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`Cutting Edge: Transcutaneous Immunization
`with Cholera Toxin Protects Mice Against
`Lethal Mucosal Toxin Challenge
`Gregory M. Glenn, Tanya Scharton-Kersten, Russell Vassell,
`Corey P. Mallett, Thomas L. Hale and Carl R. Alving
`
`1998; 161:3211-3214; ;
`J Immunol(cid:160)
`http://www.jimmunol.org/content/161/7/3211
`
`References
`
`cites 16 articles
`This article
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`http://www.jimmunol.org/content/161/7/3211.full#ref-list-1
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` is published twice each month byThe Journal of Immunology
`
`The American Association of Immunologists, Inc.,
`9650 Rockville Pike, Bethesda, MD 20814-3994.
`Copyright © 1998 by The American Association of
`Immunologists All rights reserved.
`Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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`FIGURE 2. Mortality studies in C57BL/6 mice following immunization
`with CT by the transcutaneous route and i.n. challenge with native toxin at
`3 wk after immunization. The number of mice per group is indicated in
`parentheses (total survivors/number of mice in study).
`
`sera from mice exposed twice to CT at 0 and 3 wk exhibited
`significantly augmented IgG titers at 3 wk after the second trans-
`cutaneous application (Fig. 3A). CT-specific IgG was also detected
`in five of five lung wash samples and in eight of nine stool sample
`homogenates from the single exposure groups (Fig. 3, B and C).
`Further analysis of the samples revealed a potent IgA response,
`albeit lower than the IgG titers, in the sera, lung wash, and stool
`(Fig. 3, D–F). In contrast, lung wash samples from animals ex-
`posed to an irrelevant protein, ricin A-subunit, failed to exhibit
`detectable anti-CT IgG or IgA levels (Fig. 3, B and E, F), and stool
`samples from unimmunized mice had ,0.2 IgG OD units at a 1/2
`dilution and no detectable IgA (Fig. 3, C and F, F). Neither IgM
`nor IgE anti-CT Abs were detected in the sera of transcutaneously
`immunized mice.
`
`CT Ab responses in the sera of orally and transcutaneously
`immunized mice
`It was conceivable that animals vaccinated by TCI might, through
`normal grooming, ingest small amounts of Ag and orally expose
`themselves to CT. To exclude this possibility, we compared the
`immune response using 100 mg of CT by TCI with oral gavage
`using 25 mg of CT, a log greater than the amount that was esti-
`mated to be left on the skin after washing using 125I-labeled CT
`(data not shown). As shown in Figure 4, the magnitude of the
`anti-CT IgG response at 4 wk after immunization was significantly
`higher in sera from mice in which CT was introduced by the trans-
`cutaneous route (geometric mean 5 19,973 EU) compared with
`
`Table I. Passive transfer of protection by sera in an i.n. cholera toxin
`challenge modela
`
`Survival at 9 Days
`
`Experimental Group
`
`Preimmune sera
`Immune sera
`Hyperimmune sera
`No sera
`
`n
`
`4
`3
`5
`7
`
`n
`
`0/4
`3/3
`5/5
`1/7
`
`%
`
`0
`100
`100
`14
`
`a Sera was collected from C57BL/6 mice either before immunization (preim-
`mune), at 6 wk following TCI with 100 mg of CT at 0 and 3 wk (immune), or at 9 wk
`following TCI with 100 mg of CT at 0 and 3 wk and i n challenge with 20 mg of CT
`at 6 wk (hyperimmune) Individual sera were pooled within each group, and recipient
`mice were injected i v with 0 5 ml Mice were challenged i n with 30 mg of CT (1
`mg/ml) at ;1 h after the passive transfer of sera and observed for morbidity and
`mortality
`
`FIGURE 1. CT-specific Ab responses in BALB/c mice (n 5 5) immu-
`nized transcutaneously with CT (100 mg) at 0, 8 (arrow), and 18 (arrow)
`wk. The results shown are the geometric mean and SEM of CT-specific
`IgG measured in sera from five individual animals and reported in EU,
`which is the inverse dilution at which the absorbance is equal to 1.0 at 405
`nm. The inlay displays the Ab titers induced after the 18-wk boost on a
`linear scale. p, a statistically significant increase (p , 0.05) in Ab titer
`between the 18- and 23-wk anti-CT titers. Essentially identical results were
`observed in three independent experiments.
