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The vaccination campaign against SARS-CoV-2 relies on the world-wide availability of effective vaccines, with a potential need of 20 billion vaccine doses to fully vaccinate the world population. To reach this goal, the manufacturing and logistic processes should be affordable to all countries, irrespectively of economical and climatic conditions.Outer membrane vesicles (OMVs) are bacterial-derived vesicles that can be engineered to incorporate heterologous antigens. Given the inherent adjuvanticity, such modified OMVs can be used as vaccine to induce potent immune responses against the associated protein. Here we show that OMVs engineered to incorporate peptides derived from the receptor binding motif (RBM) of the spike protein from SARS-CoV-2 elicit an effective immune response in immunized mice, resulting in the production of neutralizing antibodies. The immunity induced by the vaccine is sufficient to protect K18-hACE2 transgenic mice from intranasal challenge with SARS-CoV-2, preventing both virus replication in the lungs and the pathology associated with virus infection. Furthermore, we show that OMVs can be effectively decorated with RBM peptides derived from a different genetic variant of SARS-CoV-2, inducing a similarly potent neutralization activity in vaccinated mice. Altogether, given the convenience associated with ease of engineering, production and distribution, our results demonstrate that OMV-based SARS-CoV-2 vaccines can be a crucial addition to the vaccines currently available.

Regulation of alternative splicing events is an essential step required for the expression of functional cytoskeleton and sarcomere proteins in cardiomyocytes. About 3% of idiopathic dilated cardiomyopathy cases present mutations in the RNA binding protein RBM20, a tissue specific regulator of alternative splicing. Transcripts expressed preferentially in skeletal and cardiac muscle, including TTN, CAMK2D, LDB3, LMO7, PDLIM3, RTN4, and RYR2, are RBM20-dependent splice variants. In the present study, we investigated the RBM20 involvement in post-transcriptional regulation of splicing variants expressed by Formin homology 2 domain containing 3 (FHOD3) gene. FHOD3 is a sarcomeric protein highly expressed in the cardiac tissue and required for the assembly of the contractile apparatus. Recently, FHOD3 mutations have been found associated with heart diseases. We identified novel FHOD3 splicing variants differentially expressed in human tissues and provided evidences that FHOD3 transcripts are specific RBM20 and PTBP1 targets. Furthermore, we demonstrated that the expression of RBM20 and PTBP1 promoted the alternative shift, from inclusion to exclusion, of selected FHOD3 exons. These results indicate that RBM20 and PTBP1 play a role in the actin filament functional organization mediated by FHOD3 isoforms and suggest their possible involvement in heart diseases.

We profiled antibody responses in a cohort of recipients of the BTN162b2 mRNA vaccine who were either immunologically naïve (n=50) or had been previously infected with SARS-CoV-2 (n=51). Of the previously infected, 25 and 26 were infected during the first and second pandemic waves in Italy, respectively; the majority of those from the first wave had corresponding waning immunity with low to undetectable levels of anti-S antibodies and low anti-N antibodies. We observed in recipients who had been previously infected that spike-specific IgG and pseudovirus neutralization titers were rapidly recalled by a single vaccine dose to higher levels than those in naïve recipients after the second vaccine dose, irrespective of waning immunity. In all recipients, a single vaccine dose was sufficient to induce a potent IgA response that was not associated with serum neutralization titers.

