Andrea Savarino

Istituto Superiore di Sanità | ISS · Department of Infectious, Parasitic and Immune-mediated Diseases

Andrea Savarino and Marina Lusic made equal contributions. ABSTRACT Combinations of direct-acting antivirals are needed to minimize drug resistance mutations and stably suppress replication of RNA viruses. Currently, there are limited therapeutic options against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and testing of a numb...

Background We developed a personalized Monocyte-Derived Dendritic-cell Therapy (MDDCT) for HIV-infected individuals on suppressive antiretroviral treatment and evaluated HIV-specific T-cell responses. Methods PBMCs were obtained from 10 HIV ⁺ individuals enrolled in trial NCT02961829. Monocytes were differentiated into DCs using IFN-α and GM-CSF....

The present invention relates to cobicistat and its derivatives or prodrugs for use in the prophylaxis and/or treatment of severe acute respiratory syndrome coronavirus type 2 (SARS- CoV-2) infection, severe acute respiratory syndrome coronavirus (SARS-CoV) infection and/or Middle East respiratory syndrome coronavirus (MERS-CoV) infection. The pres...

Background We developed a personalized Monocyte-Derived Dendritic Therapy (MDDCT) for HIV-infected individuals on suppressive antiretroviral treatment and evaluated HIV-specific T-cell responses. Methods PBMCs were obtained from 10 HIV⁺ individuals enrolled in trial NCT02961829. Monocytes were differentiated into DCs using IFN-α and GM-CSF. After s...

HIV-1 infects lymphoid and myeloid cells, which can harbor a latent proviral reservoir responsible for maintaining lifelong infection. Glycolytic metabolism has been identified as a determinant of susceptibility to HIV-1 infection, but its role in the development and maintenance of HIV-1 latency has not been elucidated. By combining transcriptomic,...

Combinations of direct-acting antivirals are needed to minimize drug-resistance mutations and stably suppress replication of RNA viruses. Currently, there are limited therapeutic options against the Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) and testing of a number of drug regimens has led to conflicting results. Here we show tha...

HIV-1 infects lymphoid and myeloid cells, which can harbor a latent proviral reservoir responsible for maintaining lifelong infection. Glycolytic metabolism has been identified as a determinant of susceptibility to HIV-1 infection, but its role in the development and maintenance of HIV-1 latency has not been elucidated. By combining transcriptomic,...

Background and Objectives Chloroquine/hydroxychloroquine has recently been the subject of intense debate regarding its potential antiviral activity against SARS-Cov-2, the etiologic agent of COVID-19. Some report possible curative effects; others do not. Therefore, the objective of this study was to simulate possible scenarios of response to hydrox...

The pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is having serious consequences on health and the economy worldwide. All evidence-based treatment strategies need to be considered to combat this new virus. Drugs need to be considered on scientific grounds of efficacy, safety and cost. Chloroquine (CQ) and hydro...

Computational prediction of immunogenic epitopes is a promising platform for therapeutic and preventive vaccine design. A potential target for this strategy is human immunodeficiency virus (HIV-1), for which, despite decades of efforts, no vaccine is available. In particular, a therapeutic vaccine devised to eliminate infected cells would represent...

Chloroquine/hydroxychloroquine has recently been the subject of intense debate in regard to its potential antiviral activity against SARS-Cov-2, the etiological agent of COVID-19. Some report possible curative effects, others do not. In order to shed some light on this rather controversial topic, we used mathematical modelling to simulate possible...

HIV-1 persists in a latent form during antiretroviral therapy, mainly in CD4+ T cells, thus hampering efforts for a cure. HIV-1 infection is accompanied by metabolic alterations, such as oxidative stress, but the effect of cellular antioxidant responses on viral replication and latency is unknown. Here, we show that cells survive retroviral replica...

Gold compounds have a long history of use as immunosuppressants, but their precise mechanism of action is not completely understood. Using our recently developed liver-on-a-chip platform we now show that gold compounds containing planar N-heterocyclic carbene (NHC) ligands are potent ligands for the aryl hydrocarbon receptor (AHR). Further studies...

Recently, a second individual (the “London patient”) with HIV-1 infection and concomitant leukemia was cured of both diseases by a conditioning myeloablative regimen followed by transplantation of stem cells bearing the homozygous CCR5 Δ32 mutation. The substantial risks and cost associated with this procedure render it unfeasible on a large scale....

Antiretroviral therapy (ART) is typically composed of a combination of three antiretroviral drugs and is the treatment of choice for people with human immunodeficiency virus type 1/acquired immune deficiency syndrome (HIV-1/AIDS). However, it is unable to impact on viral reservoirs, which harbour latent HIV-1 genomes that are able to reignite the i...

