Rapid, sensitive, and specific lateral-flow immunochromatographic point-of-care device for detection of varicella-zoster virus immunoglobulin G antibodies in fingerstick blood.

Varicella zoster virus (VZV) causes childhood chickenpox, becomes latent in sensory ganglia and reactivates years later to cause shingles (Zoster) and postherpetic neuralgia in the elderly and immunosuppressed individuals. Serologic IgG tests can be used to determine if a person has antibodies to VZV from past varicella infection or had received varicella or zoster (shingles) vaccination. Commercial enzyme-linked immunosorbent assays (ELISAs) are currently used for the detection of VZV IgG antibodies in patient serum samples. However, ELISA tests require collection and processing of blood samples in a CLIA laboratory to separate serum or plasma for further testing. In this paper, we describe the development and testing of an antibody based Lateral Flow Immunochromatographic assay (LFA) device for the detection of VZV IgG in fingerstick whole blood. Analytical and clinical analyses were performed to compare the performance characteristics of the Viro VZV IgG LFA (VZV LFA) and the Diamedix VZV IgG ELISA. Analytical studies demonstrated the higher sensitivity of the VZV LFA compared to the ELISA by testing dilutions of the WHO VZV IgG serum International Standard. Clinical performance characteristics of the VZV LFA fingerstick whole blood assay were assessed at three point of care (POC) facilities by untrained users testing samples from 300 prospectively enrolled study subjects. VZV LFA results were compared with results obtained by testing serum samples obtained from the same study participants by the Diamedix VZV IgG ELISA. Two specimens with invalid results by the LFA assay were not included in the LFA performance calculations and nine equivocal ELISA results were included as positive for IgG results. The results from all three POC clinical sites demonstrated the higher sensitivity/positive percent agreement (PPA) (99.26%, 95% CI: 97.34-99.80) of the VZV LFA compared to the Diamedix VZV IgG ELISA (94.08%, 95% CI: 90.72-96.27). The specificity/negative percent agreement (NPA) of the VZV LFA compared to the ELISA test was calculated initially to be 39.29% (95% CI: 23.57-57.59) with 19 discordant test results out of 298 test results between the two assays (17 LFA positive/ELISA negative and two LFA negative/ELISA positive). The PPA and true NPA of the VZV LFA were determined by testing all 298 samples, including the discordant (19) and all concordant negative and positive (279) study subject serum samples, before and after blocking VZV gE antibody sites in the samples by spiking with VZV LFA gE capture antigen. The NPA improved to 100% (95% CI: 74.12-100) after the procedure when compared to the ELISA test results. The comparator ELISA PPA based on the spiking/blocking study remained as 94.08%, (95% CI: 90.72-96.27), comparable to test results from untreated samples. The VZV LFA has been demonstrated to be simple and sufficiently robust for use in CLIA-waived POC facilities by untrained healthcare professionals and to detect VZV IgG in 20 min from fingerstick whole blood. The VZV LFA therefore provides a fast, reliable, and highly sensitive method of determining prior VZV viral infection or varicella and zoster vaccination status.

T cells control the generation of nanomolar-affinity anti-glycan antibodies.

Vaccines targeting glycan structures at the surface of pathogenic microbes must overcome the inherent T cell-independent nature of immune responses against glycans. Carbohydrate conjugate vaccines achieve this by coupling bacterial polysaccharides to a carrier protein that recruits heterologous CD4 T cells to help B cell maturation. Yet they most often produce low- to medium-affinity immune responses of limited duration in immunologically fit individuals and disappointing results in the elderly and immunocompromised patients. Here, we hypothesized that these limitations result from suboptimal T cell help. To produce the next generation of more efficacious conjugate vaccines, we have explored a synthetic design aimed at focusing both B cell and T cell recognition to a single short glycan displayed at the surface of a virus-like particle. We tested and established the proof of concept of this approach for 2 serotypes of Streptococcus pneumoniae. In both cases, these vaccines elicited serotype-specific, protective, and long-lasting IgG antibodies of nanomolar affinity against the target glycans in mice. We further identified a requirement for CD4 T cells in the anti-glycan antibody response. Our findings establish the design principles for improved glycan conjugate vaccines. We surmise that the same approach can be used for any microbial glycan of interest.

The identification of the endogenous ligands of natural killer T cells reveals the presence of mammalian α-linked glycosylceramides.

Glycosylceramides in mammalian species are thought to be present in the form of ß-anomers. This conclusion was reinforced by the identification of only one glucosylceramide and one galactosylceramide synthase, both ß-transferases, in mammalian genomes. Thus, the possibility that small amounts of α-anomers could be produced by an alternative enzymatic pathway, by an unfaithful enzyme, or spontaneously in unusual cellular compartments has not been examined in detail. We approached the question by taking advantage of the exquisite specificity of T and B lymphocytes and combined it with the specificity of catabolic enzymes of the sphingolipid pathway. Here, we demonstrate that mammalian immune cells produce constitutively very small quantities of α-glycosylceramides, which are the major endogenous ligands of natural killer T cells. Catabolic enzymes of the ceramide and glycolipid pathway tightly control the amount of these α-glycosylceramides. The exploitation of this pathway to manipulate the immune response will create new therapeutic opportunities.