Background: IgG antibodies specific for the SARS-CoV-2 Spike protein are under intensive investigation for the urgent need to identify a correlate of protective immunity in individuals vaccinated with licensed and candidate COVID-19 vaccines. Limited data are available on vaccine-elicited IgM antibodies and their potential implication in immunity to SARS-CoV-2. Methods: We performed a longitudinal study to quantify IgG and IgM antibodies specific for the SARS-CoV-2 spike protein (IgG-S and IgM-S) in 1873 health care worker (HCW) recipients of the BNT162b2 (Comirnaty) vaccine. Samples were collected before administration (T0), at the second dose (T1) and three weeks after the second dose (T2). The cohort included 1584 immunologically naive to SARS-CoV-2 (IN) and 289 had a history of previous infection (PI). Findings: In IN we identified three patterns of responses: (a) IgG positive/IgM negative (36.1%), (b) coordinated IgM-S/IgG-S responses appearing three weeks after the first dose (37.4%) and (c) delayed IgM appearing after IgG (26.3%). Coordinated IgM-S/IgG-S responses were associated with higher IgG titers. Of the 289 PI vaccinees, 64.5% were IgM-S positive before vaccination, whereas 32% and 3.5% developed IgM-S after the first and second vaccine dose respectively. IgM-S positive sera had higher pseudovirus neutralization titers compared to the IgM-S negative. Similar results were observed in individuals who received either Moderna or Astrazeneca. Interpretation: Coordinated expression of IgG-S and IgM-S after vaccination was associated with a significantly more efficient response in both antibody titers and virus-neutralizing activity, representing a potential correlate of protection. Instead, the unconventional IgG-S positive/IgM-S negative responses may be suggestive of a recruitment of cross coronaviruses immunity by vaccination, warranting further investigation. Funding Information: This work was supported by the Italian Ministry of Health under “Fondi Ricerca Corrente”- L1P5 and “Progetto Ricerca Finalizzata COVID-2020-12371675” to IRCCS Sacro Cuore Don Calabria Hospital, by FUR 2020 Department of Excellence 2018-2022, MIUR, Italy and by The Brain Research Foundation Verona.

The antibody response to SARS-CoV-2 mRNA vaccines in individuals with waning immunity generated by a previous SARS-CoV-2 infection, as well as the patterns of IgA and IgM responses in previously infected and in naive individuals are still poorly understood. We performed a serology study in a cohort of BTN162b2 mRNA vaccine recipients who were immunologically naive (N, n = 50) or had been previously infected with SARS-CoV-2 (P.I., n = 51) during the first (n = 25) or second (n = 26) pandemic waves in Italy, respectively. We measured IgG, IgM and IgA antibodies against the SARS-CoV-2 Spike (S) and IgG against the nucleocapsid (N) proteins, as well as the neutralizing activity of sera collected before vaccination, after the first and second dose of vaccine. Most P.I. individuals from the first pandemic wave who showed declining antibody titres responded to the first vaccine dose with IgG-S and pseudovirus neutralization titres that were significantly higher than those observed in N individuals after the second vaccine dose. In all recipients, a single dose of vaccine was sufficient to induce a potent IgA response that was not associated with serum neutralization titres. We observed an unconventional pattern of IgM responses that were elicited in only half of immunologically naive subjects even after the second vaccine dose. The response to a single dose of vaccine in P.I. individuals is more potent than that observed in N individuals after two doses. Vaccine-induced IgA are not associated with serum neutralization. Dalle Carbonare et al. perform a serology study in participants with a prior infection of SARS-CoV-2 and those who are SARS-CoV-2-naive, who received two doses of the Pfizer-BioNTech BNT162b2 vaccine. After a single dose they observe a quicker recall of pseudovirus neutralization titres in previously-infected participants and a potent IgA response in both groups that was not associated with serum neutralization titres. Antibodies are proteins produced by the immune system that are released into the bloodstream and help fight infections. To understand how the immune system responds to COVID-19 vaccination in individuals who have had a previous SARS-CoV-2 infection, we compared the types and levels of antibodies produced after first and second doses of the vaccine with those of individuals who had never been infected. We found that one dose of vaccine, even several months after the infection, was sufficient to boost a very efficient response by eliciting specific types of antibodies, some of which were and some that were not able to neutralize the virus. We also observed an unusual antibody profile in almost half of individuals who had not been infected. These findings may help better understand the immune response to COVID-19 vaccines.

The GNA15 gene is ectopically expressed in human pancreatic ductal adenocarcinoma cancer cells. The encoded Gα15 protein can promiscuously redirect GPCR signaling toward pathways with oncogenic potential. We sought to describe the distribution of GNA15 in adenocarcinoma from human pancreatic specimens and to analyze the mechanism driving abnormal expression and the consequences on signaling and clinical follow-up. We detected GNA15 expression in pre-neoplastic pancreatic lesions and throughout progression. The analysis of biological data sets, primary and xenografted human tumor samples, and clinical follow-up shows that elevated expression is associated with poor prognosis for GNA15, but not any other GNA gene. Demethylation of the 5′ GNA15 promoter region was associated with ectopic expression of Gα15 in pancreatic neoplastic cells, but not in adjacent dysplastic or non-transformed tissue. Down-modulation of Gα15 by shRNA or CRISPR/Cas9 affected oncogenic signaling, and reduced adenocarcimoma cell motility and invasiveness. We conclude that de novo expression of wild-type GNA15 characterizes transformed pancreatic cells. The methylation pattern of GNA15 changes in preneoplastic lesions coincident with the release a transcriptional blockade that allows ectopic expression to persist throughout PDAC progression. Elevated GNA15 mRNA correlates with poor prognosis. In addition, ectopic Gα15 signaling provides an unprecedented mechanism in the early steps of pancreas carcinogenesis distinct from classical G protein oncogenic mutations described previously in GNAS and GNAQ/GNA11.