Metabolic alterations, such as oxidative stress, are hallmarks of HIV-1 infection. However, their influence on the development of viral latency, and thus on HIV-1 persistence during antiretroviral therapy (ART), have just begun to be explored. We analyzed omics profiles of in-vitro and in-vivo models of infection by HIV-1 and its simian homolog SIV...

Zika virus (ZIKV) infection in utero might lead to microcephaly and other congenital defects. Since no specific therapy is available thus far, there is an urgent need for the discovery of agents capable of inhibiting its viral replication and deleterious effects. Chloroquine is widely used as an antimalarial drug, anti-inflammatory agent, and it al...

Although the use of antioxidants for the treatment of cancer and HIV/AIDS has been proposed for decades, new insights gained from redox research have suggested a very different scenario. These new data show that the major cellular antioxidant systems, the thioredoxin (Trx) and glutathione (GSH) systems, actually promote cancer growth and HIV infect...

Background Administration of antiretroviral therapy and two experimental drugs, auranofin and buthionine sulfoximine (BSO), was previously shown to be followed by drug-free control of chronic SIVmac251 infection, decreased immune activation and increased cell-mediated anti-Gag responses.Methods Phylogeny was analysed with Phylogeny.fr. Entropy was...

The restoration of the immune system prompted by antiretroviral therapy (ART) has allowed drastically reducing the mortality and morbidity of HIV infection. However, one main source of clinical concern is the persistence of immune hyperactivation in individuals under ART. Chronically enhanced levels of T-cell activation are associated with several...

Off-therapy control of viremia by HIV-infected individuals has been associated with two likely players: a restricted viral reservoir and an efficient cell-mediated immune response. We previously showed that a combination of highly suppressive antiretroviral therapy and two experimental drugs, i.e., auranofin and buthionine sulfoximine, was able to...

Activation of HIV-1 replication causes oxidative stress, which in turn potentiates HIV-1 replication. The common basis for the compounds of the present invention is: A) the capacity of reactivating HIV-1 from latency, and B) the ability to counteract the cellular machinery which activates in order to limit the effects of oxidative stress. In this w...

Central memory (TCM) and transitional memory (TTM) CD4(+) T cells are known to be the major cellular reservoirs for HIV, as these cells can harbor a transcriptionally silent form of viral DNA that is not targeted by either the immune system or current antiretroviral drug regimens. In the present study, we explored the molecular bases of the anti-HI...

Despite the huge clinical success of antiretroviral therapy, several factors such as side effects, requirement of life-long adherence, high cost, incomplete access to therapies and development of drug resistance make the quest for an ultimate cure of HIV/AIDS a worldwide priority of biomedical research. In this respect, several sterilizing or funct...

Background HIV infection persists despite antiretroviral treatment (ART) and is reignited as soon as therapies are suspended. This vicious cycle is fueled by the persistence of viral reservoirs that are invulnerable to standard ART protocols, and thus therapeutic agents able to target these reservoirs are needed. One such agent, auranofin, has rece...

Our inability to cure HIV/AIDS is related to the ability of the virus to establish reservoirs during treatment. In order to develop new strategies, it is certainly essential that a suitable animal model be implemented. In the recent work of Shytaj et al., it has been possible to inhibit viral replication to levels below the assay detection limit in...

Structural analysis of SIVmac251 susceptibility to darunavir. Panel A: Sequence alignment of the protease of HIV-1 subtype B [PDB: 2HS1,V32I Mutant], HIV-2 [PDB: 3ECG], and SIVmac251 [PDB: 2SAM]. The sequence alignment is based on a structural alignment performed using the VAST algorithm. Regions showing significant structural alignment are present...

Examples of SIVmac251 infection course in rhesus macaques. Depicted is the progression of viremia in a cohort of seven SIVmac251 infected rhesus macaques. In red are the macaques displaying similar viral loads as those of the animals enrolled in the pilot study (see text). (TIF)

Starting data for the numerical simulations of the viral load and reservoir dynamics. (TIF)

Treatment with H-iART recovers CD4+ T-cell counts decreased by pathogenic SIVmac251 infection. Five macaques are considered for which pre-infection and pre-treatment CD4+ T-cell counts were available. Values during H-iART refer to a median period of 89 days (range: 83–89 days). Data have been analyzed using one-way ANOVA followed by Newmann-Keuls t...

Activation function. The step function f(t) which determines the activation of resting latently infected CD4+ T-cells. Times between two activation periods follow a Poisson distribution with a mean of 50 days. The length of activation periods follows a uniform distribution over an interval of 4 to 6 days. (TIF)

Viro-immunological background and therapeutic regimens of the SIVmac251 infected macaques employed in the study. MHC alleles analyzed are the following: Mamu A*01; A*02; A*08; A*11; B*01; B*03; B*04; B*08; B*17. The CD4 nadir is chosen as the lowest pre-therapy T-CD4 count available. Therapeutic regimens described in the present article are highlig...

