoNToxoid, botulinum neurotoxoid; CT, cholera toxin; iLP, intestinal lamina propria; LT, labile toxin; mCT, mutant of CT; NALT, nasopharyngeal-associated lymphoreticular tissue; NP, nasal passage; S-IgA, secretory IgA.
Received for publication August 31, 2004. Accepted for publication November 8, 2004.
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The threat posed by botulism, classically a food- and waterborne disease with a high morbidity and mortality, has increased exponentially in an age of bioterrorism. Because botulinum neurotoxin (BoNT) could be easily disseminated by terrorists using an aerosol or could be used to contaminate the food or water supply, the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases has classified it as a category A agent. Although clearly the development of a safe and effective mucosal vaccine against this toxin should be a high priority, essentially no studies to date have assessed mucosal immune responses to this disease. To bridge this gap in our knowledge, we immunized mice weekly for 4 wk with nasal doses of BoNT type A toxoid and a mutant of cholera toxin termed E112K. We found elevated levels of BoNT-specific IgG Abs in plasma and of secretory IgA Abs in external secretions (nasal washes, saliva, and fecal extracts). When mice given nasal BoNT vaccine were challenged with 4 x 103 LD50 of BoNT type A (BoNT/A) via the i.p. route, complete protection was seen, while naive mice given the same dosage died within 2 h. To further confirm the efficacy of this nasal BoNT vaccine, an oral LD50 was determined. When mice were given an oral challenge of 5 μg (2 x oral LD50) of progenitor BoNT/A, all immunized mice survived beyond 5 days, while nonimmunized mice did not. The fecal extract samples from nasally vaccinated mice were found to contain neutralizing secretory IgA Abs. Taken together, these results show that nasal BoNT/A vaccine effectively prevents mucosal BoNT intoxication.
Introduction
It is now clear that parenteral immunization usually fails to elicit protective mucosal immune responses (1). Various alternate routes of Ag-vaccine delivery have been suggested by researchers, but none has shown more promise for the induction of Ag-specific Ab responses in the upper mucosal compartments (e.g., the respiratory tract) than nasal administration or in the gastrointestinal (GI)3 tract than oral administration (2, 3, 4, 5, 6, 7). Our own previous studies have shown that gut-associated lymphoreticular tissue-directed oral immunization effectively induces Ag-specific secretory IgA (S-IgA) Ab responses in the small intestinal lamina propria (iLP) and the submandibular glands, but not in the lung or the lamina propria of the nasal or reproductive tracts (5, 6, 7, 8, 9). In most current nasal immunization studies, the vaccine is instilled into each nostril (usually 5 μl/nostril), and normal inhalation results in the effective delivery of the vaccine, presumably into nasopharyngeal-associated lymphoreticular tissues (NALT). Such NALT-mediated nasal immunization has been shown to elicit significant immunity in the respiratory, GI, and reproductive tracts as well as in the nasal and oral cavities. These findings suggest that the nasal route of Ag delivery may possess potential advantages in the induction of mucosal immunity.
Native cholera toxin (CT) and mutants of CT (mCTs) are effective mucosal adjuvants and have been widely used for nasal immunization with protein Ags, bacterial components, viruses, or virus-related peptides for the induction of protective immunity associated with S-IgA and plasma IgG Ab responses (4, 10, 11, 12). Nasal immunization with the weakly immunogenic OVA along with mCT as adjuvant resulted in S-IgA anti-OVA Ab responses in various mucosal external secretions (4). Furthermore, mice nasally immunized with pneumococcal surface protein A Ag plus mCT revealed pneumococcal surface protein A-specific S-IgA Ab responses associated with effective protection against capsular serotype 3 Streptococcus pneumoniae A66 (10). These Ag-specific S-IgA Ab responses were associated with polarized Th2-type responses in cervical lymph nodes (4, 10). Nasal immunization with diphtheria toxoid plus mCT E112K has also been shown to induce protective immunity to the diphtheria exotoxin (11). Furthermore, our recent studies showed that young adult as well as aged mice given a nasal vaccine of tetanus toxoid along with a chimera of mCT-A E112K and heat labile toxin (LT) B subunit were protected when mice were challenged via the systemic route with tetanus toxin (12, 13).