The Coronavirus Disease 2019 (COVID-19) has already caused hundreds of thousands of deaths worldwide in a few months. Cardiovascular disease, hypertension, diabetes and chronic lung disease have been identified as the main COVID-19 comorbidities. Moreover, despite similar infection rates between men and women, the most severe course of the disease is higher in elderly and co-morbid male patients. Therefore, the occurrence of specific comorbidities associated with renin–angiotensin system (RAS) imbalance mediated by the interaction between angiotensin-converting enzyme 2 (ACE2) and desintegrin and metalloproteinase domain 17 (ADAM17), along with specific genetic factors mainly associated with type II transmembrane serine protease (TMPRSS2) expression, could be decisive for the clinical outcome of COVID-19. Indeed, the exacerbated ADAM17—mediated ACE2, TNF-α, and IL-6R secretion emerges as a possible underlying mechanism for the acute inflammatory immune response and the activation of the coagulation cascade. Therefore, in this review, we focus on the main pathophysiological aspects of ACE2, ADAM17, and TMPRSS2 host proteins in COVID-19. Additionally, we discuss a possible mechanism to explain the deleterious effect of ADAM17 and TMPRSS2 over-activation in the COVID-19 outcome.

The serological responses to both SARS-CoV-1 and SARS-CoV-2 virus have some unique characteristics that suggest cross-reactive priming by other human coronaviruses (hCoVs). The early kinetics and magnitude of these responses are, in some cases, associated with worse clinical outcomes in SARS and COVID-19. Cross-reactive hCoV antibody responses have been detected in both SARS and COVID-19 patients. There is also evidence that pre-existing T cell immunity to common cold coronaviruses can prime the response to SARS-CoV-2. Studies in non-human primates show that SARS-CoV-1 S-protein vaccine-induced antibodies are associated with acute lung injury in macaques challenged with SARS-CoV-1. Here we discuss the potential of cross-reactive immunity to drive the immunopathogenesis of COVID-19 and its implications for current efforts to develop immune-based therapies and vaccines.

Alternative splicing is a regulatory mechanism essential for cell differentiation and tissue organization. More than 90% of human genes are regulated by alternative splicing events, which participate in cell fate determination. The general mechanisms of splicing events are well known, whereas only recently have deep-sequencing, high throughput analyses and animal models provided novel information on the network of functionally coordinated, tissue-specific, alternatively spliced exons. Heart development and cardiac tissue differentiation require thoroughly regulated splicing events. The ribonucleoprotein RBM20 is a key regulator of the alternative splicing events required for functional and structural heart properties, such as the expression of TTN isoforms. Recently, the polypyrimidine tract-binding protein PTBP1 has been demonstrated to participate with RBM20 in regulating splicing events. In this review, we summarize the updated knowledge relative to RBM20 and PTBP1 structure and molecular function; their role in alternative splicing mechanisms involved in the heart development and function; RBM20 mutations associated with idiopathic dilated cardiovascular disease (DCM); and the consequences of RBM20-altered expression or dysfunction. Furthermore, we discuss the possible application of targeting RBM20 in new approaches in heart therapies.

Ectopic expression of RUNX2 has been reported in several tumors. In melanoma cells, the RUNT domain of RUNX2 increases cell proliferation and migration. Due to the strong link between RUNX2 and skeletal development, we hypothesized that the RUNT domain may be involved in the modulation of mechanisms associated with melanoma bone metastasis. Therefore, we evaluated the expression of metastatic targets in wild type (WT) and RUNT KO melanoma cells by array and real-time PCR analyses. Western blot, ELISA, immunofluorescence, migration and invasion ability assays were also performed. Our findings showed that the expression levels of bone sialoprotein (BSP) and osteopontin (SPP1) genes, which are involved in malignancy-induced hypercalcemia, were reduced in RUNT KO cells. In addition, released PTHrP levels were lower in RUNT KO cells than in WT cells. The RUNT domain also contributes to increased osteotropism and bone invasion in melanoma cells. Importantly, we found that the ERK/p-ERK and AKT/p-AKT pathways are involved in RUNT-promoted bone metastases. On the basis of our findings, we concluded that the RUNX2 RUNT domain is involved in the mechanisms promoting bone metastasis of melanoma cells via complex interactions between multiple players involved in bone remodeling.

