Additional file 3: of A vaccine consisting of Schistosoma mansoni cathepsin B formulated in Montanide ISA 720 VG induces high level protection against murine schistosomiasis

Parasite burden reduction in experimental mice compared to adjuvant control mice. The experimental mice were immunized with a formulation of 20 μg Sm-cathepsin B + Montanide ISA 720 VG. Statistically significant reductions in all forms of parasite burden were observed in the experimental mice compared to the adjuvant control mice (mice that were immunized with Montanide ISA 720 VG alone). (PDF 128 kb

Data_Sheet_1_Immune Mechanisms Involved in Schistosoma mansoni-Cathepsin B Vaccine Induced Protection in Mice.PDF

<p>A vaccine against schistosomiasis would contribute to a long-lasting decrease in disease spectrum and transmission. Our previous protection studies in mice using Schistosoma mansoni Cathepsin B (Sm-Cathepsin B) resulted in 59 and 60% worm burden reduction with CpG oligodeoxynucleotides and Montanide ISA720 VG as adjuvants, respectively. While both formulations resulted in significant protection in a mouse model of schistosomiasis, the elicited immune responses differed. Therefore, in this study, we aimed to decipher the mechanisms involved in Sm-Cathepsin B vaccine-mediated protection. We performed in vitro killing assays using schistosomula stage parasites as targets for lung-derived leukocytes and serum obtained from mice immunized with Sm-Cathepsin B adjuvanted with either Montanide ISA 720 VG or CpG and from non-vaccinated controls. Lung cells and immune sera from the Sm-Cathepsin B + Montanide group induced the highest killing (63%) suggesting the importance of antibodies in cell-mediated parasite killing. By contrast, incubation with lung cells from Sm-Cathepsin B + CpG immunized animals induced significant parasite killing (53%) independent of the addition of immune serum. Significant parasite killing was also observed in the animals immunized with Sm-Cathepsin B alone (41%). For the Sm-Cathepsin B + Montanide group, the high level killing effect was lost after the depletion of CD4⁺ T cells or natural killer (NK) cells from the lung cell preparation. For the Sm-Cathepsin B + CpG group, high parasite killing was lost after CD8⁺ T cell depletion, and a reduction to 39% was observed upon depletion of NK cells. Finally, the parasite killing in the Sm-Cathepsin B alone group was lost after the depletion of CD4⁺ T cells. Our results demonstrate how the different Sm-Cathepsin B formulations influence the immune mechanisms involved in parasite killing and protection against schistosomiasis.</p

High rates of chronic HBV genotype E infection in a group of migrants in Italy from West Africa: Virological characteristics associated with poor immune clearance

<div><p>Hepatitis B virus (HBV) genotype E almost exclusively occurs in African people, and its presence is more commonly associated with the development of chronic HBV (CHB) infection. Moreover, an epidemiological link has been found between the distribution of HBV genotype E infection and African countries with high incidences of hepatocellular carcinoma. As part of a programme for the health assessment of migrants, we evaluated 358 young African subjects for HBV infection; 58.1% (208/358) were positive for an HBV marker, and 54 (25.5%) had CHB. Eighty-one percent of the CHB subjects were infected with HBV genotype E, with a median serum HBV-DNA of 3.2 (IQR: 2.7–3.6) logIU/ml. All patients had high serum HBsAg titres (10,899 [range 5,359–20,272] IU/ml), and no correlation was found between HBsAg titres and HBV-DNA plasma levels. RT sequence analysis showed the presence of a number of immune escape mutations: strains from all of the patients had a serine at HBsAg position 140; 3 also had T116N, Y100C, and P142L+S143L substitutions; and 1 had a G112R substitution. Six (18%) patients had stop-codons at position 216. In 5 of the 9 (26.5%) CHB patients, ultrasound liver biopsy, quantification of total intrahepatic HBV-DNA and cccDNA, and RT/HBsAg sequencing were performed. The median (IQR) total intrahepatic HBV-DNA was 766 (753–1139) copies/1000 cells, and the median (IQR) cccDNA was 17 (10–27) copies/1000 cells. Correlations were observed for both total intrahepatic HBV-DNA and cccDNA with serum HBV-DNA, while no correlation was found for the HBsAg titres. A difference of 2.5/1,000 nucleotides was found in the HBsAg sequences obtained from plasma and from liver tissue, with 3 cases of possible viral anatomical compartmentalization. In conclusion, a high rate of CHB infection due to the E genotype was demonstrated in a group of immigrants from Western Africa. An analysis of the viral strains obtained showed the virological characteristics of immune escape, which may be the cause of viral replication persistence. Moreover, a fair percentage of stop codon mutations were found. The lack of correlation between HBsAg titres and plasma or intrahepatic HBV-DNA found in these subjects suggests a pathway of virus production that is not linked to HBsAg secretion. Studies with a larger number of patients with CHB due to the E genotype are advisable to corroborate these observations.</p></div

Putative structure of HBsAg reporting the immune-escape mutations and stop-codons (grey box) and HBsAg positions with wild-type amino acids different from genotype D (white box) observed in the group of 34 HBV genotype E infected patients.

<p>Putative structure of HBsAg reporting the immune-escape mutations and stop-codons (grey box) and HBsAg positions with wild-type amino acids different from genotype D (white box) observed in the group of 34 HBV genotype E infected patients.</p

Phylogentic tree reporting the clustering of HBsAg sequences from plasma and liver tissue obtained from the 5 HBV genotype E infected patients with available liver biopsies.

<p>Phylogenetic tree was built by using HBsAg sequences from plasma and liver tissue obtained from the 5 HBV genotype E infected patients, and HBsAg sequences from HBV genotype E isolated from Genbank (Accession number:FN594748).</p

Characteristics of HBsAg-positive patients.

<p>Characteristics of HBsAg-positive patients.</p

Characteristics of genotype E infected patients.

<p>Characteristics of genotype E infected patients.</p

Graphs reporting the correlation between cccDNA and HBsAg titer (A) and serum HBV-DNA (B), and between intrahepatic total HBV-DNA and HBsAg titer (c) and serum HBV-DNA (D) in the 5 HBV genotype E infected patients with available liver biopsies.

<p>Graphs reporting the correlation between cccDNA and HBsAg titer (A) and serum HBV-DNA (B), and between intrahepatic total HBV-DNA and HBsAg titer (c) and serum HBV-DNA (D) in the 5 HBV genotype E infected patients with available liver biopsies.</p

Study population.

<p>Study population.</p