Effects of Influenza Vaccine on the Immune Responses to SARS-CoV-2 Vaccination.

A number of studies have suggested that influenza vaccination can provide protection against COVID-19, but the underlying mechanisms that could explain this association are still unclear. In this study, the effect of the 2021/2022 seasonal influenza vaccination on the immune response to the booster dose of anti-SARS-CoV-2 vaccination was evaluated in a cohort of healthy individuals. A total of 113 participants were enrolled, 74 of whom had no prior COVID-19 diagnosis or significant comorbidities were considered for the analysis. Participants received the anti-influenza tetravalent vaccine and the booster dose of the anti-SARS-CoV-2 vaccine or the anti-SARS-CoV-2 vaccine alone. Blood was collected before and 4 weeks after each vaccination and 12 weeks after SARS-CoV-2 vaccination and analyzed for anti-flu and anti-spike-specific antibody titers and for in vitro influenza and SARS-CoV-2 neutralization capacity. Results indicated an increased reactivity in subjects who received both influenza and SARS-CoV-2 vaccinations compared to those who received only the SARS-CoV-2 vaccine, with sustained anti-spike antibody titers up to 12 weeks post-vaccination. Immune response to the influenza vaccine was evaluated, and individuals were stratified as high or low responders. High responders showed increased antibody titers against the SARS-CoV-2 vaccine both after 4 and 12 weeks post-vaccination. Conversely, individuals classified as low responders were less responsive to the SARS-CoV-2 vaccine. These data indicate that both external stimuli, such as influenza vaccination, and the host's intrinsic ability to respond to stimuli play a role in the response to the vaccine.

Detection and full genomic sequencing of rare hepatitis E virus genotype 4d in Italian wastewater, undetected by clinical surveillance.

Hepatitis E is a liver disease caused by the hepatitis E virus (HEV), primarily transmitted through contaminated water or food. There are four different HEV genotypes in humans, with genotypes 1 and 2 being the most widespread. Genotypes 3 and 4 are found in animals and can also infect humans. Genotype 4 is prevalent in Asia, mainly in China. In Italy, only one outbreak of HEV-4 has been documented, which occurred in 2011, involving five patients. In 2013, HEV G4 was also detected in a pig farm. Since then, no further evidence of HEV genotype 4 has been found in the country. This study describes the first detection of HEV genotype 4, subtype d, in wastewater in central Italy, despite a lack of any clinical case reported in the area. By using a multiplex PCR protocol and two sequencing strategies, Illumina and ONT, the virus's complete genome was sequenced and characterized as subtype 4d. These findings shed light on the potential of environmental surveillance for infectious agents to improve our understanding of epidemiology and support public health efforts.

Correction to: Genetic Diversity Among Genogroup II Noroviruses and Progressive Emergence of GII.17 in Wastewaters in Italy (2011-2016) Revealed by Next-Generation and Sanger Sequencing.

The original version of this article unfortunately contained a mistake. The presentation of Table 1 was incorrect. The corrected table is given below. The original article has been corrected.

Genetic Diversity Among Genogroup II Noroviruses and Progressive Emergence of GII.17 in Wastewaters in Italy (2011-2016) Revealed by Next-Generation and Sanger Sequencing.

Noroviruses (NoV) are a major cause of gastroenteritis worldwide. Recently, a novel variant of NoV GII.17 (GII.P17_GII.17 NoV), termed Kawasaki 2014, has been increasingly reported in NoV outbreaks in Asia, and has also been described in Europe and North America. In this study, sewage samples were investigated to study the occurrence and genetic diversity of NoV genogroup II (GII) along a 6-year period. Moreover, the spread of GII.17 strains (first appearance and occurrence along time) was specifically assessed. A total of 122 sewage samples collected from 2011 to 2016 from four wastewater treatment plants in Rome (Italy) were initially tested using real-time RT-(q)PCR for GII NoV. Positive samples were subsequently subjected to genotypic characterization by RT-nested PCRs using broad-range primes targeting the region C of the capsid gene of GII NoV, and specific primers targeting the same region of GII.17 NoV. In total, eight different genotypes were detected with the broad-range assay: GII.1 (n = 6), GII.2 (n = 8), GII.3 (n = 3), GII.4 (n = 13), GII.6 (n = 3), GII.7 (n = 2), GII.13 (n = 2), and GII.17 (n = 3), with the latter two genotypes detected only in 2016. Specific amplification of GII.17 NoV was successful in 14 out of 110 positive samples, spanned over the years 2013-2016. The amplicons of the broad-range PCR, pooled per year, were further analyzed by next-generation sequencing (NGS) for a deeper analysis of the genotypes circulating in the study period. NGS confirmed the circulation of GII.17 NoV since 2013 and detected, beyond the eight genotypes identified by Sanger sequencing, three additional genotypes regarded as globally uncommon: GII.5, GII.16, and GII.21. This study provides evidence that GII.17 NoV Kawasaki has been circulating in the Italian population before its appearance and identification in clinical cases, and has become a major genotype in 2016. Our results confirm the usefulness of wastewater surveillance coupled with NGS to study the molecular epidemiology of NoV and to monitor the emergence of NoV strains.