Runx2 is a transcription factor involved in melanoma cell migration and proliferation. Here, we extended the analysis of Runt domain of Runx2 in melanoma cells to deepen understanding of the underlying mechanisms. By the CRISPR/Cas9 system we generated the Runt KO melanoma cells 3G8. Interestingly, the proteome analysis showed a specific protein signature of 3G8 cells related to apoptosis and migration, and pointed out the involvement of Runt domain in the neoangiogenesis process. Among the proteins implicated in angiogenesis we identified fatty acid synthase, chloride intracellular channel protein-4, heat shock protein beta-1, Rho guanine nucleotide exchange factor 1, D-3-phosphoglycerate dehydrogenase, myosin-1c and caveolin-1. Upon querying the TCGA provisional database for melanoma, the genes related to these proteins were found altered in 51.36% of total patients. In addition, VEGF gene expression was reduced in 3G8 as compared to A375 cells; and HUVEC co-cultured with 3G8 cells expressed lower levels of CD105 and CD31 neoangiogenetic markers. Furthermore, the tube formation assay revealed down-regulation of capillary-like structures in HUVEC co-cultured with 3G8 in comparison to those with A375 cells. These findings provide new insight into Runx2 molecular details which can be crucial to possibly propose it as an oncotarget of melanoma.

The identification of new and even more precise technologies for modifying and manipulating the genome has been a challenge since the discovery of the DNA double helix. The ability to modify selectively specific genes provides a powerful tool for characterizing gene functions, performing gene therapy, correcting specific genetic mutations, eradicating diseases, engineering cells and organisms to achieve new and different functions and obtaining transgenic animals as models for studying specific diseases. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology has recently revolutionized genome engineering. The application of this new technology to stem cell research allows disease models to be developed to explore new therapeutic tools. The possibility of translating new systems of molecular knowledge to clinical research is particularly appealing for addressing degenerative diseases. In this review, we describe several applications of CRISPR/Cas9 to stem cells related to degenerative diseases. In addition, we address the challenges and future perspectives regarding the use of CRISPR/Cas9 as an important technology in the medical sciences.

IFI16, member of the IFN-inducible PYHIN-200 gene family, modulates proliferation, survival and differentiation of different cell lineages. In particular, IFI16 expression, which is regulated during the differentiation of B cells, was recently studied in B-CLL as well. Here, we compared IFI16 expression in several lymphomas including Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma and mantle cell lymphoma with respect to normal cell counterparts. We observed that IFI16 expression was significantly deregulated only in mantle cell lymphoma (p < 0.05). Notably, IFI16 was associated with the expression of genes involved in interferon response, cell cycle, cell death and proliferation and, interestingly, lipid and glucose metabolism, suggesting that IFI16 deregulation might be associated with relevant changes in cell biology. In our group of mantle cell lymphoma samples a correlation between patient survival and IFI16 expression was not detected even though mantle cell lymphoma prognosis is known to be associated with cell proliferation. Altogether, these results suggest a complex relationship between IFI16 expression and MCL which needs to be analyzed in further studies., Molecular biology; Cell biology; Biochemistry; Oncology; Pathology; Cancer research; Mantle cell lymphoma; Immunohistochemistry; IFI16; Proliferation; Gene expression

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The risk of developing adult T-cell leukemia/lymphoma (ATLL) in individuals infected with human T-cell lymphotropic virus 1 (HTLV-1) is about 3–5%. The mechanisms by which the virus triggers this aggressive cancer are still an area of intensive investigation. The viral protein Tax-1, together with additional regulatory proteins, in particular HTLV-1 basic leucine zipper factor (HBZ), are recognized as relevant viral factors required for both viral replication and transformation of infected cells. Tax-1 deregulates several cellular pathways affecting the cell cycle, survival, and proliferation. The effects of Tax-1 on the NF-κB pathway have been thoroughly studied. Recent studies also revealed the impact of Tax-1 and HBZ on microRNA expression. In this review, we summarize the recent progress in understanding the contribution of HTLV-1 Tax- and HBZ-mediated deregulation of NF-κB and the microRNA regulatory network to HTLV-1 pathogenesis.