Validation of the real-time PCR assay for SIVmac251 DNA quantification in PBMCs and lymph node biopsies. The limit of detection of the assay is 2 copies/5*105 cells. As a control of the assay variability and to exclude PCR inhibition, spiked DNA measurements for each sample were used. (TIF)

Variability of the quantitative real-time RT-PCR assay for measurement of viral RNA. Panel A: Standard curves run on three different occasions. Panel B: Coefficients of variation at different starting RNA concentrations. The coefficients of variation were calculated as the standard deviation of each group of values (starting from the same RNA conce...

Viral loads of SIVmac251-infected macaques before and during treatment with maraviroc, tenofovir and emtricitabine. Asterisks show the significant differences between values at start of follow-up and during treatment [P<0.05, Bonferroni's post test following significant (P<0.05) repeated-measures ANOVA]. (TIF)

Bioinformatic analyses and molecular modeling studies. (DOCX)

CD4+ T-cell counts of six uninfected and four SIVmac251 infected macaques. Individual data points, as well as means (± SEM), are shown for each group. (TIF)

Maraviroc decreases T-cell proliferation in vitro. The percentage inhibition of proliferation induced by 0.1 µM MRV in CD4+ T-cells activated with αCD3/αCD28 is shown. Data are shown as mean + SEM and are derived from two experiments. CM: central memory; TM: transitional memory; EM: effector memory. P = 0.0417; Friedman's test. (TIF)

ELISPOT analysis of the number of interferon-γ secreting cells/1.5 * 105 PBMCs. The analyses refers to A: macaque P252 and B: macaque P177. The time points selected are shown as days from the zero point adopted in figure 8 of the main text. (TIF)

Mathematical modeling and numerical simulations. (DOCX)

Retrospective analysis of the response of SIV-mac251 to maraviroc in vivo . (DOCX)

In vitro measurement of the effect of MRV on T-cell proliferation. (DOCX)

Stably suppressed viremia during ART is essential for establishing reliable simian models for HIV/AIDS. We tested the efficacy of a multidrug ART (highly intensified ART) in a wide range of viremic conditions (10³-10⁷) viral RNA copies/mL) in SIVmac251-infected rhesus macaques, and its impact on the viral reservoir. Eleven macaques in the pre-AIDS...

A small pool of long-lived memory CD4 T cells harboring the retroviral genome is one main obstacle to HIV eradication. We tested the impact of the gold compound, auranofin, on phenotype and viability of CD4 T cells in vitro, and on persistence of lentiviral reservoir cells in vivo. In-vitro and in-vivo study. The pro-differentiating effect of auran...

Structural alignment of the integrase catalytic core domains (IN CCDs) HIV-1 subtype B (PDB: 1BL3) and SIVmac251 (PDB: 1C6V). The alignment was conducted on structures deposited in the NCBI database using the VAST algorithm embedded in the website. The structures were then visualised using Cn 3D v. 4.1 (available freely from NCBI). The video was cr...

IFD binding mode of raltegravir at the SIVmac251 catalytic site in complex with proviral DNA. Molecular surfaces are shown for IN (gray), catalytic loop (residues 140-149; cyan), metal ions (magenta), 3'-DNA strand (green), and 5'-DNA strand (yellow). This figure was prepared using PyMOL [73].

Sequence alignment of the integrase catalytic core domains from several lentiviruses. For the sequences adopted, see caption of Figure 6.

Three-dimensional coordinates of a theoretical model for raltegravir docking at the SIVmac251 integrase/proviral DNA interface. Three-dimensional coordinates of a theoretical model for raltegravir docking at the SIVmac251 integrase/proviral DNA interface

Correlation between inhibition of p24 production and inhibition of syncytium formation in acutely HIV-1-infected CEMx174 cells. Cells were infected with HIV-1 (IIIB), washed and incubated for five days in the presence or absence of a range of concentrations of raltegravir in a 96-well plate. HIV-1 p24 was quantified in supernatants by commercially...

In this study we successfully created a new approach to ART in SIVmac251 infected nonhuman primates. This drug regimen is entirely based on drugs affecting the pre-integration stages of replication and consists of only two nucleotidic/nucleosidic reverse transcriptase inhibitors (Nt/NRTIs) and raltegravir, a promising new drug belonging to the inte...

Plasmodium parasites lacking plasmepsin 4 (PM4), an aspartic protease that functions in the lysosomal compartment and contributes to hemoglobin digestion, have only a modest decrease in the asexual blood-stage growth rate; however, PM4 deficiency in the rodent malaria parasite Plasmodium berghei results in significantly less virulence than that for...

