Novel LysM motifs for antigen display on lactobacilli for mucosal immunization.

We characterized two LysM domains of Limosilactobacillus fermentum, belonging to proteins Acglu (GenBank: KPH22907.1) and Pgb (GenBank: KPH22047.1) and bacterium like particles (BLP) derived from the immunomodulatory strain Lacticaseibacillus rhamnosus IBL027 (BLPs027) as an antigen display platform. The fluorescence protein Venus fused to the novel LysM domains could bind to the peptidoglycan shell of lactobacilli and resisted harsh conditions such as high NaCl and urea concentrations. Acglu with five LysM domains was a better anchor than Pgb baring only one domain. Six-week-old BALB/c mice were nasally immunized with the complex Venus-Acglu-BLPs027 at days 0, 14 and 28. The levels of specific serum IgG, IgG1 and IgG2a and the levels of total immunoglobulins (IgT) and IgA in broncho-alveolar lavage (BAL) were evaluated ten days after the last boosting. Venus-Acglu-BLPs027, nasally administered, significantly increased specific BAL IgT and IgA, and serum IgG levels. In addition, spleen cells of mice immunized with Venus-Acglu-BLPs027 secreted TNF-α, IFN-γ and IL-4 when stimulated ex vivo in a dose-dependent manner. We constructed a Gateway compatible destination vector to easily fuse the selected LysM domain to proteins of interest for antigen display to develop mucosal subunit vaccines.

A Novel In-House Enzyme-Linked Immunosorbent Assay for Genotype 3 Hepatitis E Virus Reveals High Seroprevalence in Blood Donors in Northern Argentina.

The Hepatitis E virus (HEV) is an emergent virus that causes acute hepatitis in immunocompetent hosts and chronic hepatitis in immunocompromised hosts. In Latin America, the main circulating genotype HEV-3 is usually of zoonotic origin. Diagnosis and seroprevalence studies mainly rely on the detection of specific antibodies. There are scarce data on the seroprevalence of HEV infection in Latin America mainly due to the lack of awareness of HEV circulation. Furthermore, in some countries, like Argentina, HEV testing is not included in routine assays. In order to provide tools to deepen the knowledge on HEV epidemiology in South America, we designed a new in-house ELISA based on the native recombinant protein ORF2 aa112-608 and demonstrated its potential for detecting anti-HEV immunoglobulin G (IgG) in human serum samples. The following conditions were determined: an optimal antigen concentration of 0.25 µg/ml, a serum dilution of 1:80, gelatin as a blocking agent, and a secondary antibody dilution of 1:2000. A relative sensitivity of 93.33% (95% CI: 77.9-99.2%) and a relative specificity of 99.4% (95% CI: 96.7-100%) were determined using a panel of previously characterized sera and a gold standard (HEV IgG ELISA, DIA.PRO, Italy). Further, we obtained a very good agreement (κ index = 0.94, 95% CI: 0.87-1.00) with the gold standard. We screened 813 blood donor samples with this newly developed ELISA and found a seroprevalence of 9.23% (95% confidence interval, 7.33-11.43%). We show for the first time evidence of past HEV infection in Tucuman, the most populated city in northern Argentina. We expect that this study will raise the interest of health decision makers who should intercede to include indirect testing of HEV in regular diagnostic protocols. In conclusion, the in-house ELISA developed in this work shows a very good agreement with an already licensed commercial HEV IgG ELISA (DIA.PRO, ITALY), which can be used as an epidemiologic tool for HEV surveillance.