Induction Of Primary Antibody Response Research Articles

Flagellin’s hypervariable D2/D3 domain is required for robust anti-flagellin primary antibody responses (VAC11P.1065)

Abstract Bacterial flagellin is a well-known agonist of the innate immune system. Several clinical trials investigating flagellin fusion proteins have demonstrated promising results for inducing protective immunity towards influenza virus, which has been largely attributed to flagellin activation of TLR5. Our lab recently demonstrated that the Salmonella enterica serovar Typhimurium flagellin protein, FliC, induces antibody responses in mice through a third pathway that is independent of TLR5, Casp1, and MyD88. Herein, we define the structural features of FliC that contribute to this third pathway. By destroying the Naip5/6 (D0 domain) or TLR5 (D1 domain) recognition sites, we demonstrate that neither affected the TLR5-, inflammasome- and MyD88-independent antibody responses towards FliC. In contrast, deletion of the D2/D3 domain eliminated primary anti-flagellin antibody responses. Optimal primary and secondary anti-flagellin antibody responses required TLR5- and inflammasome-mediated recognition, and FliC’s D2/D3 domain. We demonstrate FliC’s D2/D3 domain is required for induction of primary antibody responses and robust secondary antibody responses. Our results indicate that FliC’s D2/D3 domain is essential for flagellin’s robust immunogenicity and suggest that the D2/D3 domain interacts with the host immune system to augment antibody production. Thus, the D2/D3 domain of FliC may also be critical for optimal immunogenicity and antibody responses in flagellin based vaccines.

In vitro Induction of primary antibody responses to particulate and soluble protein antigens in T cell-replaced murine spleen cell cultures

Specific antibody responses could be induced in serum, free condition. Specific anti-SRBC or anti-SRBC ghost antibody were induced from anti-Thy treated (T-depleted) murine spleen cells in serum-free culture in the presence of Con A conditioned medium. This induction system may facilitate the study of lymphokine functions on antigen triggered B cells.In T cell-replaced cultures, the antibody responses of B cells could be successfully induced when soluble SRBC membrane proteins were used as antigens. It thus indicates that antigen together with lymphokines are sufficient to drive B cells to become antibody secreting cells in the absence of T cells. The T cell-replaced system provides a more stable way for in vitro immunization and may be applied to monoclonal antibody production when in vivo immunization is difficult to be carried out.

In vitro induction of specific immune responses in the absence of serum: requirement for nonspecific T or B cell mitogens.

AbstractA mixed lymphocyte culture reaction (MLC) between histoincompatible mouse lymphocytes could be induced in the absence of serum in vitro. There was a steady increase of DNA synthesis for 7 days in the absence of serum, whereas peak activity occurred on day 4 in the presence of serum. Analogous results were obtained when the secondary responses to SRC were determined by stimulation of DNA synthesis.It was not possible to induce a primary humoral antibody response to SRC in the absence of serum. However, if the serum‐free cultures were supplemented with optimal concentrations of T (concanavalin A) or B (lipopolysaccharide, LPS) cell mitogens, a primary specific antibody response to sheep red cells (SRC) could be induced and increased in magnitude for 4 days in culture. Also, a MLC reaction permitted the induction of primary antibody responses to SRC. In the presence of fetal calf serum (FCS), optimal concentrations of T cell mitogens, as well as a MLC reaction, suppressed the induction of a primary antibody response to SRC, whereas suboptimal doses of the mitogen stimulated it.It was possible to induce specific secondary IgM and IgG antibody responses to SRC in the absence of serum.In contrast to the findings with SRC, a primary – as well as secondary – antibody response could be induced to LPS, which is a relatively thymus‐independent antigen.Taken together the results suggest that:a) FCS is necessary for the induction of primary antibody responses, because it contains B and possibly T cell mitogens.b) Primary antibody responses to SRC can be induced in the absence of FCS if nonspecific B cell activation is provided by the addition of either B cell mitogens or B cell activating factors, obtained by activating T cells by specific mitogens or by a MLC reaction.c) Secondary responses to T cell‐dependent antigens are possible in the absence of FCS because of the large number of specifically activated T cells present in the culture system.d) Primary antibody responses to LPS are possible in the absence of FCS because LPS by itself is a B cell mitogen.

DNA replication as a prerequisite for the induction of primary antibody response

Studies were carried out to test whether the induction of a primary production of antibodies is determined by DNA replication. An in vitro system for the induction of antibody to dinitrophenyl (DNP) determinants following immunization with α-DNP-poly- l-lysine was employed. Cytosine arabinoside (CA), at doses which inhibited DNA synthesis, yet did not affect viability of cells, was applied at different time intervals following antigenic stimulation. Inhibition of DNA synthesis during 36–48 hr following antigenic stimulation prevented the appearance of antibody producing cells. On the other hand, CA applied during the first 36 hr or at 48 hr did not prevent antibody production. Thus, one critical cycle of DNA replication seems to determine antibody production. The lag period prior to the CA-sensitive period is antigen determined. Spleens of animals which were preimmunized with rabbit serum albumin (RSA), then stimulated in vitro with DNP-RSA, showed the same pattern of sensitivity to CA. It is proposed that one cycle of DNA replication is essential for the differentiation of precursor B cells to antibody producing cells. We have previously found that one critical replication determines the carrier effect. A model of the replicating event of T and B cells during the induction of an immune response to hapten-carrier conjugates is suggested.