LT-related ETEC disease accounts for considerable morbidity among travelers and mortality in weanling infants (32
). LT is both an immunogenic and key pathogenic factor for LT-related ETEC disease and is thus an ideal vaccine antigen. However, LT is difficult to deliver by traditional vaccine delivery routes due to its strong effects on cells via the ADP-ribosyl transferase pathway. This results in diarrhea if LT is ingested as an oral antigen in buffer, or it results in inflammation in the nose if LT is given intranasally (17
) or at the site of injection if LT is given parenterally (5
). Delivery to the skin by TCI is a safe method for delivery and results in an ideal immune environment, resulting in strong antitoxin immune responses (11
), as demonstrated in the present study.
The stratum corneum is the skin's principal barrier to penetration of molecules and is an effective yet fragile barrier (12
). TCI is possible with minimal intervention to the skin, even with no physical stratum corneum disruption, by simple application of a wet, occlusive patch with antigen (12
). An occlusive patch hydrates the stratum corneum and allows small molecules (generally less than 500 Da), such as drugs used in transdermal patches, to penetrate through the skin, and this method has also been shown to allow much larger molecules (e.g., 86,000-Da LT), such as antigens, to move into the skin. However, follow-up patch vaccine delivery studies have indicated that even modest physical disruption of the stratum corneum can significantly improve the efficiency of TCI for a given dose (11
). In these studies, classic methods for stratum corneum disruption, including tape stripping and EKG prep pad abrasives, were employed. Using an EKG prep pad (emery paper), a 50-fold increase in the IgG titer was achieved with a 50-μg LT patch. When hydration was used as a pretreatment, a 29-fold increase in the LT IgG response was achieved using 400 μg of LT in a patch after two doses, indicating that physical disruption of the stratum corneum resulted in almost doubling of the increase in the LT IgG response using 87.5% less LT, an approximately 10-fold improvement in efficiency (11
In the present study, TEWL measurement was conducted after skin pretreatment, prior to application of the patch. TEWL analysis is a well-developed method that is used in both transdermal drug delivery development and skin studies, and it provides an accurate measurement of stratum corneum disruption (8
). However, there have been no studies correlating TEWL and the immune response after TCI. In early pilot studies, we first demonstrated that there was a graduated increase in the TEWL based on the number of nonwoven abrasive pad strokes used on the skin (data not shown) and determined that 15 strokes were well tolerated and could consistently increase the TEWL in humans. Although we recognize that this method is not a practical bedside method for an ETEC vaccine, it is a medically accepted method for skin pretreatment that we believe can be modified so that it is suitable for use in a product. We then used this regimen as a pretreatment in a vaccination regimen with a 50-μg LT patch applied for 6 h. Immune responses, indicated by individual increases in anti-LT antibody titers, correlated with the individual TEWL measurements, suggesting that the magnitude of stratum corneum disruption and delivery are related. In a separate study, skin biopsies after the same pretreatment regimen resulted in loss of 29% of the cell layers in the stratum corneum. Together, these data indicated that stratum corneum disruption was modest but important since the increase in TEWL correlated with immune responses and, therefore, delivery.
There were no differences in the immunogenicity and safety of the three lots of LT, the primary endpoint. The immune response after pretreatment and patch application appeared to be robust, both compared to previous data (11
) and compared to postinfection immune responses and to antitoxin responses to an oral cholera vaccine. The immune responses to LT delivered via TCI were greater than the immune responses observed for sera in subjects after oral challenge with virulent LT+
ETEC. In the challenge study, subjects' diarrhea may have been caused by exposure to both LT and ST. However, 88% of the subjects in the challenge group seroconverted to LT, indicating that there was LT exposure at the level of the gut, suggesting that the diarrhea was in part mediated by LT. In animal studies, this level of exposure and the subsequent immune responses are acutely accompanied by measurable gut fluid accumulation. The immune responses to LT in the human challenge study suggest that LT played a role in the virulent diarrhea seen in subjects and that TCI resulted in immunity that was superior to the immunity generated by LT exposure in the gut that accompanies ETEC disease (i.e., postinfection immunity). Similarly, the LT cross-reacting anti-CTB immune responses to the recombinant CTB/cholera whole-cell vaccine, which has been shown protect against LT+
ETEC in field studies, were approximately 50% of the responses produced by TCI. Although it has been shown that TCI elicits a mucosal immune response (12
), studies of the mucosal immunity for both live infectious challenge and recombinant CTB/cholera vaccination have not been performed, and the mucosal responses may be important contributors to protection. However, the systemic immune responses to live infectious challenge and an existing vaccine provide important benchmarks for immune responses elicited by TCI and suggest that the anti-LT IgG elicited by skin immune responses is robust.