`
`Statistical analysis
`
`A comparison between Ab titers in groups was performed using the Stu-
`dent t test. For challenge studies, the groups were compared by Fisher’s
`exact test (SigmaStat, SPSS, Chicago, IL).
`
`Results
`Kinetics of anti-CT serum IgG response induced by TCI
`Placing CT on the skin induced a rise in detectable anti-CT Abs
`from ,10 ELISA units (EU) before immunization to .10,000 EU
`after a single application (Fig. 1). Elevated CT titers were apparent
`within 2 wk of Ag exposure and persisted for $8 wk. Subsequent
`immunizations at 8 and 18 wk following the primary immunization
`induced ;30-fold (Fig. 1) and 3-fold (Fig. 1, inlay) increases in the
`CT-specific Ab titers.
`
`Induction of protective host immunity by transcutaneous
`vaccination with native CT
`Challenging C57BL/6J mice i n. with CT induces fatal cytotoxic
`pulmonary lesions that are characterized by suppurative interstitial
`pneumonia with marked perivascular edema, fibrin deposition, and
`hemorrhage (our unpublished observations). We used this chal-
`lenge model to assess the significance of the antitoxin response
`induced by TCI. Mice were immunized on the skin with native CT
`and challenged i n. with a lethal dose of CT. Following a single
`immunization, only 11% (1 of 9) of control mice survived the
`challenge compared with 80% (12 of 15) of the vaccinated animals
`(Fig. 2, p 5 0.002). Older mice (20 wk) immunized twice and
`similarly challenged were 100% protected vs 54% of control mice
`(p,0.007). The passive transfer of sera from either transcutane-
`ously immunized or immunized and challenged mice (hyperim-
`mune) resulted in 100% protection in this model (Table I).
`
`Characterization of transcutaneously induced mucosal IgG and
`IgA responses
`To characterize the nature of the immune response induced by
`TCI, sera, lung washes, and stool samples were collected and an-
`alyzed for CT-specific IgG and IgA. The titer of anti-CT IgG Abs
`increased by .3 logs following a single immunization (Fig. 3A);
`
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`FIGURE 3. Sera (A and D), mucosal lung (B and E), and stool (C and F) Ab responses to CT after TCI. A and D, C57BL/6 mice (17–22 animals per
`group) were immunized transcutaneously at 0 or at 0 and 3 wk with 100 mg of CT. Sera were collected at 3 and 6 wk. Data shown are the geometric mean 6
`SEM for ELISA measurements from five individual animals. p, a statistically significant (p , 0.05) difference between the titers measured in the 13 and
`23 immunization groups. B and E, C57BL/6 mice were immunized transcutaneously at 0 wk; lung washes were collected from vaccinated, unchallenged
`mice (n 5 5) on the day of challenge (3 wk). F, indicates the OD detected from control lung washes from mice immunized with an irrelevant protein. IgG
`and IgA levels were assessed by ELISA; the titers (OD 5 405 nm) from individual animals are shown. C and F, C57BL/6 mice were immunized
`transcutaneously at 0 wk. Single stool pellets were collected immediately after defecation on the day before toxin challenge (6 wk). IgG and IgA levels
`were assessed in fecal homogenates by ELISA; the dilution curves from eight (F) or nine (C) individual animals are shown. F, the maximal level of anti-CT
`Ig or anti-CT IgA Ab detected in 1/2 dilutions of stool from unimmunized mice (background).
`
`the oral route (geometric mean 5 395 EU). Moreover, while TCI
`induced a full complement of IgG subclasses (IgG1, IgG2a, IgG2b,
`and IgG3), only IgG1 (four of five animals) and to a lesser extent
`IgG2b (three of five animals) were detected in the sera from the
`orally exposed mice. In a separate experiment, oral feeding with 10
`mg of CT in saline at 0 and 3 wk induced a 6-wk mean IgG Ab
`response of ,1,000 EU, whereas TCI with 100 mg of CT resulted
`in an anti-CT response of 39,828 EU. Similar results were obtained
`using 25 mg of CT on the unshaved ear, which is less accessible
`than the back for grooming, as compared with 25 mg of CT ad-
`ministered by oral feeding (34,426 vs 2,829 EU, respectively).