Molecular characterization of human adenoviruses in urban wastewaters using next generation and Sanger sequencing.

Human adenoviruses (HAdVs) are of major public health importance and are associated with a variety of clinical manifestations, including gastroenteritis, respiratory, ocular and urinary tract infections. To study the occurrence, prevalence and diversity of HAdV species and types circulating in Italy, we conducted a large-scale molecular-epidemiological investigation, a yearlong monitoring of 22 wastewater treatment plants, covering 10 Italian regions, representative of northern, central, and southern Italy. A total of 141 raw sewage samples were collected from January to December 2013, and processed to detect and characterize by phylogenetic analysis a fragment of the hexon coding region of HAdVs. Nested PCR results showed the presence of HAdVs in 85 out of 141 samples (60% of samples). Fifty-nine samples were characterized by conventional Sanger sequencing as belonging to four HAdV species and four types: A (type 12, 5 samples), B (type 3, 8 samples), C (type 5, 1 sample) and F (type 41, 45 samples). The remaining 26 samples could not be characterized because of uninterpretable (mixed) electropherograms suggesting the presence of multiple species and/or types. Pools of characterized and uncharacterized PCR amplicons were further analyzed by next-generation sequencing (NGS). NGS results revealed a marked HAdV diversity with 16 additional types detected beyond the four types found by Sanger sequencing. Overall, 19 types were identified, belonging to HAdV species A-F: types 12 and 31 (species A), type 3 (species B), types 1, 2, and 5 (species C), types 9, 17, 24, 26, 37, 38, 42, 44, 48, and 70 (species D), type 4 (species E), and types 40 and 41(species F). An untypeable HAdV was also detected, showing similar percentages of identity with more than one prototype (types 15, 30, 56, and 59). Our findings documented the circulation of a wide variety of species and types in raw sewage, potentially able to affect other surface water environments and hence human health. Next-generation sequencing proved to be an effective strategy for HAdV genotyping in wastewater samples. It was able to detect a wide range of "less prevalent" types unidentified by conventional Sanger sequencing, confirming that studies based on conventional technologies may grossly underestimate the existence of some, possibly less common, types. Knowledge of the distribution of HAdV species and types would improve our understanding of waterborne HAdV-related health risks.

Strong CD8+ T cell antigenicity and immunogenicity of large foreign proteins incorporated in HIV-1 VLPs able to induce a Nef-dependent activation/maturation of dendritic cells.

Virus-like particles (VLPs) are excellent tools for vaccines against pathogens and tumors. They can accommodate foreign polypeptides whose incorporation efficiency and immunogenicity however decrease strongly with the increase of their size. We recently described the CD8(+) T cell immune response against a small foreign antigen (i.e., the 98 amino acid long human papilloma virus E7 protein) incorporated in human immunodeficiency virus (HIV)-1 based VLPs as product of fusion with an HIV-1 Nef mutant (Nef(mut)). Here, we extended our previous investigations by testing the antigenic/immunogenic properties of Nef(mut)-based VLPs incorporating much larger heterologous products, i.e., human hepatitis C virus (HCV) NS3 and influenza virus NP proteins, which are composed of 630 and 498 amino acids, respectively. We observed a remarkable cross-presentation of HCV NS3 in dendritic cells challenged with Nef(mut)-NS3 VLPs, as detected using a NS3 specific CD8(+) T cell clone as well as PBMCs from HCV infected patients. On the other hand, when injected in mice, Nef(mut)-NP VLPs elicited strong anti-NP CD8(+) T cell and CTL immune responses. In addition, we revealed the ability of Nef(mut) incorporated in VLPs to activate and mature primary human immature dendritic cells (iDCs). This phenomenon correlated with the activation of Src tyrosine kinase-related intracellular signaling, and can be transmitted from VLP-challenged to bystander iDCs. Overall, these results prove that Nef(mut)-based VLPs represent a rather flexible platform for the design of innovative CD8(+) T cell vaccines.

Activation of the ER stress gene gadd153 by hepatitis C virus sensitizes cells to oxidant injury.