There were several limitations in this study. The primary endpoints, safety, and immunogenicity of the three lots of LT precluded measuring TEWL for the first immunization. It was important to immunize without any additional procedures or skin interventions and to compare the immunogenicities of the different lots of LT, which could be evaluated after a single dose. TEWL measurements were obtained for the second dose. Similarly, the biopsy was separate from the immunization, although the same pretreatment protocol was used and appeared to be a highly consistent procedure, as suggested by TEWL measurements obtained for the three treatment groups. Furthermore, a control group without pretreatment would have provided additional information, but this was not within the scope of the study. However, there have been no previous correlations of TEWL, a common measure of stratum corneum disruption, and delivery of antigens to the skin, and there have been no previous comparisons of the LT immune response to the results of comparable regimens that can elicit anti-LT antibodies.
In the present study, we were able to demonstrate robust, persistent immune neutralizing antitoxin responses to LT with a two-dose regimen using TCI, suggesting that toxin immunity may be feasible as an approach to an ETEC vaccine. In early studies of enteric vaccines workers used a toxin-based vaccine strategy, which is mirrored by several current, highly successful licensed vaccines (vaccines for diphtheria, pertussis, tetanus). In animals, compelling data for protection were generated using LT (22
), ST (18
), and CT/toxoids (14
). The concept was further explored using LT-ST conjugates, which were fully protective in challenges with strains producing both toxins (19
). In the past, use of these toxins as vaccine antigens presented difficulties since the native antigens can cause untoward reactions when delivery modes (e.g., intranasal, oral, or parenteral) other than TCI are used. Additionally, the chemically produced toxoid antigen for cholera failed in the field, and together these problems cast doubt on the viability of the toxin-based approach for dealing with enteric disease.
In concert with extensive preclinical data showing that antitoxin immunity is protective, experimental ETEC infections and field data have shown that active immunity and passive immunity are protective (23
). Field data have shown that antitoxin neutralizing titers to LT play a key role in protection against ETEC disease in the face of natural exposure to repeated infections. In a 2-year follow-up study of 200 neonates, neonates infected with ETEC were protected 47% of the time from repeat LT+
ETEC infection, and the protection appeared to be based on immunity to the toxin (33
). Several studies have shown the protective effects of breast milk antibodies to LT and CT against diarrheal disease (10
). In developing countries, ETEC illness decreases over time (2
), and in long-term travelers, illness decreases with length of stay in an area where ETEC is endemic (6
). Experimental challenge with an ETEC strain protected against rechallenge (7
), although a small challenge study cast disproportionate doubt on the potential for antitoxin immunity to be protective (22
). By contrast, antitoxin neutralizing titers have been shown to play a key role in protection against ETEC disease in large field trials of an oral cholera vaccine containing CTB, which is homologous to LT and induces cross-reacting anti-LT antibodies for both LT and LT+
; Bourgeois et al., 45th ICAAC). In a live-organism challenge, recipients of LT-expressing ETEC strains develop LT ELISA titers that are lower than those seen with TCI. The robust, LT-specific (holotoxin) immunity that can exceed the immunity seen after live infectious challenge and that compares well to the immunity obtained with an oral CTB-based vaccine that has shown field efficacy suggests that an anti-LT toxin vaccine could be efficacious in the field for both LT and LT+
ETEC is a burden on the health of people who live in or travel to areas where ETEC is endemic. Although some workers have suggested that an ETEC LT vaccine provides only narrow coverage against the host of enteric organisms, lessons learned from studying vaccines against other mucosal pathogens, such as diphtheria and pertussis vaccines, suggest that focused targets are more likely to succeed in vaccine development programs. The burden of disease due to LT-related ETEC is very large, both in travelers and in children in the poorest countries (25
). The prospect of elimination of several hundred million cases of dehydrating diarrhea in infants and hundreds of thousands of deaths with a vaccine that is needle-free, stable at room temperature, and easy to administer has led to a vigorous program to develop a patch and pretreatment regimen suitable for such an application.