`
`Discussion
`In this study, we present data indicating that TCI with CT can
`induce Abs detectable both in the systemic and mucosal compart-
`ments and can confer protection against toxin-mediated disease.
`The model described induces a lethal toxin-mediated disease that
`can be prevented by passive transfer of immune sera containing
`antitoxin Ig (Table I). This protective anti-CT immune response to
`
`TCI may be relevant to other toxin-mediated diseases active at the
`mucosal level.
`CT is exquisitely sensitive to degradation in the low pH of the
`stomach and is generally administered orally with a buffer to in-
`duce a mucosal response (8). Therefore, it is unlikely that ingestion
`of CT by grooming causes the dramatic rise in Ab titers that we
`observe following TCI. Consistent with this argument, we ob-
`served that the IgG subclass responses following oral and trans-
`cutaneous immunization differed; oral immunization induced al-
`most exclusively IgG1 and IgG2b Abs, whereas TCI induced a
`broad IgG subclass response (Fig. 4). Additionally, comparative
`immunization by either gavage or oral feeding failed to achieve Ab
`responses comparable with those induced by TCI. Moreover, TCI
`via the ear, which is a site less accessible to oral grooming, re-
`sulted in similar Ab responses, and radiolabeled CT studies sug-
`gested that the amount of Ag remaining on the skin after washing
`is negligible (our unpublished observations).
`Protection against toxin-mediated diseases such as pertussis is
`known to be mediated in large part by antitoxin Abs (9). The role
`
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`protective role of anti-CT Abs have used less toxic but less im-
`munogenic derivatives of CT such as its B subunit (CTB) (5, 16)
`and cholera toxoid (17). TCI may prove to be a powerful technique
`that elicits potent immune responses in the absence of obvious
`toxicity. Additional studies are warranted to assess the utility of
`TCI in human vaccines against infectious and toxin-mediated dis-
`eases, particularly cholera and traveler’s diarrhea.
`
`Acknowledgments
`We thank Deborah Walwender and Elaine Morrison for technical assistance.
`
`References
`1 De, S N 1959 Enterotoxicity of bacteria-free culture filtrate of Vibrio cholerae
`Nature 183:1533.
`2 Levine, M M , J B Kaper, R E Black, and M L Clemens 1983 New knowl-
`edge on pathogenesis of bacterial infections as applied to vaccine development
`Microbiol. Rev. 47:510.
`3 Snider, D P 1995 The mucosal adjuvant activities of ADP-bacterial enterotox-
`ins Crit. Rev. Immunol. 15:317.
`4 Holmgren, J , and A -M Svennerholm 1977 Mechanisms of disease and immu-
`nity in cholera: a review J. Infect. Dis. 136:S105.
`5 Clemens, J D , D A Sack, J R Harris, J Chakraborty, M R Khan,
`B F Stanton, B A Kay, M U Khan, M Yunus, W Atkinson, et al 1986 Field
`trial of oral cholera vaccines in Bangladesh Lancet 2:124.
`6 Glenn, G M , M Rao, G R Matyas, and C R Alving 1998 Cholera toxin
`opens up skin immunization route Nature 391:851.
`7 Glenn, G M , M R Rao, R L Richards, G R Matyas, and C R Alving 1995
`Murine IgG subclass antibodies to antigens incorporated in liposomes containing
`lipid A Immunol. Lett. 47:73.
`8 Clemens, J , M Jertborn, D Sack, B Stanton, J Holmgren, M R Khan, and
`S Huda 1986 Effect of neutralization of gastric acid on immune responses to an
`oral B subunit, killed whole-cell vaccine J. Infect. Dis. 154:175.
`9 Schneerson, R E , J B Robbins, J Taranger, T Lagergaard, and B Trollfors
`1996 A toxoid vaccine for pertussis as well as diphtheria? Lessons to be re-
`learned Lancet 348:1289.
