Safety and immunogenicity of an enterotoxigenic Escherichia coli vaccine patch containing heat-labile toxin: use of skin pretreatment to disrupt the stratum corneum

Abstract

Transcutaneous immunization allows safe delivery of native heat-labile enterotoxin (LT) from Escherichia coli via application of a simple patch. Physical disruption of the stratum corneum can improve the efficiency of delivery. In the current study, the stratum corneum was disrupted using an electrocardiogram prep pad prior to patch application. The effects were quantified using transepidermal water loss (TEWL) and were correlated with the immune responses. Sixty adults received 50 microg of LT from three lots of LT (20 adults per group) administered in a patch on days 0 and 21. The immunizations were well tolerated. There were no differences in the anti-LT immunoglobulin G (IgG) titers between the three LT lots; the seroconversion rate was 100%, and the mean anti-LT IgG titer was 12,185 enzyme-linked immunosorbent assay units (EU) (a 24-fold increase). TEWL measurements obtained at the time of the second immunization were found to correlate with the day 42 individual increases in the anti-LT IgG titer (r = 0.59, P < 0.001). In a comparative assessment of the immune responses, sera after an LT+ ST+ (E2447A) oral ETEC challenge, which induced moderate to severe diarrhea in 81% of the recipients, had anti-LT IgG titers of 3,245 EU (a 10.8-fold increase). Similarly, the anti-LT IgG titer after administration of an oral cholera toxin B subunit-containing cholera vaccine, which cross-reacts with LT and protects against LT and LT/heat-stable toxin ETEC disease in the field, was 6,741 EU (a 3.3-fold increase). This study confirmed that a well-tolerated regimen for stratum corneum disruption before vaccine patch application results in robust immunity comparable to natural immunity and vaccine-induced immunity and that the magnitude of stratum corneum disruption correlates with the immune response.

FIG. 1.

Immunogenicity of LT delivered by TCI. Following pretreatment of the skin with a nonwoven abrasive EKG prep pad to disrupt the stratum corneum, a wet patch containing 50 μg of one of three lots of LT was applied to the pretreated area on days 0 and 21. Serum samples obtained on days 0, 21, 42, 90, and 381 were analyzed to determine the presence of anti-LT IgG and IgA by ELISA. There were no significant differences between the anti-LT immune responses of the three groups (P < 0.05). The serum antibody levels for the combined treatment groups are expressed in ELISA units; the bars indicate geometric means, and the error bars indicate 95% confidence intervals. The percentages are percentages of seroconversion (a twofold increase compared with the baseline for IgG and a fourfold increase for IgA).

FIG. 2.

LT toxin neutralization by LT immune sera. Serum samples obtained on days 0, 21, 42, 90, and 381 from subjects vaccinated on days 0 and 21 with 50 μg of LT by TCI were tested to determine their abilities to neutralize LT in a Y-1 serum neutralization assay. The serum antibody levels for the combined treatment groups are expressed as average ED₅₀s, and the error bars indicate 95% confidence intervals.

FIG. 3.

Comparison of neutralization of LT and CT toxins by LT immune sera. Pooled serum samples obtained on days 0 and 42 from subjects vaccinated on days 0 and 21 with 50 μg of LT by TCI were tested to determine their abilities to neutralize CT in a Y-1 serum neutralization assay. Toxin activity is indicated by the reduction in OD₅₃₀, which reflects the number of neutral red-stained cells remaining after treatment. Toxin neutralization of both LT and CT by serum is shown.

FIG. 4.

Anti-LT serum IgG induced following a live infectious challenge. Subjects received an oral infectious challenge consisting of 1 × 10⁹ viable ETEC E2447A LT⁺ ST⁺ bacteria. Serum samples obtained on day 0, as well as on days 7 and 28 following the oral challenge, were analyzed to determine the presence of anti-LT IgG by ELISA. The serum antibody levels are expressed in ELISA units; the bars indicate geometric means, and the error bars indicate 95% confidence intervals. The percentages are percentages of seroconversion.

FIG. 5.

Serological recognition of LT by sera from CTB-vaccinated subjects. Sera obtained from subjects 35 days after vaccination with a single dose of an oral whole-cell cholera vaccine containing 1 mg CTB and killed V. cholerae cells were analyzed to determine their recognition of LT, using an ELISA. The serum antibody levels are expressed in ELISA units; the bars indicate geometric means, and the error bars indicate 95% confidence levels. The percentage is the percentage of seroconversion.

FIG. 6.

Correlation between net TEWL and fold increase in the IgG titer. TEWL was measured on the skin before pretreatment and after skin pretreatment but before patch application in 51 of 59 subjects at day 42. Individual net TEWL (posttreatment TEWL − baseline TEWL) at the time of vaccination is plotted against the increase in anti-LT IgG at day 42 (r = 0.59, P = 0.001).

FIG. 7.

Biopsy of control and nonwoven abrasive pad-treated human skin. After informed consent, a shave biopsy of human skin was obtained from a control site (A) and a site that was treated with 15 strokes of a nonwoven abrasive EKG pad (B). The skin was formalin fixed and stained using hematoxylin and eosin. The three layers of the skin, the stratum corneum, epidermis, and dermis, are visible, and in the group pretreated with the nonwoven abrasive pad a modest decrease in the thickness of the stratum corneum is visible.