Response to Cutaneous Immunization with Low-Molecular-Weight Subunit Keyhole Limpet Hemocyanin

Abstract

Background: This study was carried out to determine whether humoral and cellular immune responses would be provoked by cutaneous administration of keyhole limpet hemocyanin (KLH) and in particular by scarification of the skin (SS). Methods: This was an unblinded, single-center, 8-week pilot study in healthy young adults. Twenty-four subjects assigned to 4 groups completed the study. Each group was immunized twice, with a 3-week interval, either by SS or intradermally (ID), with an SS dose of 50 or 250 µg and an ID dose of 100 or 250 µg. Serum was collected for antibody assays at baseline and 3 weeks after both the first and second immunizations. Delayed-type hypersensitivity (DTH) testing was performed before the first immunization and 3 weeks after the second. Results: In the 250-µg SS group, there was a significant increase from day 0 to day 47 in anti-KLH IgG (p = 0.02; day 0: 3.46 ± 5.49 mg/dl, day 47: 7.54 ± 8.87 mg/dl) and anti-KLH IgA (p = 0.04; day 0: 4.78 ± 9.15 mg/dl, day 47: 11.42 ± 13.62 mg/dl). One subject in each treatment group showed a positive DTH test result representing 20% (50-µg SS), 10% (250-µg SS), 25% (100-µg ID) and 20% (250-µg ID) of the subjects. Conclusions: It was possible to induce both humoral and cellular immune responses by SS administration despite the limited antigenic potency of the low-molecular-weight KLH preparation. This approach may be useful for studying the mechanisms of immune response in allergic skin diseases such as atopic dermatitis.

Characterization of KLH-driven immune responses in clinical studies: A systematic review

“…Intranasal KLH inhalation has also been reported, however, sufficient penetration of KLH through the mucosal tissue likely requires higher (cumulative) KLH doses in order to exert measurable systemic immune responses (Salvaggio et al, 1969;Waldo et al, 1994;Diaz-Sanchez et al, 1999). Intradermal KLH administration is the preferential administration route when analyzing the skin response after KLH rechallenge (Salvaggio et al, 1969;Curtis and Hersh, 1972;Brunner et al, 1973;Palestine et al, 1985;Ashorn et al, 1986;Husby et al, 1994;Waldo et al, 1994;Valdez et al, 2000;Rentenaar et al, 2002;Lange et al, 2003;Smith et al, 2004a;Boelens et al, 2004;Smith et al, 2004b;Moldoveanu et al, 2004;Spazierer et al, 2009;Boulton et al, 2012;Milgrom et al, 2012;Hostmann et al, 2015;Belson et al, 2016;Saghari et al, 2020;Saghari et al, 2022). The arm is most often used for KLH Waldo FB, et al, 1994(Waldo et al, 1994) Diameter Induration 11.9 mm (0-23 mm) Husby S, et al, 1995(Husby et al, 1994) Diameter Induration 11.9 mm (0-23 mm) Ashorn RG, et al, 1986(Ashorn et al, 1986) Diameter Induration 17.5 mm (10-25 mm) Palestine AG, et al, 1985(Palestine et al, 1985 Diameter Induration 15.4 mm (8-20 mm)** Brunner CM, et al, 1973(Brunner et al, 1973 Diameter Induration 15 mm (single subject) Curtis JE, et al, 1972 (Curtis andHersh, 1972) Diameter Induration 8.7 mm (0-18 mm) Salvaggio J, et al,...…”

“…Analysis methods varied from enzyme-linked immunosorbent assay (ELISA) to hemagglutination assay (HA), radioimmunoassay (RIA), and cytometric bead array (CBA) (Table 2). The majority of the studies had used ELISA to quantify KLHspecific antibodies (Volkman et al, 1981;Palestine et al, 1985;Bird et al, 1990;Ward et al, 1990;Falconer et al, 1992;Husby et al, 1994;Waldo et al, 1994;Fijter et al, 1996;Kondratenko et al, 1997;Schuyler et al, 1997;Rentenaar et al, 2002;Lange et al, 2003;Smith et al, 2004a;Boelens et al, 2004;Smith et al, 2004b;Kraus et al, 2004;Moldoveanu et al, 2004;Miller et al, 2005;Spazierer et al, 2009;Kapp et al, 2010;Boulton et al, 2012;Milgrom et al, 2012;Hostmann et al, 2015;Poirier et al, 2016;Giesecke et al, 2018;Saghari et al, 2020;Otterhaug et al, 2021;Yang et al, 2021;Saghari et al, 2022).…”

“…The KLH-specific antibody responses were analyzed differently across the studies included in this review. The comparison of optical density (OD) values of experimental sera in precalculated dilutions to negative control and OD values of a positive control included on the same ELISA plate was used in several studies (Bird et al, 1990;Schuyler et al, 1997;Smith et al, 2004a;Smith et al, 2004b;Kraus et al, 2004;Milgrom et al, 2012;Ferbas et al, 2013;Saghari et al, 2020). Other studies analyzing the anti-KLH antibody production in sera also prepared standard curves for each studied antibody isotype with established KLH antibody concentrations in mg/L, thereby being able to calculate KLH antibody levels (Palestine et al, 1985;Fijter et al, 1996;Kondratenko et al, 1997;Diaz-Sanchez et al, 1999;Valdez et al, 2000;Boelens et al, 2004;Miller et al, 2005;Spazierer et al, 2009;Poirier et al, 2016;Giesecke et al, 2018;Otterhaug et al, 2021).…”

“…Anti-KLH IgG antibodies were measured in all studies characterizing the systemic humoral immune response, of which 8 studies also included the IgG subtypes (IgG1-4) (Bird et al, 1990;Falconer et al, 1992;Schuyler et al, 1997;Boelens et al, 2004;Miller et al, 2005;Spazierer et al, 2009;Kapp et al, 2010;Hostmann et al, 2015). Anti-KLH IgM antibodies were analyzed in 20 studies (Salvaggio et al, 1969;Brunner et al, 1973;Volkman et al, 1981;Birdsall and Rossen, 1983;Palestine et al, 1985;Ward et al, 1990;Husby et al, 1994;Waldo et al, 1994;Fijter et al, 1996;Kondratenko et al, 1997;Schuyler et al, 1997;Smith et al, 2004a;Boelens et al, 2004;Kraus et al, 2004;Moldoveanu et al, 2004;Miller et al, 2005;Spazierer et al, 2009;Kapp et al, 2010;Boulton et al, 2012;Milgrom et al, 2012;Ferbas et al, 2013;Hostmann et al, 2015;Giesecke et al, 2018;Saghari et al, 2020) and anti-KLH IgA antibodies were analyzed in 10 studies (Birdsall and Rossen, 1983;Husby et al, 1994;Waldo et al, 1994;Fijter et al, 1996;Schuyler et al, 1997;Diaz-Sanchez et al, 1999;…”

Novel mucosal adjuvant, mastoparan-7, improves cocaine vaccine efficacy

“…KLH was chosen as the carrier in this system due to its biochemical properties that promote haptenation, as well as existing evidence that it is both immunogenic and safe in humans. 26 We hypothesized that intranasal coadministration of these carrier protein conjugates with the MCactivating adjuvant compound M7 could be an effective way to evoke high and sustained anti-cocaine antibody levels in both the circulation and mucosal secretions. Here, we also aimed to optimize the route of cocaine vaccine administration and determine whether a nasal route of administration could improve mucosal responses while providing protection from the psychostimulatory effects of cocaine.…”