Clostridium botulinum is an anaerobic bacterium that produces a powerful exotoxin termed botulinum neurotoxin (BoNT), which induces flaccid paralysis (14, 15, 16, 17). The BoNTs, serologically divided into seven immunologically different types (A through G), are released from the bacterium as a single polypeptide chain (14, 15, 16, 17). Among these seven serotypes, types A, B, and E are typically associated with botulism in humans, and type C has been the most common cause of disease in domestic animals. However, both C and D induce botulism in cows (15, 16, 17, 18). As with foodborne botulism, type A is responsible for 60%, type B for 30%, and type E for 10% (18) of cases in humans. It should be emphasized that bioterrorists could potentially use any or all seven serotypes. The molecular mass of each exotoxin type is 150 kDa (14, 15, 16, 17). Each consists of two polypeptide chains, an L chain (50-kDa) and an H (100-kDa) chain. The main targets of BoNT are the peripheral cholinergic nerve endings and particularly the cholinergic neuromuscular junction. BoNT inhibits exocytosis, inducing the flaccid paralysis characteristic of botulism and eventually leading to death (19, 20, 21, 22, 23, 24, 25).
The bacterium C. botulinum and its derived BoNTs enter the body most commonly through the GI tract via tainted food. In the case of a bioterrorist event, the BoNT would most likely be disseminated by airborne or possibly waterborne routes. BoNTs are classified as one of the six highest risk agents for bioterrorism (category A agents) by the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases. Any chance of surviving this extremely potent and lethal toxin rests upon prolonged intensive care. The current pentavalent botulinum vaccine (serotypes A-E) is used for at-risk populations such as scientists researching BoNTs, health care providers dealing with their clinical manifestations, and the military (26). Furthermore, the current pentavalent vaccine is administered via the parenteral route and induces prolonged pain and swelling at the site of injection (26) (H. F. Staats, unpublished results) To minimize these side effects, several research groups have attempted to develop BoNT vaccines using recombinant C-terminal half of the H chain of BoNT peptide or synthetic epitopes (27, 28, 29, 30, 31, 32). Furthermore, a Venezuelan equine encephalomyelitis virus replication vector system provided effective protection against BoNT intoxication (33). However, given the nature of botulinum intoxication, it is essential to devise more effective mucosal vaccines to prevent the intoxication at both mucosal and systemic tissue compartments.
In this study, we have examined whether a nasal vaccine consisting of botulinum neurotoxoid type A (BoNToxoid/A) and mCT E112K as adjuvant would effectively induce protective immunity to BoNT/A challenge. Our results indicate that this nasal vaccine induced BoNT-specific Ab responses in both mucosal and systemic lymphoid tissues. Furthermore, the nasal vaccine prevented botulinum intoxication following either mucosal or parenteral challenge.
Materials and Methods
Mice
Young adult (6–8 wk old) C57BL/6 mice were purchased from the Frederick Cancer Research Facility (National Cancer Institute). Upon arrival, all mice were immediately transferred to microisolators and maintained in horizontal laminar flow cabinets and provided sterile food and water ad libitum. Experiments were performed using young adult C57BL/6 mice between 6 and 8 wk of age. The health of the mice was tested semiannually, and mice of all ages used in these experiments were free of bacterial and viral pathogens.
Preparation of BoNToxoid/A
The BoNT/A from C. botulinum type A 62 was used for toxoid preparation. The BoNT/A was purified using previously described methods (15). Briefly, purified BoNT/A was detoxified at 30°C by dialyzing against 0.2% Formalin in 0.1 M phosphate buffer (0.2 mg/ml, type A toxin containing 3.5, 106 LD50/ml). The detoxification proceeded at 30°C. The formalized toxin was sampled at intervals for mouse inoculation until it became completely nontoxic. At each interval, two mice were i.p. administered with 0.5 ml of the sample and then observed for 4 days. The formalized toxoid was shown to be nontoxic because mice neither died from intoxication nor showed any specific symptoms of intoxication such as muscle spasms, stiffening, or any other abnormal signs during the observation period. The toxoid was kept at 4°C until used (15, 34).
