Transcutaneous Immunization with Bacterial ADP-Ribosylating Exotoxins, Subunits, and Unrelated Adjuvants

Abstract

We have recently described a needle-free method of vaccination, transcutaneous immunization, consisting of the topical application of vaccine antigens to intact skin. While most proteins themselves are poor immunogens on the skin, we have shown that the addition of cholera toxin (CT), a mucosal adjuvant, results in cellular and humoral immune responses to the adjuvant and coadministered antigens. The present study explores the breadth of adjuvants that have activity on the skin, using diphtheria toxoid (DTx) and tetanus toxoid as model antigens. Heat-labile enterotoxin (LT) displayed adjuvant properties similar to those of CT when used on the skin and induced protective immune responses against tetanus toxin challenge when applied topically at doses as low as 1 g. Interestingly, enterotoxin derivatives LTR192G, LTK63, and LTR72 and the recombinant CT B subunit also exhibited adjuvant properties on the skin. Consistent with the latter finding, non-ADPribosylating exotoxins, including an oligonucleotide DNA sequence, as well as several cytokines (interleukin-1␤ [IL-1␤] fragment, IL-2, IL-12, and tumor necrosis factor alpha) and lipopolysaccharide also elicited detectable anti-DTx immunoglobulin G titers in the immunized mice. These results indicate that enhancement of the immune response to topical immunization is not restricted to CT or the ADP-ribosylating exotoxins as adjuvants. This study also reinforces earlier findings that addition of an adjuvant is important for the induction of robust immune responses to vaccine antigens delivered by topical application.Worldwide rates of immunization for diphtheria, pertussis, tetanus, polio, measles, and tuberculosis have increased dramatically over the last 20 years. Barriers to mass immunization vary widely among economic markets, but common constraints include the requirement for trained personnel, the association of vaccines with needle-related diseases and injuries, and coldchain and transport issues. Development of less-invasive and more readily administered vaccines has thus become a priority for public health agencies and is associated with an emergence of new technologies in the areas of vaccine delivery vehicles and routes of administration (1,15,19,25).Researchers have recently described transcutaneous immunization (TCI), a needle-free method for delivering vaccines to the host by simple application of adjuvant and antigen to the exterior skin surface (15,16,17,18,19,29). Our previous studies demonstrated that cholera toxin (CT) applied to the skin could be used as an adjuvant to elicit serum immunoglobulin G (IgG) responses against itself and coadministered antigens, including bovine serum albumin, diphtheria toxoid (DTx), hen egg lysozyme, tetanus fragment C, and tetanus toxoid (TTx). The humoral responses elicited by TCI are boosted by sequential applications, and antibodies are detectable in lung and stool exudates (16, 17), suggesting the potential for eliciting mucosally relevant immunity by this method. Moreover, we have reported that application of CT...

Transcutaneous Immunization with Cross-Reacting Material CRM 197 of Diphtheria Toxin Boosts Functional Antibody Levels in Mice Primed Parenterally with Adsorbed Diphtheria Toxoid Vaccine

“…However, we show that the toxin mutant LTR72 used in this study significantly enhances postboost functional antibody levels, an effect that was not seen using the fully enzymatically active CT holotoxin. This suggests that full ADP-ribosylating activity is not required for significant enhancement of the immune response to a topically coadministered antigen and is consistent with previous findings where several adjuvants with no ADP-ribosylating activity were shown to enhance antibody responses to a topically coapplied diphtheria toxoid (28). However, the presence of some enzymatic activity appears to be important for optimum adjuvanticity, and the spiking of a recombinant CTB subunit (devoid of enzyme activity) with a small amount of holotoxin was shown to induce immune responses that were comparable to those obtained with the native CT (28).…”

“…This suggests that full ADP-ribosylating activity is not required for significant enhancement of the immune response to a topically coadministered antigen and is consistent with previous findings where several adjuvants with no ADP-ribosylating activity were shown to enhance antibody responses to a topically coapplied diphtheria toxoid (28). However, the presence of some enzymatic activity appears to be important for optimum adjuvanticity, and the spiking of a recombinant CTB subunit (devoid of enzyme activity) with a small amount of holotoxin was shown to induce immune responses that were comparable to those obtained with the native CT (28). LTR72 which retains residual enzymatic activity is a more effective mucosal adjuvant than the related LT derivative LTK63 which is devoid of enzymatic activity (7).…”

Recombinant Cholera Toxin B Subunit Activates Dendritic Cells and Enhances Antitumor Immunity

“…However, oral administration of CTB chemically coupled to antigens is known to induce tolerance, such as inhibition of acute GVHD and prevention of autoimmune diabetes (26,31). Recently it was demonstrated that needle-free transcutaneous immunization by CT enhanced antibody response against diphtheria toxoid (1). In this study we demonstrated that rCTB induced maturation of DC and tumor growth was inhibited by s.c. vaccination of DC pulsed with rCTB and tumor lysate.…”

Layer-by-Layer-Assembled Multilayer Films for Transcutaneous Drug and Vaccine Delivery

“…In the context of vaccination, this order of dose/area is well within a meaningful range as antibody responses have been reported for transcutaneous antigen doses as low as 3 μg. [42][43][44] When dried as-assembled (Poly-1/ova) n PEM films on glass were rehydrated in phosphatebuffered saline (PBS, pH 7.4), films prepared across all deposition conditions exhibited rapid protein release, as illustrated in Figure 1c. Characterization of (Poly-1/ova) 40 film thickness vs. time following rehydration revealed rapid dissolution of the films coinciding with protein release.…”