In this study, we show that low-molecular-weight KLH by ID or SS administration stimulates both humoral and cellular responses in some subjects. Ours is the first report of immune responses to scarification with a protein antigen in human subjects. We base this on an exhaustive PubMed search. Systemic immunization with KLH has been very useful for the assessment of the immune function of human subjects for many years. Our immunization strategy used doses of KLH in a range similar to earlier studies in healthy volunteers. We observed less potent immune responses to ID immunization than previously reported [14
]. We attribute these results to the lower molecular weight and greater purity of the current clinical-grade KLH preparation. Our findings may be enhanced by the use of a more potent KLH preparation; even so, any immune signal after SS with a protein antigen such as KLH warrants further inquiry.
Hemocyanin obtained from the marine mollusk giant keyhole limpet, Megathura crenulata
, has been in use as a crude or partially purified product for over 40 years [17
]. Native KLH is composed of 12 subunits with a molecular weight of 9,000–10,000 kDa. Earlier studies with KLH acting as an immunogen used preparations consisting in large measure of native KLH manufactured by chemical suppliers. Intracel KLH, unavailable during our study, preserves the high-molecular-weight structure with greater than 95% purity [18
]. The low-molecular-weight subunit preparation of KLH appears to be inherently less immunogenic than the high-molecular-weight native or partially purified KLH [19
]. Miller et al. [19
] induced potent IgM, IgG1 and IgG2 responses with high-molecular-weight Intracel KLH in healthy volunteers within 1 month of administration. When they switched to low molecular-weight Biosyn KLH, 1 mg administered subcutaneously, they were unable to induce significant humoral or cellular responses. However, when they emulsified Biosyn KLH with a synthetic incomplete Freund's adjuvant (Montanide ISA-51, Seppic Inc., Fairfield, N.J., USA), full immunogenicity was restored with 100% of the healthy volunteers developing a humoral response [19
]. The fact that they were able to restore full immunogenicity with the use of an adjuvant suggests that the lack of response was not due to a loss of important epitopes, but rather that the intact high molecular-weight KLH product comprises adjuvant properties [19
The route and the dose of the administered antigen determine the response. In a recent study, low-dose oral KLH induced an antigen-specific systemic CD4+ T cell response with a shift toward a Th2 cytokine pattern. Later parenteral immunization boosted B cell responses and shifted the cytokine pattern of KLH-specific CD4+ T cells from a Th1 towards a Th2 pattern, suggesting a novel effect of oral immunomodulation [20
Cutaneous immunization is being explored as an alternative to conventional IM or subcutaneous vaccination [5
]. Liu et al. [6
] recently immunized mice by SS with live vaccinia virus. These mice generated superior humoral responses to those immunized by conventional routes. Kenney et al. [7
] and Belshe et al. [8
] found that, in human volunteers, a fraction of the standard-dose-administered ID elicited immune responses similar or superior to those of a full dose of vaccine given IM. These results and ours, all using protein immunogens tested in humans, showed the best responses with ID administration [7
], while the live virus immunization (even if capable of only limited replication), used by Liu et al. [6
] in mice, was most effective by SS.
Although this was a small pilot study, we demonstrated that it is possible to induce both humoral and cellular immune responses in some healthy subjects by either ID or SS administration of KLH. Both ID and SS administration of antigen may be useful in probing mechanisms that underlie the variability amongst human subjects in skin immune responses to different stimuli. An understanding of the range and dynamics of these responses is indispensable for the development of new therapies against cancer and autoimmune diseases. The information gained may also be used to develop new vaccines against important pathogens. Lastly, it may be possible to use immunization by SS to study the defects of immune protection in the skin.