Immunity to viruses: learning from successful human vaccines.

For more than a century, immunologists and vaccinologists have existed in parallel universes. Immunologists have for long reveled in using 'model antigens', such as chicken egg ovalbumin or nitrophenyl haptens, to study immune responses in model organisms such as mice. Such studies have yielded many seminal insights about the mechanisms of immune regulation, but their relevance to humans has been questioned. In another universe, vaccinologists have relied on human clinical trials to assess vaccine efficacy, but have done little to take advantage of such trials for studying the nature of immune responses to vaccination. The human model provides a nexus between these two universes, and recent studies have begun to use this model to study the molecular profile of innate and adaptive responses to vaccination. Such 'systems vaccinology' studies are beginning to provide mechanistic insights about innate and adaptive immunity in humans. Here, we present an overview of such studies, with particular examples from studies with the yellow fever and the seasonal influenza vaccines. Vaccination with the yellow fever vaccine causes a systemic acute viral infection and thus provides an attractive model to study innate and adaptive responses to a primary viral challenge. Vaccination with the live attenuated influenza vaccine causes a localized acute viral infection in mucosal tissues and induces a recall response, since most vaccinees have had prior exposure to influenza, and thus provides a unique opportunity to study innate and antigen-specific memory responses in mucosal tissues and in the blood. Vaccination with the inactivated influenza vaccine offers a model to study immune responses to an inactivated immunogen. Studies with these and other vaccines are beginning to reunite the estranged fields of immunology and vaccinology, yielding unexpected insights about mechanisms of viral immunity. Vaccines that have been proven to be of immense benefit in saving lives offer us a new fringe benefit: lessons in viral immunology.

Systems biological approaches to measure and understand vaccine immunity in humans.

Recent studies have demonstrated the utility of using systems approaches to identify molecular signatures that can be used to predict vaccine immunity in humans. Such approaches are now being used extensively in vaccinology, and are beginning to yield novel insights about the molecular networks driving vaccine immunity. In this review, we present a broad review of the methodologies involved in these studies, and discuss the promise and challenges involved in this emerging field of "systems vaccinology."

Estrogen-related receptor alpha is critical for the growth of estrogen receptor-negative breast cancer.

Expression of estrogen-related receptor alpha (ERRalpha) has recently been shown to carry negative prognostic significance in breast and ovarian cancers. The specific role of this orphan nuclear receptor in tumor growth and progression, however, is yet to be fully understood. The significant homology between estrogen receptor alpha (ERalpha) and ERRalpha initially suggested that these receptors may have similar transcriptional targets. Using the well-characterized ERalpha-positive MCF-7 breast cancer cell line, we sought to gain a genome-wide picture of ERalpha-ERRalpha cross-talk using an unbiased microarray approach. In addition to generating a host of novel ERRalpha target genes, this study yielded the surprising result that most ERRalpha-regulated genes are unrelated to estrogen signaling. The relatively small number of genes regulated by both ERalpha and ERRalpha led us to expand our study to the more aggressive and less clinically treatable ERalpha-negative class of breast cancers. In this setting, we found that ERRalpha expression is required for the basal level of expression of many known and novel ERRalpha target genes. Introduction of a small interfering RNA directed to ERRalpha into the highly aggressive breast carcinoma MDA-MB-231 cell line dramatically reduced the migratory potential of these cells. Although stable knockdown of ERRalpha expression in MDA-MB-231 cells had no effect on in vitro cell proliferation, a significant reduction of tumor growth rate was observed when these cells were implanted as xenografts. Our results confirm a role for ERRalpha in breast cancer growth and highlight it as a potential therapeutic target for estrogen receptor-negative breast cancer.

Tumor shedding of laminin binding protein modulates angiostatin production in vitro and interferes with plasmin-derived inhibition of angiogenesis in aortic ring cultures.

The growth of solid tumors is largely controlled by the process of angiogenesis. A 67 kDa protein, the laminin binding protein (LBP), is shed from malignant cells in significant amounts and binds to laminin-1 (Starkey et al., Cytometry 1999;35:37-47; Karpatová et al., J Cell Biochem 1996;60:226-34). However, the functions of shed LBP are not fully understood. We hypothesize that matrix-bound LBP could modulate local tumor angiogenesis. In support of this hypothesis, we demonstrate that shed LBP exhibits sulfhydryl oxidase-like activities, and modifies the production of angiostatins from plasmin in vitro. The molecular weights of the autocatalytic products of lys-plasmin incubated with LBP in vitro suggest that PMDs (plasmin A chains attached to degraded B chains) (Ohyama et al., Eur J Biochem 2004;271:809-20) are preferentially generated. Using rat aortic ring assays, we also show that shed LBP reverses plasmin-dependent inhibition of vascular outgrowth. To elucidate which LBP region(s) are active in modulating angiogenesis, limited proteolysis experiments were conducted to determine stable rLBP domains likely to fold correctly, and these were cloned, expressed and purified. The stable LBP fragments were tested for binding to laminin-1 and for competition with shed LBP activity in the aortic ring assay. Results of these studies suggest that the active LBP domains lie within the 137-230 amino acid sequence, a region known to contain 2 bioactive sequences. Since this fragment binds to laminin-1 and modulates angiogenesis, it appears likely that binding of shed LBP to matrix laminin-1 is related to its functions in tumor angiogenesis. The findings presented in this manuscript suggest that LBP shedding could provide a useful therapeutic target.

Comparative modeling of the N-terminal domain of the 67kDa laminin-binding protein: implications for putative ribosomal function.

Laminin-binding protein/p40 (LBP/p40) precursor appears to be involved in two seemingly unrelated activities-cell adhesion and ribosomal biogenesis. Analysis of primary structure revealed a two-domain organization of the LBP/p40. The N-terminal portion of LBP is similar to the S2 family of prokaryotic ribosomal proteins, while the C-terminus is unique for Metazoa and is involved in extraribosomal functions. To gain insight into putative ribosomal functions of LBP we performed comparative modeling of the N-terminal domain using crystal structures of S2p from Thermus thermophilus. The LBP model assumes an alpha-beta sandwich fold similar to that of S2. Modeling revealed the loss of a significant portion of ribosomal RNA (rRNA) interaction domain, lack of conservation of many residues involved in interactions with rRNA, and a major shift in surface charge distribution (compared to the S2 protein). The overall stability of the fold argues against a proposed transmembrane domain in the central part of the protein. Partial overlap in S2 and laminin-binding domains suggests that ribosomal and surface receptor functions would be mutually exclusive. The possible biological role of LBP/p40 bifunctionality is discussed.