Nasal immunization and sample collection
Mice were nasally immunized at weekly intervals for 4 wk with 20 μg of BoNToxoid/A and 5 μg of mCT E112K in PBS (4, 10, 11, 12, 13). Plasma and mucosal external secretions (nasal washes, saliva, and fecal extracts) were collected on day 28. Saliva was obtained from mice following i.p. injection with 100 μg of sterile pilocarpine (7). Fecal pellets (100 mg) were suspended into 1 ml of PBS containing 0.1% sodium azide and were then extracted by vortexing for 5 min. The samples were spun at 10,000 x g for 5 min, and the supernatants were collected as fecal extracts (5, 6, 7). The mice were sacrificed 7 days after the last immunization. The nasal washes were obtained by injecting 1 ml of PBS on three occasions into the posterior opening of the nasopharynx with a hypodermic needle (13).
Ab assays
Ab titers in plasma and mucosal secretions were determined by an ELISA (4, 5, 6, 7, 11, 12, 13). Briefly, Falcon microtest assay plates (BD Biosciences) were coated with an optimal concentration of purified BoNT/A (100 μl of 2 μg/ml) in PBS overnight at 4°C (35). Two-fold serial dilutions of samples were added after blocking with 1% BSA. To detect BoNT/A-specific Ab levels, HRP-conjugated, goat anti-mouse or , H chain-specific Abs were used (Southern Biotechnology Associates). For IgG Ab subclass determinations, biotinylated mAbs specific for IgG1 IgG2a, IgG2b, and IgG3 (BD Pharmingen) and peroxidase-conjugated goat anti-biotin Ab were used, as described elsewhere (11, 12, 13). Endpoint titers were expressed as the last dilution yielding an OD at 414 nm (OD414) of >0.1 U above negative control values after a 15-min incubation.
Enumeration of Ab-forming cells (AFCs)
The spleens were removed aseptically and single cell suspensions were prepared, as described elsewhere (4, 10, 13). For isolation of mononuclear cells from nasal passages (NPs) and iLP, a modified dissociation method was used. This method was based upon a previously described protocol using collagenase type IV (0.5 mg/ml; Sigma-Aldrich) enzymatic dissociation to obtain single cell preparations (4, 10, 13). Mononuclear cells were purified using a discontinuous Percoll gradient (Pharmacia Fine Chemicals). Mononuclear cells at the interface between the 40 and 75% layers were removed, washed, and resuspended in RPMI 1640 (Mediatech) supplemented with HEPES buffer (15 mM), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% FCS (complete medium). An ELISPOT assay was used to detect cells producing IgG and IgA Abs (5, 6, 7, 12, 13). Ninety-six-well nitrocellulose plates (Millititer HA; Millipore) were coated with 10 μg/ml BoNT/A for analysis of anti-BoNT/A-specific AFCs.
BoNT/A challenge
A 100-μl aliquot (2 μg/ml) of progenitor BoNT/A (2 x 107 i.p. LD50/mg; WAKO) diluted in 0.2% gelatin/PBS was given to each mouse via the i.p. route. For oral challenge, mice were starved for 12 h before oral delivery of BoNT/A and were then gastrically intubated with a 7.5% sodium bicarbonate isotonic solution 30 min before actual intubation of BoNT/A (5, 6, 7). The various doses (25 ng, 250 ng, and 2.5 μg/mouse) of BoNT/A were gastrically intubated into the duodenum of each mice (six mice/group) using a 22-gauge ball-tip intubation needle to establish the oral LD50 for BoNT/A (5, 6, 7). Individual mice were monitored daily for paralysis and death.
S-IgA Ab neutralization assay
Undiluted fecal extract samples (200 μl) from mice given either BoNT/A vaccine or a nonrelevant Ag (OVA) were incubated with 50 pg of progenitor BoNT/A (WAKO) in 0.2% gelatin/PBS for 30 min at room temperature (36). The solution mixture was then injected into mice via the i.p. route, and individual mice were monitored daily for paralysis and death.