Structural superimposition of MC2211 (carbon backbone in cyan) and SAHA (vorinostat; carbon backbone in yellow) docking at the HDAC2 catalytic site. SAHA, a non-selective HDACI, displays an amide group in a conformation that does not match that of the class I-selective HDACIs (Figure 3). The other molecular players are displayed in the same fashion...

To study the HDACI response in a cell population, we used quiescently infected T-lymphoid Jurkat cell clones. Two types of cell clones were used: 1) A1, and A2, which have an integrated GFP/Tat construct under control of the HIV-1 LTR; 2) 6.3, and 8.4, which contain the entire HIV-1 genome under control of the LTR and have the GFP gene replacing ne...

Structures and HDAC inhibiting activity of the cited HDACIs. Where data on human HDACs are unavailable, data on maize HD1-B (homologous with human class I HDACs) and HD1-A (homologous with human class II HDACs), or relevant references, are provided.

Cell infection by HIV-1 is inhibited by both the expression of CD38 and a soluble peptide (sCD38p) corresponding to its extracellular membrane-proximal amino acid sequence (amino acids 51 - 74). We show here the effects of PEG conjugation to sCD38p and provide new insights into the mechanisms behind the anti-HIV-1 effects of CD38 and derived peptid...

The purpose of this study was to evaluate the serological response to the recombinant phospholipase D protein of Chlamydophila pneumoniae (CpPLD) in patients with acute coronary syndromes (ACS) and infected by C. pneumoniae. We assessed 100 serum samples from ACS patients and 100 sera from healthy blood donors, all categorized by microimmunefluores...

After the approval of suberoylanilide hydroxamic acid (SAHA, vorinostat, Zolinza) for the treatment of cutaneous T cell lymphoma (CTCL), a number of HDAC inhibitors (HDACi) are currently in Phase II or III clinical trials (alone or in combination) for the treatment of a great number of tumors. In addition to these cancer uses, HDACi can be successf...

Latently infected, resting memory CD4+ T cells and macrophages represent a major obstacle to the eradication of HIV-1. For this purpose, "shock and kill" strategies have been proposed (activation of HIV-1 followed by stimuli leading to cell death). Histone deacetylase inhibitors (HDACIs) induce HIV-1 activation from quiescence, yet class/isoform-se...

The feline AIDS model for HIV-1 treatment failed in the 1990s, due to structural features resembling protease inhibitor (PI) resistant HIV-1 variants. Widespread drug-resistance to PIs now invokes the possibility of rescuing feline immunodeficiency virus (FIV) as a model for PI treatment. We here analyzed susceptibility of FIV to second generation...

Ramachandran plot for the homology-based model of FIV integrase catalytic core domain. The output of an analysis conducted using MolProbity (see Ref. [41]) is shown.

Sensitivity and reproducibility of the real-time quantitative assay and melting curve profile of specific amplicons. The text describes the experiments devised for validation of the real-time PCR assays adopted in the present study.

Real-time quantitative assay. Sensitivity and reproducibility of the test (Panel A) and melting curve profile (Panel B). Panel A: Graphical representation of the DNA standard curve (ranging from 107 to 102 copies per reaction) based on the recombinant plasmid pGEM-T easy vector carrying the specific 159 bp integrase core fragment. The corresponding...

A two-metal model of HIV-1 integrase. This file contains the 3D coordinates of the two-metal model of HIV-1 integrase described in the paper. Although the extension is .txt, it complies with a .pdb format. It can thus be opened with either programs such as MS Word or 3D molecular viewers such as the Swiss-PDB Viewer.

L-731,988 in complex with HIV-1 integrase catalytic core domain. This file contains the 3D coordinates of diketo acid, L-731,988, as docked at the model of HIV-1 integrase core domain in Additional file 1. Its format is the same as that of Additional file 1, and can be opened in combination with it so as to obtain a full view of this compound in co...

L-870,812 in complex with HIV-1 integrase catalytic core domain. This file contains the 3D coordinates of naphthyridine carboxamide, L-870,812, as docked at the model of HIV-1 integrase core domain in Additional file 1. Format and instructions for opening are the same as for Additional file 2.

L-731,988 in complex with HIV-1 integrase catalytic core domain. This file contains the 3D coordinates of diketo acid analogue, S-1360, as docked at the model of HIV-1 integrase core domain in Additional file 1. Format and instructions for opening are the same as for Additional file 2

L-870,810 in complex with HIV-1 integrase catalytic core domain. This file contains the 3D coordinates of naphthyridine carboxamide, L-870,810, as docked at the model of HIV-1 integrase core domain in Additional file 1 Format and instructions for opening are the same as for Additional file 2.

GS-9137 in complex with HIV-1 integrase catalytic core domain. This file contains the 3D coordinates of quinolone integrase inhibitor GS-9137, as docked at the model of HIV-1 integrase core domain in Additional file 1. Format and instructions for opening are the same as for Additional file 2.