Washing processes cannot fully remove interfering species that remain on biosensing surfaces when a sample solution contains a high concentration of interfering species. This study reports an immunosensing scheme employing electroreduction-based electrochemical-chemical (EC) redox cycling that allows sensitive detection of vaccinia virus (VV) in a solution containing a high concentration of L-ascorbic acid (AA). To obtain high signal amplification, an enzymatic reaction by β-D-galactosidase (Gal) is combined with electroreduction-based EC redox cycling by an oxidant. Among the four possible oxidants (KIO3, NaClO, Ag2O, and H2O2), KIO3 shows the highest signal-to-background ratio and is chosen. During an incubation period of 10 min, Gal converts β-D-galactopyranoside into p-aminophenol (AP), which is oxidized to p-quinone imine (QI) by KIO3. When -0.05 V vs. Ag/AgCl is applied to an immunosensing electrode, QI is reduced to AP, and the regenerated AP is then reoxidized by KIO3. The electroreduction-based EC redox cycling is induced. An indium-tin oxide electrode modified with reduced graphene oxide and an applied potential of -0.05 V are used to achieve low and reproducible background currents, slow O2 reduction, and fast electroreduction of QI. KIO3 favorably converts AA into noninterfering species during the incubation period. The detection limit for VV in commercial 50% mandarin juice (AA concentration = 0.7 mM) is 4 × 10(3) plaque-forming unit (PFU) per mL. The new EC redox cycling scheme is promising for sensitive detection of proteins, viruses, and bacteria in solutions containing high concentrations of AA.

Although studies investigating the nature of Ab-secreting cells (ASCs) during acute infection with influenza or dengue virus found that the ASC response was dominated by virus-specific IgG secretion, the Ag specificity and phenotype of ASCs during primary acute viral infection were not identified. To this end, we investigated the nature of ASCs in direct ex vivo assays from patients with acute hepatitis A caused by primary infection with hepatitis A virus (HAV). We found that the frequency of CD27(high)CD38(high) ASCs was markedly increased in the peripheral blood during the acute phase of HAV infection. Moreover, substantial numbers of ASCs were non-HAV-specific and dominantly secreted IgM. We detected HAV-specific ASCs by staining with fluorochrome-tagged HAV-VP1 protein. As compared with HAV-specific ASCs, non-HAV-specific ASCs were Ki-67(low)CD138(high)CD31(high)CD38(high), demonstrating that non-HAV-specific ASCs had a bone marrow plasma cell-like phenotype whereas HAV-specific ASCs had a phenotype typical of circulating plasmablasts. These data suggest that non-HAV-specific ASCs might be mobilized plasma cells from the bone marrow or the spleen, whereas HAV-specific ASCs were newly generated plasmablasts. In this study, we provide evidence that pre-existing plasma cells are released into the circulation and contribute to Ag-nonspecific secretion of IgM during primary HAV infection.

Genital papilloma is caused by human papilloma virus (HPV) infection and recurs frequently. Although T cells are known to play a critical role in the control of HPV infection and papilloma development, the function and phenotype of these cells in the lesion remain to be elucidated. In the present study, we examined the function and phenotype of CD4(+) T cells isolated from the lesions of primary (n = 9) and recurrent (n = 11) genital papillomas. In recurrent papillomas, the frequency of proliferating (Ki-67(+)) CD4(+) T cells was significantly reduced compared with primary papillomas. Cytokine production was evaluated by intracellular cytokine staining in anti-CD3/anti-CD28-stimulated CD4(+) T cells. CD4(+) T cells from recurrent lesions showed impaired production of IL-2, IFN-γ, and TNF-α. Of interest, the frequency of cytokine-producing CD4(+) T cells significantly correlated with the frequency of Ki-67(+)CD4(+) T cells. We also studied expression of programmed death-1 (PD-1), a T-cell exhaustion marker. The frequency of PD-1(+)CD4(+) T cells was significantly increased in recurrent lesions and inversely correlated with the frequency of cytokine-producing CD4(+) T cells. The functional significance of PD-1 expression was determined in blocking assays with anti-PD-L1, which restored cytokine production of CD4(+) T cells from recurrent lesions. Taken together, in recurrent genital papilloma lesions, proliferation, and cytokine production by CD4(+) T cells are impaired and the PD-1/PD-L1 interaction is responsible for the functional impairment of CD4(+) T cells.

PurposeT cell-mediated immune responses, and particularly activation of polyfunctional T cells that simultaneously produce multiple cytokines, are necessary for the control of Mycobacterium tuberculosis. In the present study, we examined if DNA immunization of Mycobacterium tuberculosis resuscitation-promoting factor B (RpfB) elicits polyfunctional T cell responses in mice.Materials and MethodsC57BL/6 mice were immunized intramuscularly three times, at 3-week intervals, with RpfB-expressing plasmid DNA. For comparison, protein immunization was performed with recombinant RpfB in control mice. After immunization, RpfB-specific T cell responses were assessed by interferon-γ (IFN-γ) enzyme-linked immunosorbent spot assay and intracellular cytokine staining (ICS), and T cell polyfunctionality was assessed from the ICS data.ResultsRpfB DNA immunization induced not only humoral immune responses, but also CD8+ and CD4+ T cell responses. Immunodominant T-cell epitopes were identified within RpfB by assays with overlapping peptides. RpfB DNA immunization elicited a polyfunctional CD8+ T cell response that was dominated by a functional phenotype of IFN-γ+/TNF-α+/IL-2-/CD107a+.ConclusionRpfB DNA immunization elicits polyfunctional CD8+ T cell responses, suggesting that RpfB DNA immunization might induce protective immunity against tuberculosis.