Statistics
The data are expressed as the mean ± SEM, and mouse groups were compared with control mice using a Mann-Whitney U test with Statview II software (Abacus Concepts) designed for Macintosh computers. A p value of <0.05 or less was considered significant.
Results
Induction of BoNT-specific S-IgA Ab responses
To date, no studies have assessed BoNT-specific mucosal IgA Ab responses. To bridge this gap in our knowledge, we first examined BoNT/A-specific mucosal immune responses in mice given nasal BoNToxoid/A plus mCT E112K as mucosal adjuvant. Nasal washes, saliva, and fecal extracts were collected 1 wk after the last nasal immunization with BoNToxoid/A plus mCT E112K and were then subjected to BoNT/A-specific ELISA. Significant S-IgA Ab responses were induced in external secretions of mice given nasal BoNToxoid/A plus mCT E112K; however, mice given nasal BoNToxoid alone did not exhibit BoNT-specific S-IgA Ab responses (Fig. 1). Interestingly, higher levels of BoNT/A-specific S-IgA Abs were detected in fecal extracts than in other mucosal secretions such as saliva and nasal washes. In contrast, nasal washes contained significant levels of anti-BoNT/A-specific IgG Abs (reciprocal log2 titer 5). As one might expect, mice systemically immunized with BoNToxoid/A plus mCT E112K showed high titers of BoNT/A-specific plasma IgG (reciprocal log2 titer 22), but not S-IgA Abs (data not shown). These results are the first evidence that BoNT/A-specific S-IgA Ab responses can be induced by nasal immunization with BoNToxoid plus an appropriate mucosal adjuvant (e.g., mCT E112K).
FIGURE 1. Comparison of BoNT/A-specific IgA Ab responses in nasal washes, fecal extracts, and saliva of mice given nasal BoNT vaccine with () or without () mCT E112K. Each mouse group was nasally immunized once/wk for 4 consecutive weeks with 20 μg of BoNToxoid alone or in together with 5 μg of mCT E112K as mucosal adjuvant. Seven days after the last nasal immunization, S-IgA Ab levels in nasal washes, fecal extracts, and saliva were determined by a BoNT/A-specific ELISA. The values shown are the mean ± SEM for 12 mice in each experimental group. The asterisks indicate a significant difference between the mean of groups given BoNT/A vaccine in the presence or absence of adjuvant; *, p < 0.05.
BoNT/A-specific plasma Abs in mice given the nasal vaccine
Since it has been shown that nasal immunization induces Ag-specific immune responses in systemic as well as mucosal sites, we sought to assess BoNT/A-specific plasma Ab levels after nasal vaccine delivery and found that significant responses were induced (Fig. 2A). The level of BoNT/A-specific IgG Abs in mice given nasal vaccine (reciprocal log2 titer 23) was comparable to that of systemically immunized mice (reciproal log2 titer 22). Furthermore, mice given the nasal vaccine with mCT E112K as mucosal adjuvant also showed elevated BoNT/A-specific IgG Ab levels in plasma (Fig. 2A), while mice given nasal BoNToxoid alone exhibited only minimally detectable levels of BoNT/A-specific plasma IgG or IgA Ab responses (Fig. 2A). Furthermore, mice given nasal BoNToxoid/A plus mCT E112K showed significant levels of plasma IgG1, IgG2a, and IgG2b, but not IgG3 Ab responses (Fig. 2B). However, anti-BoNT/A IgG2a Ab levels were lower than IgG1 and IgG2b subclass Ab responses (Fig. 2B). These results clearly show that our mucosal BoNT vaccine evokes two layers of BoNT/A-specific immune responses: S-IgA Abs in mucosal external secretions, and IgG and IgA Abs in plasma.
FIGURE 2. Comparison of BoNT/A-specific IgG, IgA, and IgG subclass Ab responses in plasma of mice given nasal BoNT vaccine alone () or together with mCT E112K as adjuvant (). Each mouse group was nasally immunized once/wk for 4 consecutive weeks with 20 μg of BoNToxoid alone or together with 5 μg of mCT E112K as mucosal adjuvant. Seven days after the last nasal immunization, IgG or IgA (A) and IgG subclass (B) Ab levels in plasma were determined by a BoNT/A-specific ELISA. The values shown are the mean ± SEM for 12 mice in each experimental group. The asterisks indicate a significant difference between the mean of groups given BoNT/A vaccine in the presence or absence of adjuvant; *, p < 0.01.