HCV induces endoplasmic reticulum (ER) stress which correlates with transcriptional induction of ER stress genes. Previously, we reported that expression of HCV structural proteins activates the ER stress and pro-apoptotic gene gadd153 which plays a relevant role in cell death induced by oxidative stress. In the present study, using human hepatic cell lines Huh7 carrying a full-length HCV replicon, we demonstrated that replication and expression of the complete set of HCV proteins were associated with elevated expression of gadd153. Analysis of gadd153 promoter activity revealed that both the ATF4 and the ATF6 pathways, which are typically induced during ER stress response, contribute to the induction of gadd153 in HCV replicon cells. Activation of the ATF4 pathway was confirmed by identification of increased levels of ATF4 protein in replicon cells. Importantly, we showed that, following H2O2 treatment, gadd153 gene reached higher levels of expression in replicon cells. Consistent with the marked induction of the pro-apoptotic gene gadd153, HCV replicon cells showed an increased vulnerability to oxidant injury. Treatment of replicon cells with a specific small interfering RNA, targeted to gadd153 gene, reduced basal expression of gadd153 and decreased cell death following H2O2. These findings suggest that gadd153 may play a major role in sensitivity of HCV replicon cell to oxidative stress.

Activation of endoplasmic reticulum stress response by hepatitis C virus proteins.

Flaviviruses utilize the endoplasmic reticulum (ER) as the main site for replication and protein synthesis and cause some level of ER stress. In the present study, we evaluated the ability of HCV proteins to induce ER stress response by using a tetracycline-regulated cell line expressing a region of HCV genome containing the structural genes. In this system different levels of HCV protein expression could be obtained by varying the concentration of tetracycline in the medium. Real Time PCR and Western blotting assay demonstrated that HCV mRNA and protein levels reach a maximum value at 24-48 h and decrease at 72 h postinduction. Cell proliferation analysis indicated that HCV synthesis causes cell growth inhibition. The effect was also observed in cells expressing lower levels of HCV proteins. The expression profile of specific genes, which are markers of ER stress response, revealed the upregulation of the chaperone GRP78 and the transcription factor GADD153. Induction of GADD153 correlates with the downregulation of the antiapoptotic Bcl-2 gene suggesting that synthesis of HCV proteins may influence cell fate through the activation of ER stress signaling pathway.

The transmembrane domain of hepatitis C virus E1 glycoprotein induces cell death.

The E1 protein of hepatitis C virus (HCV) shows the ability to induce cell lysis by the alteration of membrane permeability when expressed in Escherichia coli cells. This function seems to be an intrinsic property of a C-terminal hydrophobic region of E1 as permeability changes and cell lysis can be blocked by mutagenesis of specific amino acids in this domain. To establish whether the expression of E1 protein and its C-terminal domain was able to induce cell death also in eukaryotic cell, we cloned HCV sequences expressing the full-length E1 (E383), the C-terminal domain (SVP) and a mutant lacking the C-terminal region (E340) in the pRC/CMV expression vector. HepG2 cell line was co-transfected with empty vector or HCV expression plasmids and a reporter vector that expressed beta-galactosidase (beta-gal) to visualize co-transfected blue cells. At 60 h after transfection, the loss of blue cells, considered as a measure of cell death, was 31.5 and 64.3% for the E1 and SVP clones. On the contrary, the number of blue cells after transfection with E340 plasmid was similar to that observed with the control vector. The analysis by the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay revealed an increased number of apoptotic cells at 48 h after transfection with E1 and SVP clones. Furthermore, cells transfected with SVP revealed a typical internucleosomal DNA fragmentation and the activation of caspase-3-like proteases as the specific inhibitor Ac-DEVD-CHO peptide partially blocked SVP apoptosis. These data indicate that the intracellular expression of HCV E1 protein and its C-terminal domain induces an apoptotic response in human hepatoma cell line.

Mutagenesis of hepatitis C virus E1 protein affects its membrane-permeabilizing activity.

