Comparison of ELISA with electro-chemiluminescence technology for the qualitative and quantitative assessment of serological responses to vaccination.

BACKGROUND: Profiling immune responses induced by either infection or vaccination can provide insight into identification of correlates of protection. Furthermore, profiling of serological responses can be used to identify biomarkers indicative of exposure to pathogens. Conducting such immune surveillance requires readout methods that are high-throughput, robust, and require small sample volumes. While the enzyme-linked immunosorbent assay (ELISA) is the classical readout method for assessing serological responses, the advent of multiplex assays has significantly increased the throughput and capacity for immunoprofiling. This report describes the development and assay performance (sensitivity, linearity of detection, requirement for multiple dilutions for each sample, intra- and inter-assay variability) of an electro-chemiluminescence (ECLIA)-based multiplex assay. METHODS: The current study describes the development of a multiplex ECLIA-based assay and characterizes the sensitivity, linear range, and inter- and intra-assay variability of the ECLIA platform and its agreement with the traditional ELISA. Special emphasis was placed on potential antigenic competition when testing closely related antigens in the multiplex format. RESULTS: Multiplexing of antigens in ECLIA provides significant practical benefits in terms of reducing sample volume requirements and experimental time. Beyond the practical advantages of multiplexing, the ECLIA provides superior assay performance when compared to the ELISA. Not only does ECLIA show good agreement with the ELISA assay, but the linear range of ECLIA is also sufficiently wide to permit single-dilution measurements of concentration without the need to do serial dilutions. The lack of antigenic competition allows the simultaneous testing of closely related antigens, such as plate antigens representing different alleles of the same protein, which can inform about cross-reactivities-or lack thereof-of serological responses. CONCLUSION: The advantages of the newly developed tool for assessing the antigen profiles of serological responses may ultimately lead to the identification of biomarkers associated with various disease stages and or protection against disease.

Proteomic characterization and functional analysis of outer membrane vesicles of Francisella novicida suggests possible role in virulence and use as a vaccine.

We have isolated and characterized outer membrane vesicles (OMVs) from Francisella. Transport of effector molecules through secretion systems is a major mechanism by which Francisella tularensis alters the extracellular proteome and interacts with the host during infection. Outer membrane vesicles produced by Francisella were examined using TEM and AFM and found to be 43-125 nm in size, representing another potential mechanism for altering the extracellular environment. A proteomic analysis (LC-MS/MS) of OMVs from F. novicida and F. philomiragia identified 416 (F. novicida) and 238 (F. philomiragia) different proteins, demonstrating that OMVs are an important contributor to the extracellular proteome. Many of the identified OMV proteins have a demonstrated role in Francisella pathogenesis. Biochemical assays demonstrated that Francisella OMVs possess acid phosphatase and hemolytic activities that may affect host cells during infection, and are cytotoxic toward murine macrophages in cell culture. OMVs have been previously used as a human vaccine against Neisseria meningitidis . We hypothesized that Francisella OMVs could be useful as a novel Francisella vaccine. Vaccinated BALB/C mice challenged with up to 50 LD50 of Francisella showed statistically significant protection when compared to control mice. In the context of these new findings, we discuss the relevance of OMVs in Francisella pathogenesis as well as their potential use as a vaccine.

Breadth of humoral immune responses to the C-terminus of the circumsporozoite protein is associated with protective efficacy induced by the RTS,S malaria vaccine.

The circumsporozoite protein (CSP) is the main surface antigen of malaria sporozoites, a prime vaccine target, and is known to have polymorphisms in the C-terminal region. Vaccines using a single allele may have lower efficacy against genotypic variants. Recent studies have found evidence suggesting the efficacy of the CSP-based RTS,S malaria vaccine may be limited against P. falciparum CSP alleles that diverge from the 3D7 vaccine allele, particularly in this polymorphic C-terminal region. In order to assess the breadth of the RTS,S-induced antibody responses against CSP C-terminal antigenic variants, we used a novel multiplex assay to measure reactivity of serum samples from a recent RTS,S study against C-terminal peptides from 3D7 and seven additional CSP alleles that broadly represent the genetic diversity found in circulating P. falciparum field isolates. We found that responses to the variants showed, on average, a ~ 30-fold reduction in reactivity relative to the vaccine-matched 3D7 allele. The extent of this reduction, ranging from 21 to 69-fold, correlated with the number of polymorphisms between the variants and 3D7. We calculated antibody breadth of each sample as the median relative reactivity to the seven CSP variants compared to 3D7. Surprisingly, protection from 3D7 challenge in the RTS,S study was associated with higher C-terminal antibody breadth. These findings suggest CSP C-terminal-specific avidity or fine-specificity may play a role in RTS,S-mediated protection and that breadth of C-terminal CSP-specific antibody responses may be a marker of protection.