BoNT/A-specific AFCs in mice given the nasal vaccine
To confirm that BoNT/A-specific Ab responses were induced in both mucosal and systemic lymphoid tissues, mononuclear cells from NP, iLP, and spleen were taken from mice nasally immunized with BoNToxoid/A plus mCT E112K as mucosal adjuvant. These lymphoid cells were then subjected to a BoNT/A-specific ELISPOT assay to determine the numbers and isotypes of AFCs present. Mice given nasal vaccine exhibited high numbers of BoNT/A-specific AFCs in IgA effector sites such as NPs and iLP (Fig. 3). Increased numbers of BoNT/A-specific IgG AFCs were noted in NP and iLP of mice given mCT E112K as nasal adjuvant when compared with the group of mice given BoNToxoid/A alone. Furthermore, mice given BoNToxoid/A and mCT E112K as nasal adjuvant showed significant numbers of anti-BoNT IgA and IgG AFCs in spleen, while mice given BoNToxoid alone showed only minimal numbers of BoNT/A-specific IgA or IgG AFCs (Fig. 3). These results clearly show that BoNT/A-specific immunity mediated through the NALT immune system is robustly induced in both mucosal and systemic lymphoid compartments.
FIGURE 3. Analysis of BoNT/A-specific AFCs in mice given nasal BoNT vaccine with () or without () mCT E112K as adjuvant. Seven days after the last nasal immunization, mononuclear cells isolated from the NP, iLP, and spleen were examined using a BoNT/A-specific ELISPOT assay to determine the numbers of IgG and IgA AFCs. The results represent the mean values ± SEM for 15 mice in each experimental group. The asterisks indicate a significant difference between the mean of groups given BoNT/A vaccine in the presence or absence of adjuvant; *, p < 0.01.
Induction of protective systemic immunity by the nasal BoNT vaccine
Because nasal immunization with BoNToxoid/A plus mCT E112K as mucosal adjuvant induced both mucosal and systemic Ab responses, it was important to determine whether these Ag-specific Ab responses could protect mice from BoNT/A intoxication. To make this determination, groups of mice were immunized nasally with BoNToxoid/A plus mCT E112K or BoNToxoid without adjuvant at weekly intervals for 4 consecutive weeks. One week after the last immunization, the mice were then challenged i.p. with a lethal dose (0.2 μg/mouse: 4000 x i.p. LD50) of BoNT/A, which induces paralysis and death within 30 min. Mice given nasal BoNToxoid/A plus mCT E112K as mucosal adjuvant were completely protected for the first 24 h (Fig. 4), while mice given BoNToxoid alone were not protected from paralysis and died within 24 h of challenge (Fig. 4). These findings clearly suggest that BoNT/A-specific, plasma IgG Abs in mice induced by the BoNT/A nasal vaccine were protective.
FIGURE 4. Nasal immunization with BoNToxoid/A together with mCT E112K as mucosal adjuvant elicits protection against systemic challenge with BoNT/A. Mice were immunized nasally with either 15 μg of BoNToxoid/A and 5 μg of mCT E112K (?) or 20 μg of BoNToxoid without adjuvant () three times at weekly intervals. Mice were challenged with a lethal dose (4000 x i.p. LD50) of BoNT/A in 0.1 ml of PBS including 0.2% gelatin via the i.p. route. Each group consisted of six mice, and the data are representative of three separate experiments.