The E1 glycoprotein of hepatitis C virus is a transmembrane glycoprotein with a C-terminal anchor domain. When expressed in Escherichia coli, E1 induces a change in membrane permeability that is toxic to the bacterial cell. The C-terminal hydrophobic region (aa 331-383) of E1 is mainly responsible for membrane association and for inducing changes in membrane permeability. These observed changes are similar to those produced in E. coli by influenza virus M2, human immunodeficiency virus gp41 and poliovirus 3AB proteins, whose hydrophobic domains are thought to cause pore formation in biological membranes. To further characterize the activity of E1 at a molecular level, the membrane-permeabilizing ability of a second internal hydrophobic region (aa 262-291) was examined by expressing different deletion mutants of E1 in an E. coli system that is widely used for analysing membrane-active proteins from other animal viruses. Moreover, highly conserved amino acids in the C-terminal hydrophobic region were mutated to identify residues that are critical for inducing changes in membrane permeability. Analysis of cell growth curves of recombinant cultures and membrane-permeability assays revealed that synthesis of this fragment increased the flux of small compounds through the membrane and caused progressive cell lysis, suggesting that this domain has membrane-active properties. Furthermore, analysis of C-terminal mutants indicated that the conserved amino acids Arg(339), Trp(368) and Lys(370) play a critical role in protein function, as both cell lysis and changes in membrane permeability induced by the wild-type clone could be blocked by substitutions in these positions.

Expression and membrane association of hepatitis C virus envelope 1 protein.

The expression of hepatitis C virus (HCV) E1 protein is toxic for Escherichia coli cells. For this reason, we have cloned the E1 gene in the pET3a vector and analyzed the inducible expression of the protein in two strains of E. coli characterised by a different level of reduction of basal synthesis. The results indicated that synthesis of E1 was supported only by the BL21(DE3)pLysS strain which provides a tightest control of protein expression before the induction. The BL21(DE3)pLysS cells were then used for the expression of E1 gene, varying at its carboxy terminus in order to retain (E1, aa 192-383) or delete (Elt, aa 192-340) a C-terminal hydrophobic region that may be involved in membrane association. Following cell fractionation, E1 protein was found associated with the membrane fraction. By contrast, the truncated mutant E1t, was identified in the soluble phase suggesting a direct role for the C-terminal domain in E1 membrane association.

Hepatitis C virus E1 protein induces modification of membrane permeability in E. coli cells.

The E1 gene of hepatitis C virus (HCV) has been cloned and expressed in BL21(DE3)pLys Escherichia coli strain by pET3a vector to analyze changes in membrane permeability produced by this protein. We showed that the expression of E1 (aa 192-383), as well as of two C-terminal fragments (aa 331-383 and aa 341-383) corresponding to the transmembrane (TM) region of this protein, induced a rapid lysis of cells. On the contrary, the expression of a mutant of E1 (aa 192-340), lacking the last 40 amino acids, did not cause cell lysis. The analysis of permeability changes revealed that modification of membrane permeability to several compounds were observed only in clones expressing E1 and C-terminal fragments, while the synthesis of the C-terminal-deleted mutant had little or no effect on permeability. These findings demonstrate that the TM domain of E1 protein has membrane-active properties that may be involved in some aspects of virus-cell interaction.

Secretion and purification of HCV E1 protein forms as glutathione-S-transferase fusion in the baculovirus insect cell system.

We have expressed the E1 protein of Hepatitis C Virus (HCV) in a new recombinant form by using a baculovirus transfer vector directing the expression of proteins fused to the carboxy-terminus of glutathione-S-transferase (GST). The E1 domain was expressed varying at its carboxy terminus in order to retain (GST-E1) or delete (GST-E1b) the C-terminal hydrophobic region that may be involved in membrane association. Following infection with the recombinant virus, GST-E1b was efficiently secreted into the culture media and could be purified in a single step with the minimum of denaturation by glutathione affinity chromatography. The purified product was specifically immunoprecipitated by HCV positive human sera suggesting the maintenance of an immuno-relevant tertiary structure despite removal of the hydrophobic anchor. By contrast, cells infected with a recombinant baculovirus expressing GST-E1 gave a fusion protein with an appropriate molecular weight but also a series of polypeptides of lower molecular weight consistent with cleavage at the C-terminus of E1. GST-E1 was not secreted into the medium and was associated predominantly with the membrane fraction following cell disruption; the lower molecular weight forms were soluble and secreted.

Evaluation of anionic collagen membranes in the treatment of class II furcation lesions: an histometric analysis in dogs.

This study aims to evaluate the effect of using anionic collagen membranes in guided tissue regeneration treatment of Class II furcation lesions in dogs. The defects were created in the buccal furcation of 16 mandibular premolars of four dogs. After 56 days without plaque control, the sites were scaled and divided into two groups according to the treatment applied: control sites, open flap debridement; and test sites, guided tissue regeneration treatment. The animals were killed after 3 months. Histological and histometrical analyses showed that the collagen membrane was better than open flap debridement in terms of newly formed cementum and epithelial migration prevention. It provided effective blockade of epithelial tissue and promoted regeneration of lost periodontal tissues, suggesting that the membrane warrants further study.