Role of Opsonophagocytosis in Immune Protection against Malaria.

The quest for immune correlates of protection continues to slow vaccine development. To date, only vaccine-induced antibodies have been confirmed as direct immune correlates of protection against a plethora of pathogens. Vaccine immunologists, however, have learned through extensive characterizations of humoral responses that the quantitative assessment of antibody responses alone often fails to correlate with protective immunity or vaccine efficacy. Despite these limitations, the simple measurement of post-vaccination antibody titers remains the most widely used approaches for vaccine evaluation. Developing and performing functional assays to assess the biological activity of pathogen-specific responses continues to gain momentum; integrating serological assessments with functional data will ultimately result in the identification of mechanisms that contribute to protective immunity and will guide vaccine development. One of these functional readouts is phagocytosis of antigenic material tagged by immune molecules such as antibodies and/or complement components. This review summarizes our current understanding of how phagocytosis contributes to immune defense against pathogens, the pathways involved, and defense mechanisms that pathogens have evolved to deal with the threat of phagocytic removal and destruction of pathogens.

Cigarette smoke extract induces differential expression levels of beta-defensin peptides in human alveolar epithelial cells.

BACKGROUND: The damaging effects of cigarette smoke on the lungs are well known in terms of cancer risks. Additional molecular changes within the lung tissue can also occur as a result of exposure to cigarette smoke. The human ß-defensin (hBD) class of antimicrobial peptides is the focus of our research. In addition to antimicrobial activity, ß-defensins also have immunomodulatory functions. Over 30 previously unrecognized ß-defensin genes have recently been identified in the human genome, many with yet to be determined functions. We postulated that altered ß-defensin production may play a role in the pathogenesis observed in the lungs of smokers. Our hypothesis is that cigarette smoke exposure will affect the expression of ß-defensins in human lung alveolar epithelial cells (A549). METHODS: We exposed A549 cells to cigarette smoke extract (CSE) and measured the changes in mRNA levels of several antimicrobial peptides by quantitative real-time PCR, and directly observed peptide expression in cells by immunofluorescence (IF) microscopy. RESULTS: We found that hBD3, hBD5, and hBD9 gene expression was upregulated in A549 cells exposed to CSE. HBD1, hBD8, hBD18 and LL-37 gene expression did not significantly change upon exposure to CSE. Expression of hBD3 and hBD4 peptides was visualized by IF. CONCLUSIONS: This differential expression suggests that hBD3, hBD5, and hBD9 may play a role in the changes to the lung tissue observed in smokers. Establishing differential ß-defensin expression following CSE treatment will add to our understanding of the molecular response of the lung alveolar epithelium to cigarette smoke exposure.

Francisella philomiragia biofilm formation and interaction with the aquatic protist Acanthamoeba castellanii.

The bacterium Francisella philomiragia has been isolated from environmental samples originating from around the globe. F. philomiragia-related strains cause francisellosis of both farmed and wild fish. In addition, occasional human infections caused by F. philomiragia are found in victims of near-drowning and patients with chronic granulomatous disease. We have shown that F. philomiragia forms in vitro biofilms with increased formation at 25 °C over 37 °C conditions. We found that F. philomiragia can form a biofilm in a co-culture with live Acanthamoeba castellanii, an aquatic amoeba. Interestingly, amoeba-conditioned supernatant has an inhibitory effect on production of biofilm by F. philomiragia, whereas Francisella-conditioned supernatant has no effect on growth of amoebae. We have shown that F. philomiragia can infect A. castellanii after only 5 days of co-incubation and that it infects A. castellanii more quickly than the related species F. novicida does. Our studies point to a potentially overlooked interaction between F. philomiragia and Acanthamoeba. This relationship in the marine lifecycle of F. philomiragia may support the persistence of the bacterium in waterways and its ability to infect fish. An understanding of the persistence of this organism in aquatic systems through biofilm formation and its interaction with Acanthamoeba will be important in developing prevention strategies for this pathogen.