Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast

<p>Abstract</p> <p>Background</p> <p>The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in <it>in vitro </it>and <it>in vivo </it>detection of CD expression in GDEPT/ADEPT studies.</p> <p>Results</p> <p>An enzymatic active recombinant CD from yeast (yCD) was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU.</p> <p>Conclusion</p> <p>The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.</p

Endogenous CCL2 neutralization restricts HIV-1 replication in primary human macrophages by inhibiting viral DNA accumulation

Macrophages are key targets of HIV-1 infection. We have previously described that the expressionof CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is furtherup-modulated by HIV-1 exposure. Moreover, CCL2 acts as an autocrine factor that promotes viral replication ininfected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibitsHIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restrictionfactors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoproteinB mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members.Results:CCL2 neutralization potently reduced the number of p24 Gag+cells during the course of either productive orsingle cycle infection with HIV-1. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thusdemonstrating that the restriction involves post-entry steps of the viral life cycle. Notably, the accumulation of viralDNA, both total, integrated and 2-LTR circles, was strongly impaired by neutralization of CCL2. Looking for correlates ofHIV-1 DNA accumulation inhibition, we found that the antiviral effect of CCL2 neutralization was independent of themodulation of SAMHD1 expression or function. Conversely, a strong and selective induction of APOBEC3A expression,to levels comparable to those of freshly isolated monocytes, was associated with the inhibition of HIV-1 replicationmediated by CCL2 blocking. Interestingly, the CCL2 neutralization mediated increase of APOBEC3A expression was typeI IFN independent. Moreover, the transcriptome analysis of the effect of CCL2 blocking on global gene expressionrevealed that the neutralization of this chemokine resulted in the upmodulation of additional genes involved in thedefence response to viruses.Conclusions:Neutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primaryMDM affecting post-entry steps of the viral life cycle with a mechanism independent of SAMHD1. In addition, CCL2blocking is associated with induction of APOBEC3A expression, thus unravelling a novel mechanism which mightcontribute to regulate the expression of innate intracellular viral antagonistsin vivo. Thus, our study may potentially leadto the development of new therapeutic strategies for enhancing innate cellular defences against HIV-1 and protecting macrophages from infection

SAMHD1-independent inhibition of HIV-1 DNA accumulation by endogenous CCL2 neutralization: possible role of APOBEC3A

Macrophages are key targets of HIV-1 infection. We have previously described that the expression of CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is further up-modulated by HIV-1 exposure. Furthermore, CCL2 acts as an autocrine factor that promotes viral replication in infected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibits HIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restriction factors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members. We found that CCL2 neutralization potently reduced viral DNA accumulation and the number of p24 Gag+ cells during the course of either a productive or a single cycle infection with HIV-1BaL or (VSV-G) HIV-1, respectively. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thus demonstrating that the restriction involves post-entry steps of the viral life cycle. Looking for correlates of HIV-1 DNA accumulation inhibition, we found that SAMHD1 expression was not affected by CCL2 neutralization. Furthermore, CCL2 blocking reduced the percentage of p24 Gag+ cells both in the absence and in the presence of exogenous dNTPs, and restricted the transduction of macrophages with Vpx+ lentiviral particles, which determined a strong down-modulation of SAMHD1 expression. These results demonstrated that altered SAMHD1 expression or function cannot account for the CCL2 neutralization-mediated restriction of HIV-1 replication in macrophages. Conversely, a strong and selective induction of APOBEC3A transcript and protein expression was associated with the inhibition of HIV-1 replication mediated by CCL2 neutralization. Interestingly, the level of CCL2 blocking induced APOBEC3A expression was comparable to those of freshly isolated monocytes. Finally, induction of A3A was type I IFN independent, since neutralizing Abs to IFN-α or IFN-β did not abolish CCL2 blocking-mediated A3A expression. Overall, these data demonstrate that neutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primary MDM affecting post-entry steps of the viral life cycle likely involving the up-regulated expression of APOBEC3A. These results highlight a novel mechanism which may contribute to regulate the expression of innate intracellular viral antagonists in vivo. Thus, our study may potentially lead to the development of new therapeutic strategies for enhancing innate cellular defenses against HIV-1 and protecting macrophages from infection

The role of IL-15 in challenging Acquired Immunodeficiency Syndrome

Objective: To determine the functions of in vitro primed Natural Killer (NM) cells in Human Immunodeficiency Virus (HIV-1) infection and the role of IL-2, IL-12 and IL-15 in enhancing the NM survival and activity in terms of viral suppression and of purging of HIV provirus. Methods: Peripheral Blood Mononuclear Cells (PBMCs) and CD4+ T lymphocytes cells obtained from eight healthy donors were infected in vitro with HIV-1 and p24 was measured with and without IL-2, IL-12 and IL-15. We studied the effect of NM pulsed in vitro with IL-2, IL-12 and IL-15 on HIV replication by measurement of p-24 and DNA-provirus load when added into the culture of PBMCs and CD4+ T lymphocytes cells infected in vitro. We evaluated the effect of NK cells pulsed with IL-2, IL-12 and IL-15 on HIV replication and DNA-load into the culture of CD4+ T lymphocytes cells and PBMCs by trans-well chamber. Results: We found high levels of p24 in the supernatants of PBMCs and CD4+ T lymphocytes cells cultured with IL-2, IL-12, and IL-15. We observed a significant reduction of p24 in the culture both of infected PBMCs and CD4+ T lymphocytes cells in which was added NK pulsed with IL-15. We did not obtain the some results with NM pulsed with IL-2 and IL-12. We observed a power effect of NK pulsed with IL-15 on HIV-DNA. The trans-well chamber experiments showed that the effect of NM is both direct and both mediated by realizing of soluble factors. Conclusions: This study highlights some important effects of IL 15 on NK in HIV patients anyway our results are preliminary and descriptive and others studies will be needed to provide rationale for immune therapies. (C) 2011 Elsevier Ltd. All rights reserved