`10 Clemens, J D , D A Sack, J R Harris, J Chakraborty, P K Neogy,
`B F Stanton, N Huda, M U Khan, B A Kay, M R Khan, M Ansaruzzaman,
`M D Yunus, M R Rao, A -M Svennerholm, and J Holmgren 1988 Cross-
`protection by B subunit-whole cell cholera vaccine against diarrhea associated
`with heat-labile toxin-producing enterotoxigenic Escherichia coli: results of a
`large-scale field trial J. Infect. Dis. 158:372.
`11 Pierce, N F , W C Cray, and P F Engel 1980 Antitoxic immunity to cholera
`in dogs immunized orally with cholera toxin Infect. Immun. 27:632.
`12 Holmgren, J , A -M Svennerholm, O Ouchterlony, A Anderson, G Wallerstrom,
`and U Westerberg-Berndtsson 1975 Antitoxic immunity in experimental chol-
`era: comparison of immunity induced perorally and parenterally in mice J. Infect.
`Dis. 12:463.
`13 Pierce, N F , and H Y Reynolds 1974 Immunity to experimental cholera:
`protective effect of humoral IgG antitoxin demonstrated by passive immuniza-
`tion J. Immunol. 113:1017.
`14 Pierce, N F , E A Kaniecki, and R S Northrup 1972 Protection against ex-
`perimental cholera by antitoxin J. Infect. Dis. 126:606.
`15 Holmgren, J , A -M Svennerholm, O Ouchterlony, A Anderson, G Walletstrom,
`and U Westerberg-Berndtsson 1975 Antitoxic immunity in experimental chol-
`era: protection and serum and local antibody responses in rabbits after enteric and
`parenteral immunization Infect. Immun. 12:463.
`16 Clemens, J D , D A Sack, J R Harris, F Van Loon, J Chakraborty, F Ahmed,
`M R Rao, M R Khan, M D Yunus, N Huda, B F Stanton, B A Kay, S
`Walter, R Eeckels, A M Svennerholm, and J Holmgren 1990 Field trial of oral
`cholera vaccines in Bangladesh: results from three-year follow up Lancet 335:
`27.
`17 Feeley, J C , G T Curlin, K M Aziz, G L Wiggins, and W L Albritton 1979
`Response of children in Bangladesh to adult-type tetanus-diphtheria toxoid (Td)
`administered during a field trial of cholera toxoid J. Biol. Stand. 3:249.
`
`FIGURE 4. Characterization of sera Ab responses induced by oral (A
`and B) or transcutaneous (C and D) exposure to CT. BALB/c mice (n 5 5)
`were immunized with 25 mg of CT by oral gavage or with 100 mg of CT
`by transcutaneous application to the back. Sera was collected after 4 wk,
`and the levels of CT-specific IgG, IgG1, IgG2a, IgG2b, and IgG3 were
`assessed by ELISA. The results shown are measurements from five indi-
`vidual animals (M, A and C; E, B and D). Solid symbols indicate the
`geometric mean value for each cohort of animals. p, the mean value de-
`tected in prebleed sera of the mice.
`
`of antitoxin immunity in protection against human cholera is not
`entirely clear (5, 10), but antitoxin immunity can be completely
`protective in animals (11–13) and clearly contributes to immunity
`in resistant humans (10). For example, dogs parenterally immu-
`nized with CT or cholera toxoid (14) or administered anti-CT IgG
`Abs parenterally (13) are protected against intragastric challenge
`with CT-producing strains of V. cholerae. Moreover, anti-CT IgA
`reduces rabbit ileal loop secretory responses to CT (15). Based on
`studies such as these, it is tempting to speculate that the Abs de-
`tected at the mucosa that are induced by TCI confer protection
`against
`toxin challenge. Consistent with this hypothesis,
`lung
`washes and stool samples from transcutaneously immunized mice
`exhibited elevated anti-CT IgG and IgA Ab levels (Fig. 3), and
`passive Ab transfer to naive mice was clearly protective (Table I).
`The toxicity of CT administered via the mucosal route has lim-
`ited its use as a vaccine component; consequently, studies on the
`
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