Mucosal BoNT/A-specific S-IgA Abs prevent enteric botulism
Because our nasal BoNT/A vaccine induced BoNT/A-specific S-IgA Ab responses in mucosal sites, it was important to determine whether these mucosal S-IgA Abs also provided protection against mucosal BoNT/A intoxication. To do so, we initially established an oral challenge system using gastric intubation. Because nasal washes, but not fecal extracts, contained significant levels of BoNT/A-specific IgG Abs, it was logical to devise an oral challenge system to elucidate the role of BoNT-specific S-IgA Abs for mucosal intoxication. Mice were starved and pretreated orally with 7.5% sodium bicarbonate isotonic solution before oral challenge. When various doses (25 ng, 250 ng, and 2.5 μg/mouse) of BoNT/A were administered into the duodenum of each group of mice (six mice/group), three of six mice given the highest dose (2.5 μg/mouse) died within 24 h. All mice in the other groups survived until the end of the experiment (7 days) (Table I). Thus, we established that 2.5 μg was a LD50 for oral challenge. Based upon these results, mice given nasal vaccine consisting of BoNToxoid/A and mCT E112K were orally challenged with a dose of 2 x LD50 of BoNT/A 1 wk after the last immunization. All mice in this mucosally vaccinated group survived for 7 days after oral challenge with BoNT/A (Fig. 5). Conversely, naive mice and mice given BoNToxoid alone died within 24 h after challenge (Fig. 5). These results indicate that mucosal BoNT/A-specific S-IgA Ab responses induced by our nasal vaccine containing BoNToxoid/A and mCT E112K protect mice from mucosal BoNT/A intoxication.
Table I. Determination of the oral challenge dose for BoNT/A
FIGURE 5. Nasal immunization with BoNToxoid/A and mCT E112K as mucosal adjuvant prevents enteric intoxication. Mice were immunized nasally with 20 μg of BoNToxoid/A and 5 μg of mCT E112K (?) or 15 μg of BoNToxoid without adjuvant () three times at weekly intervals. Mice were challenged with a lethal dose (2 x oral LD50) of BoNT/A in 0.25 ml of PBS including 0.2% gelatin via the oral route. Each group consisted of six mice, and the data are representative of three separate experiments.
Mucosal BoNT/A-specific S-IgA Abs possess neutralization activity
To further establish a role for BoNT/A-specific S-IgA Abs, mice were challenged with BoNT/A pretreated with fecal extract samples. When mice were challenged with 1 x LD50 i.p. dose of BoNT/A that had been preincubated with a fecal extract sample, one-half survived for 3 days and one-third survived until the end of the experiment. In contrast, when mice were injected with BoNT/A and fecal extracts from mice nasally immunized with OVA plus mCT, three-quarters died of BoNT/A intoxication within 1 day (Fig. 6). These results indicate that BoNT/A-specific S-IgA Abs play a key role in the protection against BoNT/A intoxication.
FIGURE 6. BoNT/A-specific S-IgA Abs in fecal extracts of mice given nasal vaccine neutralize BoNT/A toxicity. Fecal extracts (200 μl) from mice given nasal BoNToxoid/A (?) or OVA () plus mCT E112K were incubated with 1 x i.p. LD50 of BoNT/A in 0.2 ml of PBS including 0.2% gelatin for 30 min at room temperature. Each mixture was then injected into mice via the i.p. route. Each group consisted of nine mice, and the data are representative of two separate experiments. The asterisks indicate a significant difference between the groups; *, p < 0.01.
Discussion
The current botulinum vaccines, both pentavalent (serotpyes A-E) and monovalent (serotype F), are used for at-risk populations such as scientists, health care providers, and the military (26, 37). Professional health care workers are well trained in avoiding mucosal BoNT intoxication while working with BoNTs. Thus, the current vaccines that induce only systemic immunity to BoNTs may be sufficient to protect these professional or military populations. However, natural botulism is disseminated by food, water, or air, and so a bioterrorist attack would most likely target mucosal surfaces. It is feared that the currently available botulinum vaccine will most likely not prevent such mucosal BoNT intoxication. Although passive immunization has been reported to prevent inhalational BoNT intoxication (38), it is not at all certain that such a systemic BoNT-specific Ab treatment would provide effective protection against food- or waterborne botulism. Furthermore, the mucosal tissue damage induced by BoNTs needs to be carefully evaluated. Our studies are aimed at developing a mucosal vaccine that can prevent mucosal BoNT intoxication as well as provide systemic immune protection comparable to that offered by the two current vaccines.