Scientific Reports 7: Article number: 40037; published online: 04 January 2017; updated: 22 May 2018 The original version of this Article contained an error in Figure 7A, where the immunoblot of flotillin from HeLa was a duplicate of the immunoblot of flotillin from HeLa-LAI. This error has been corrected in the HTML and PDF versions of the Article and the figure has been appropriately delineated.

AIDS dementia complex (ADC) and HIV-associated neurocognitive disorders (HAND) are complications of HIV-1 infection. Viral infections are risk factors for the development of neurodegenerative disorders. Aging is associated with low-grade inflammation in the brain, i.e., the inflammaging. The molecular mechanisms linking immunosenescence, inflammaging and the pathogenesis of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease, are largely unknown. ADC and HAND share some pathological features with AD and may offer some hints on the relationship between viral infections, neuroinflammation, and neurodegeneration. β2-microglobulin (β2m) is an important pro-aging factor that interferes with neurogenesis and worsens cognitive functions. Several studies published in the 80-90s reported high levels of β2m in the cerebrospinal fluid of patients with ADC. High levels of β2m have also been detected in AD. Inflammatory diseases in elderly people are associated with polymorphisms of the MHC-I locus encoding HLA molecules that, by associating with β2m, contribute to cellular immunity. We recently reported that HLA-C, no longer associated with β2m, is incorporated into HIV-1 virions, determining an increase in viral infectivity. We also documented the presence of HLA-C variants more or less stably linked to β2m. These observations led us to hypothesize that some variants of HLA-C, in the presence of viral infections, could determine a greater release and accumulation of β2m, which in turn, may be involved in triggering and/or sustaining neuroinflammation. ADC is the most severe form of HAND. To explore the role of HLA-C in ADC pathogenesis, we analyzed the frequency of HLA-C variants with unstable binding to β2m in a group of patients with ADC. We found a higher frequency of unstable HLA-C alleles in ADC patients, and none of them was harboring stable HLA-C alleles in homozygosis. Our data suggest that the role of HLA-C variants in ADC/HAND pathogenesis deserves further studies. If confirmed in a larger number of samples, this finding may have practical implication for a personalized medicine approach and for developing new therapies to prevent HAND. The exploration of HLA-C variants as risk factors for AD and other neurodegenerative disorders may be a promising field of study.

HLA-C expression is associated with a differential ability to control HIV-1 infection. Higher HLA-C levels may lead to better control of HIV-1 infection through both a higher efficiency of antigen presentation to cytotoxic T lymphocytes and the triggering of activating killer immunoglobulin-like receptors on NK cells, whereas lower levels may provide poor HIV-1 control and rapid progression to AIDS. We characterized the relative amounts of HLA-C heterotrimers (heavy chain/β2 microglobulin [β2m]/peptide) and HLA-C free heavy chains on peripheral blood mononuclear cells (PBMCs) from healthy blood donors harboring both alleles with stable or unstable binding to β2m/peptide. We analyzed the stability of HLA-C heterotrimers of different allotypes and the infectivity of HIV-1 virions produced by PBMCs with various allotypes. We observed significant differences in HLA-C heterotrimer stability and in expression levels. We found that R5 HIV-1 virions produced by PBMCs harboring unstable HLA-C alleles were more infectious than those produced by PBMCs carrying the stable variants. We propose that HIV-1 infectivity might depend both on the amounts of HLA-C molecules and on their stability as trimeric complex. According to this model, individuals with low-expression HLA-C alleles and unstable binding to β2m/peptide might have worse control of HIV-1 infection and an intrinsically higher capacity to support viral replication. IMPORTANCE Following HIV-1 infection, some people advance rapidly to AIDS while others have slow disease progression. HLA-C, a molecule involved in immunity, is a key determinant of HIV-1 control. Here we reveal how HLA-C variants contribute to the modulation of viral infectivity. HLA-C is present on the cell surface in two different conformations. The immunologically active conformation is part of a complex that includes β2 microglobulin/peptide; the other conformation is not bound to β2 microglobulin/peptide and can associate with HIV-1, increasing its infectivity. Individuals with HLA-C variants with a predominance of immunologically active conformations would display stronger immunity to HIV-1, reduced viral infectivity and effective control of HIV-1 infection, while subjects with HLA-C variants that easily dissociate from β2 microglobulin/peptide would have a reduced immunological response to HIV-1 and produce more infectious virions. This study provides new information that could be useful in the design of novel vaccine strategies and therapeutic approaches to HIV-1.

Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), an aggressive CD4+/CD25+ T-cell malignancy and of a severe neurodegenerative disease, HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The chronic activation or deregulation of the canonical and non-canonical nuclear factor kappa B (NF-κB) pathways play a crucial role in tumorigenesis. The HTLV-1 Tax-1 oncoprotein is a potent activator of the NF-κB transcription factors and the NF-κB response is required for promoting the development of HTLV-1 transformed cell lines. The homologous retrovirus HTLV-2, which also expresses a Tax-2 transforming protein, is not associated with ATL. In this review, we provide an updated synopsis of the role of Tax-1 in the deregulation of the NF-κB pathway, highlighting the differences with the homologous Tax-2. Special emphasis is directed toward the understanding of the molecular mechanisms involved in NF-κB activation resulting from Tax interaction with host factors affecting several cellular processes, such as cell cycle, apoptosis, senescence, cell proliferation, autophagy, and post-translational modifications. We also discuss the current knowledge on the role of the antisense viral protein HBZ in down-regulating the NF-κB activation induced by Tax, and its implication in cellular senescence. In addition, we review the recent studies on the mechanism of HBZ-mediated inhibition of NF-κB activity as compared to that exerted by the HTLV-2 antisense protein, APH-2. Finally, we discuss recent advances aimed at understanding the role exerted in the development of ATL by the perturbation of NF-κB pathway by viral regulatory proteins.

HLA-C expression is associated with a differential ability to control HIV-1 infection. Higher HLA-C levels may lead to better control of HIV-1 infection through both a higher efficiency of antigen presentation to cytotoxic T lymphocytes and the triggering of activating killer immunoglobulin-like receptors on NK cells, whereas lower levels may provide poor HIV-1 control and rapid progression to AIDS. We characterized the relative amounts of HLA-C heterotrimers (heavy chain/β 2 microglobulin [β 2 m]/peptide) and HLA-C free heavy chains on peripheral blood mononuclear cells (PBMCs) from healthy blood donors harboring both alleles with stable or unstable binding to β 2 m/peptide. We analyzed the stability of HLA-C heterotrimers of different allotypes and the infectivity of HIV-1 virions produced by PBMCs with various allotypes. We observed significant differences in HLA-C heterotrimer stability and in expression levels. We found that R5 HIV-1 virions produced by PBMCs harboring unstable HLA-C alleles were more infectious than those produced by PBMCs carrying the stable variants. We propose that HIV-1 infectivity might depend both on the amounts of HLA-C molecules and on their stability as trimeric complex. According to this model, individuals with low-expression HLA-C alleles and unstable binding to β 2 m/peptide might have worse control of HIV-1 infection and an intrinsically higher capacity to support viral replication. IMPORTANCE Following HIV-1 infection, some people advance rapidly to AIDS while others have slow disease progression. HLA-C, a molecule involved in immunity, is a key determinant of HIV-1 control. Here we reveal how HLA-C variants contribute to the modulation of viral infectivity. HLA-C is present on the cell surface in two different conformations. The immunologically active conformation is part of a complex that includes β 2 microglobulin/peptide; the other conformation is not bound to β 2 microglobulin/peptide and can associate with HIV-1, increasing its infectivity. Individuals with HLA-C variants with a predominance of immunologically active conformations would display stronger immunity to HIV-1, reduced viral infectivity and effective control of HIV-1 infection, while subjects with HLA-C variants that easily dissociate from β 2 microglobulin/peptide would have a reduced immunological response to HIV-1 and produce more infectious virions. This study provides new information that could be useful in the design of novel vaccine strategies and therapeutic approaches to HIV-1.

