Epitope characterization of ovalbumin in BALB/c mice using different entry routes (1)
Ovalbumin is a 385 amino acid glycoprotein of approximately 45 kDa, which constitutes about 54% of the egg white proteins. The carbohydrate moieties of ovalbumin account for about 3% of its molecular weight. Utilizing synthetic or cyanogen bromide (CNBr)-cleaved OVA peptides, subsequent studies demonstrated that the segments C11K19, I34E70 and also V41M172, G301P385 were reactive with patients’ IgE antibodies. The sequence A347P385 were capable of specifically inhibiting histamine release from human basophils in vitro cultures. The fragment I323R339 was initially identified as an immunodominant T-cell epitope. Additional studies also identified the region V41M172 and G301P385 as antigenic determinants of OVA, using specific anti-OVA monoclonal antibodies, containing IgG1 and IgM isotypes, produced from BALB/c mice. The fragment V173M196 was specifically identified as an IgG1-antigenic determinant only found on the irradiated form of OVA. A total of five distinct regions i.e. L38T49, D95A102, E191V200, V243E248 and G251N260 were finely mapped and structurally characterized.
With the orally sensitized mice sera, strong signals were detected on spot numbers 25–27[A-1], 37–39[A-2], 49–52[A-3], 63–64[A-4], 135–138[A-5], 148– 151[A-6], 161–162[A-7] and 187–188[A-8]. By intraperitoneal immunization, there were corresponding high intensity spots number 25–28[B-1] and 136–139[B-5]. Subcutaneously sensitized mice sera indicated five distinct binding regions. Spots number 24–28[C-1] and 136–140[C-5] displayed a high IgE binding intensity while spots 63–64[C-4]; 149–152[C-6] and 161–162[C-7] indicated a lower binding intensity.
The peptide sequences comprised within spots 24–28 and spots 135–140 have not only generated the highest intensity signals within 3 sera.
With orally sensitized mice sera, eight epitope regions were distributed along the length of the ovalbumin amino acid sequence. These regions were designated as I53D60, V77R84, S103E108, G127T136, E275V280, G301F306, I323A332 and A375S384 respectively.
The critical residues identified in epitope regions A1, A3, A5 and A8 are shown.
Most of the epitopes are found on the surface on the OVA native structure or partially buried, and mainly consist of β-sheets and β-turns. The two epitope regions I53D60 [A1] and V77R84 [A2] are present on α-helices (red and green colored-ribbons).
The OVA IgE epitope map obtained in mice compared to the one obtained in our previous study using human patients’ sera was compared.
All epitope regions identified in our study (oral sensitization) – except for epitope E275L280 – do in fact overlap with the reported human IgE epitopes i.e. sequences I34E70 and also V41M172, G301P385. The sequences V41M172, G301P385 and V173M196 contain structures that are very immunogenic in BALB/c mice.
Immunological adjuvants are usually classified into two groups: the first, known as “vehicle” adjuvants, create a depot effect and transport the antigen from the injection site to local lymphoid tissues. The second class of adjuvants, known as immunomodulators, can activate antigen presenting cells (APC) or other immune cells to produce cytokines in a non-antigen specific manner. CFA was capable of inducing allergen-specific antibody of additional specificity compared to dextran sulphate (DS). Orally administered antigens (ovalbumin in our study) will transit through the gastrointestinal compartment, encountering acidic pH and enzymatic activities, before reaching the intestinal mucosa where the antigen is taken up, processed and presented by the underlying immune cells. Such partial degradation or unfolding of the protein may account for the larger number of epitopes generated after oral administration (compared to the parenteral routes).