Our study provides the first evidence that nasal administration of BoNToxoid/A plus mCT E112K effectively induces BoNT/A-specific S-IgA Ab responses in addition to plasma IgG Abs. Thus, Ag-specific Ab responses were seen in plasma and mucosal secretions, while Ag-specific AFCs were detected in the NPs, the iLP, and the spleen, clearly showing that both mucosal and systemic immunity were induced in mice given the nasal BoNT vaccine. In this study, we have developed a unique oral BoNT/A challenge model that allows for investigation of protective mucosal S-IgA Ab responses. With this novel method, the levels of BoNT required for intoxication can be quantitated.
In showing that mice given a nasal BoNToxoid with mCT as mucosal adjuvant were protected from an oral challenge dose of 2 x LD50 BoNT/A, this study provided the first evidence that BoNT/A-specific S-IgA Abs play an important role in protecting against mucosally delivered BoNT/A. Furthermore, our study has also shown that BoNT/A-specific IgG Ab responses in plasma of mice given nasal vaccine also provide effective protection against i.p. BoNT/A intoxication. These results clearly demonstrate that this nasal BoNT/A vaccine establishes two layers of immune protection, one at the mucosal surfaces themselves and the second in the blood circulation via protective IgG Abs.
To assess the potential of mucosal vaccines, studies have relied on the oral delivery of mutant BoNT/B and on the nasal administration of the whole H chain component of BoNT/A. Although these reports showed successful induction of BoNT-specific Ab responses, they limited themselves to an examination of the IgG Ab isotype and did not investigate a role for S-IgA Ab responses in BoNT intoxication (39, 40). In contrast, our study has shown that BoNT/A-specific S-IgA Ab responses were induced in various mucosal secretions when mice were given a nasal BoNT/A vaccine. Indeed, like Ag-specific neutralizing plasma IgG Abs, BoNT/A-specific S-IgA Abs in fecal extract samples neutralized biologic activity and prevented BoNT/A intoxication. Furthermore, mice possessing high levels of BoNT/A-specific S-IgA Abs were completely protected against oral challenge with BoNT/A. These results indicate that intestinal mucosal BoNT/A-specific Abs, which are predominantly of the S-IgA isotype, play an important role in the prevention of GI tract botulism. To directly confirm the role of BoNT-specific S-IgA Abs in BoNT/A intoxication, we are currently testing the efficacy of nasal BoNT/A vaccine in both IgA-deficient and polymeric Ig receptor-deficient mice.
It is not enough, however, to consider the effects of BoNT on the GI tract. Because bioterrorists are more likely to disseminate BoNT by air than by food or water, it is of paramount importance to protect again nasal BoNT intoxication. For an example of terrorists’ predilection for an airborne method of delivery, one need go no farther than the failed attempt by the Japanese cult Aum Shinrikyo to disseminate BoNT in downtown Tokyo (37).
The immunopathologic threat posed by nasal BoNT intoxication may be of a different order than those posed by orally ingested BoNT, because the nasal cavity is directly connected with the CNS through the olfactory nerves and epithelium as well as the olfactory bulbs. These neuronal tissues express an abundance of different types of gangliosides, including GD1a and GT1b, which are potent binding receptors for the BoNT/B serotype (41). The other type of ganglioside-binding toxins, such as CT and heat LT of Escherichia coli, has been shown to cause significant neuronal tissue damage when administered via the nasal route (42). Such a prospect of neuronal tissue damage underlines once again how imperative it is to devise a novel mucosal vaccine for the prevention of nasal BoNT intoxication. Our current study has shown that nasal BoNToxoid/A plus mCT E112K induces significant levels of BoNT/A-specific S-IgA and IgG Ab responses in nasal washes, indicating that this nasal vaccine protocol provides potent protection against nasal BoNT intoxication as well as neuronal tissue damage. We are currently developing a nasal BoNT challenge system to examine the efficacy of this mucosal BoNT vaccine and to assess any potential BoNT toxicity for the olfactory tissues and the CNS.
