Stability studies of binding and functional anti-vaccine antibodies.

BACKGROUND: As most vaccines exert their protective capacity by eliciting pathogen-specific antibodies, antibody assays assessing immunogenicity of vaccines in development should be well characterized. Part of the validation of immunogenicity assays for vaccines is the study of stability of antibodies in serum. Materials & methods: Stability of antibodies in human serum was assessed by circumsporozoite-binding IgG ELISA designed for assessing the immunogenicity of a malaria vaccine under development, adenovirus neutralization assay, designed to assess neutralizing antibodies against adenovirus and commercially available test kits for hepatitis A and B. RESULTS: Stability studies indicated stability of serum-binding IgG antibodies and serum-neutralizing antibodies in: long-term storage below -65°C and -20°C; short-term storage; multiple freeze/thaw rounds; during shipment; and during heat inactivation. CONCLUSION: RESULTS have shown the stability of both binding and functional polyclonal antibodies in human serum under stable storage and common usage circumstances.

A peptide-based Plasmodium falciparum circumsporozoite assay to test for serum antibody responses to pre-erythrocyte malaria vaccines.

Various pre-erythrocyte malaria vaccines are currently in clinical development, and among these is the adenovirus serotype 35-based circumsporozoite (CS) vaccine produced on PER.C6 cells. Although the immunological correlate of protection against malaria remains to be established, the CS antibody titer is a good marker for evaluation of candidate vaccines. Here we describe the validation of an anti-Plasmodium falciparum circumsporozoite antibody enzyme-linked immunosorbent assay (ELISA) based on the binding of antibodies to a peptide antigen mimicking the CS repeat region. The interassay variability was determined to be below a coefficient of variation (CV) of 15%, and sensitivity was sufficient to detect low antibody titers in subjects from endemic regions. Antibody titers were in agreement with total antibody responses to the whole CS protein. Due to its simplicity and high performance, the ELISA is an easy and rapid method for assessment of pre-erythrocyte malaria vaccines based on CS.

Immunological and antitumor effects of IL-23 as a cancer vaccine adjuvant.

The promising, but modest, clinical results of many human cancer vaccines indicate a need for vaccine adjuvants that can increase both the quantity and the quality of vaccine-induced, tumor-specific T cells. In this study we tested the immunological and antitumor effects of the proinflammatory cytokine, IL-23, in gp100 peptide vaccine therapy of established murine melanoma. Neither systemic nor local IL-23 alone had any impact on tumor growth or tumor-specific T cell numbers. Upon specific vaccination, however, systemic IL-23 greatly increased the relative and absolute numbers of vaccine-induced CD8(+) T cells and enhanced their effector function at the tumor site. Although IL-23 specifically increased IFN-gamma production by tumor-specific T cells, IFN-gamma itself was not a primary mediator of the vaccine adjuvant effect. The IL-23-induced antitumor effect and accompanying reversible weight loss were both partially mediated by TNF-alpha. In contrast, local expression of IL-23 at the tumor site maintained antitumor activity in the absence of weight loss. Under these conditions, it was also clear that enhanced effector function of vaccine-induced CD8(+) T cells, rather than increased T cell number, is a primary mechanism underlying the antitumor effect of IL-23. Collectively, these results suggest that IL-23 is a potent vaccine adjuvant for the induction of therapeutic, tumor-specific CD8(+) T cell responses.

The impact of self-tolerance on the polyclonal CD8+ T cell repertoire.

TCRs possess considerable cross-reactivity toward structurally related Ags. Because the signaling threshold for negative selection is lower than that required for activation of mature T cells, the question arises as to which extent thymic deletion of self-specific T cells affects T cell responsiveness toward foreign peptides. In this study we show, in three different mouse models systems, that the polyclonal CD8(+) T cell repertoire has a marked ability to react against the majority of Ags related to self despite self-tolerance, even in cases where self and foreign differ only marginally at a single TCR-contact residue. Thus, while individual T cells are markedly cross-reactive, the ability to distinguish between closely related Ags is introduced at the polyclonal T cell level.

Can the low-avidity self-specific T cell repertoire be exploited for tumor rejection?

Can self-specific T cells that have escaped intrathymic deletion be exploited to generate antitumor immunity? To determine whether antitumor immunity to a self-Ag for which central tolerance exists can be generated, a mouse model is used in which a fragment of the influenza nucleoprotein (NP) is expressed as a transgene under the control of the H-2K promoter in C57BL/10 mice (B10NP mice). In these mice an oligoclonal population of NP-specific T cells escapes thymic and peripheral deletion and can be activated upon immunization. The main hallmark of these self-specific CD8(+) T cells is diminished avidity for the pertinent MHC/peptide complex. We show in this study that intranasal infection with influenza virus can stimulate low-avidity NP-specific T cells to recognize and destroy NP-expressing microtumors in the lung, but not NP-expressing tumors growing s.c. Only a memory NP-specific CD8(+) T cell response can suppress the growth of an s.c. growing NP-expressing tumor. This delay in tumor growth is associated with a dramatic increase in the number of circulating NP-specific CD8(+) T cells. In addition, cultured memory NP-specific T cells require approximately 100-fold less Ag to induce NP-specific lysis than primary T cells, consistent with the observation that memory T cells have an increased avidity due to affinity maturation. Finally, during an NP-specific memory response, substantial numbers of low-avidity NP-specific T cells can be recovered from s.c. growing tumors. Together, these findings indicate that, when only a low-avidity repertoire is available to generate antitumor immunity, the best strategy may be to enhance memory responses.