HLA-C has been demonstrated to associate with HIV-1 envelope glycoprotein (Env). Virions lacking HLA-C have reduced infectivity and increased susceptibility to neutralizing antibodies. Like all others MHC-I molecules, HLA-C requires β2-microglobulin (β2m) for appropriate folding and expression on the cell membrane but this association is weaker, thus generating HLA-C free-chains on the cell surface. In this study, we deepen the understanding of HLA-C and Env association by showing that HIV-1 specifically increases the amount of HLA-C free chains, not bound to β2m, on the membrane of infected cells. The association between Env and HLA-C takes place at the cell membrane requiring β2m to occur. We report that the enhanced infectivity conferred to HIV-1 by HLA-C specifically involves HLA-C free chain molecules that have been correctly assembled with β2m. HIV-1 Env-pseudotyped viruses produced in the absence of β2m are less infectious than those produced in the presence of β2m. We hypothesize that the conformation and surface expression of HLA-C molecules could be a discriminant for the association with Env. Binding stability to β2m may confer to HLA-C the ability to preferentially act either as a conventional immune-competent molecule or as an accessory molecule involved in HIV-1 infectivity.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Its clinical course is typically indolent; however, based on a series of pathobiological, clinical, genetic, and phenotypic parameters, patient survival varies from less than 5 to more than 20 years. In this paper, we show for the first time that the expression of the interferon-inducible DNA sensor IFI16, a member of the PYHIN protein family involved in proliferation inhibition and apoptosis regulation, is associated with the clinical outcome in CLL. We studied 99 CLLs cases by immunohistochemistry and 10 CLLs cases by gene expression profiling. We found quite variable degrees of IFI16 expression among CLLs cases. Noteworthy, we observed that a reduced IFI16 expression was associated with a very poor survival, but only in cases with ZAP70/CD38 expression. Furthermore, we found that IFI16 expression was associated with a specific gene expression signature. As IFI16 can be easily detected by immunohistochemistry or flow cytometry, it may become a part of phenotypic screening in CLL patients if its prognostic role is confirmed in independent series.

HIV-1 Nef interacts with several cellular proteins, among which the human peroxisomal thioesterase 8 (ACOT8). This interaction may be involved in the endocytosis regulation of membrane proteins and might modulate lipid composition in membrane rafts. Nef regions involved in the interaction have been experimentally characterized, whereas structural details of the ACOT8 protein are unknown. The lack of structural information hampers the comprehension of the functional consequences of the complex formation during HIV-1 infection. We modelled, through in silico predictions, the ACOT8 structure and we observed a high charge complementarity between Nef and ACOT8 surfaces, which allowed the identification of the ACOT8 putative contact points involved in the interaction. The predictions were validated by in vitro assays through the development of ACOT8 deletion mutants. Coimmunoprecipitation and immunofluorescence analyses showed that ACOT8 Arg45-Phe55 and Arg86-Pro93 regions are involved in Nef association. In addition, K91S mutation abrogated the interaction with Nef, indicating that Lys91 plays a key role in the interaction. Finally, when associated with ACOT8, Nef may be preserved from degradation. These findings improve the comprehension of the association between HIV-1 Nef and ACOT8, helping elucidating the biological effect of their interaction.

The hypothesis that open conformers of HLA-C on target cells might directly exert an effect on their infectability by human immunodeficiency virus (HIV) has been suggested previously. This was tested by exploiting the peculiar specificity of monoclonal antibody (mAb) L31 for HLA-C open conformers to show that normal levels of Env-driven fusion were restored in HLA-C transfectants of a major histocompatibility complex-deleted (fusion-incompetent) cell line. The physiological relevance of this finding is now confirmed in this report, where small interfering RNA (siRNA) technology was used to silence HLA-C expression in peripheral blood lymphocytes (PBLs) from 11 healthy donors. Infectability by HIV (strains IIIB and Bal and primary isolates) was significantly reduced (P=0.016) in silenced cells compared with cells that maintained HLA-C expression in 10 of the 11 PBL donors. Normal infectability was resumed, together with HLA-C expression, when the effect of siRNA interference waned after several days in culture. Additional confirmation of the HLA-C effect was obtained in several assays employing HLA-C-positive and -negative cell lines, a number of HIV strains and also pseudoviruses. In particular, viruses pseudotyped with env genes from HIV strains AC10 and QH0692.42 were assayed on siRNA-silenced lymphocytes from three healthy donors: the differences in infection with pseudoviruses were even higher than those observed in infections with normal viruses.