Although the nasal vaccine regimen helped induce two layers of Ag-specific responses in mucosal and systemic lymphoid tissues, it succeeded in doing so only with the aid of a mucosal adjuvant. Concern has been expressed that the potent toxicity of toxin-based mucosal adjuvants such as CT and LT may induce CNS damage (42). To circumvent this problem, both current and our previous studies have examined whether nontoxic mCTs as well as cytokines and chemokines provide mucosal adjuvanticity. These studies have clearly shown that nasal application of cytokines or chemokines as mucosal adjuvants together with selected Ags induces Ag-specific Ab responses in both systemic and mucosal compartments (5, 10, 11, 12, 43, 44, 45, 46, 47). Although the toxicity of these cytokines and chemokines needs to be elucidated, our most recent studies show that nasal mCT E112K does not elicit nerve growth factor-1 induction indicative of neuronal tissue damage in the CNS of nonhuman primates (48). Thus, we feel confident that our current approach may yield a nasal vaccine able to generate effective immunity against botulism.
Ag-specific plasma IgG subclass Ab responses are excellent indicators of Th1- or Th2-type cytokine responses by CD4+ T cells. Our results showed significantly increased levels of IgG1, IgG2a, and IgG2b Ab responses in plasma of mice given nasal BoNToxoid/A plus mCT E112K when compared with those mice given nasal toxoid only. Among these elevated IgG subclass Abs, levels of anti-BoNT/A IgG1 and IgG2b Abs were higher than the IgG2a subclass Ab response. A similar pattern of Ag-specific IgG subclass responses has been seen in mice given native CT or mCT as nasal adjuvants, which are known to induce Th2-type cytokine responses (3, 4). Thus, we anticipate that BoNT/A-specific Ab responses induced by nasal BoNToxoid plus mCT E112K are mediated through Th2-type cytokine responses. To our knowledge, no studies have reported potential roles of Th1- and Th2-type responses by BoNT-specific CD4+ T cells. To this end, our studies are the first to suggest that BoNT/A-specific and dominant Th2-type responses are associated with protective immunity against botulism. More precise and direct studies of BoNT vaccine-induced Th1 and Th2 cytokine responses are currently underway in our laboratory.
In summary, our studies have now provided the first evidence that nasal immunization with BoNToxoid/A plus mCT E112K induces BoNT/A-specific S-IgA Ab responses in mucosal secretions as well as plasma IgG Ab responses. Furthermore, mice given nasal BoNT/A vaccine were protected from both parenteral and oral challenge with BoNT/A. These results directly demonstrate that mucosal immunization is able to provide two layers of immunity, a first line of defense at mucosal surfaces and a second line via systemic blood circulation. The precise roles of BoNT-specific S-IgA Abs will require further investigation; however, our findings in this study show that this BoNT-based mucosal vaccine is an effective and perhaps essential strategy for the protection of the population against botulism used as a weapon of bioterrorists.
Acknowledgments
We thank Dr. Kimberly K. McGhee for editorial advice and Sheila D. Turner for the preparation of the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This research was supported by National Institutes of Health Grants DE 12242, AI 43197, AI 18958, AI 35932, AI56286, DC 04976, and DK 44240, as well as grants-in-aid from CREST of Japan Science and Technology Corporation, the Ministry of Education, Science and Sports, and the Ministry of Health and Labor, Japan.
2 Address correspondence and reprint requests to Dr. Kohtaro Fujihashi, Department of Pediatric Dentistry, Immunobiology Vaccine Center, University of Alabama at Birmingham, 845 19th Street South, BBRB Room 761, Birmingham, AL 35294-2170. E-mail address: kohtarof@uab.edu
3 Abbreviations used in this paper: GI, gastrointestinal; AFC, Ab-forming cell; BoNT, botulinum neurotoxin; BoNToxoid, botulinum neurotoxoid; CT, cholera toxin; iLP, intestinal lamina propria; LT, labile toxin; mCT, mutant of CT; NALT, nasopharyngeal-associated lymphoreticular tissue; NP, nasal passage; S-IgA, secretory IgA.
Received for publication August 31, 2004. Accepted for publication November 8, 2004.
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