Publications by authors named "Andrea Cara"

60 Publications

Integrase-Defective Lentiviral Vector Is an Efficient Vaccine Platform for Cancer Immunotherapy.

Viruses 2021 Feb 23;13(2). Epub 2021 Feb 23.

Department of Infectious Diseases, Istituto Superiore di Sanità, 00161 Rome, Italy.

Integrase-defective lentiviral vectors (IDLVs) have been used as a safe and efficient delivery system in several immunization protocols in murine and non-human primate preclinical models as well as in recent clinical trials. In this work, we validated in preclinical murine models our vaccine platform based on IDLVs as delivery system for cancer immunotherapy. To evaluate the anti-tumor activity of our vaccine strategy we generated IDLV delivering ovalbumin (OVA) as a non-self-model antigen and TRP2 as a self-tumor associated antigen (TAA) of melanoma. Results demonstrated the ability of IDLVs to eradicate and/or controlling tumor growth after a single immunization in preventive and therapeutic approaches, using lymphoma and melanoma expressing OVA. Importantly, LV-TRP2 but not IDLV-TRP2 was able to break tolerance efficiently and prevent tumor growth of B16F10 melanoma cells. In order to improve the IDLV efficacy, the human homologue of murine TRP2 was used, showing the ability to break tolerance and control the tumor growth. These results validate the use of IDLV for cancer therapy.
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http://dx.doi.org/10.3390/v13020355DOI Listing
February 2021

Therapeutic vaccination with IDLV-SIV-Gag results in durable viremia control in chronically SHIV-infected macaques.

NPJ Vaccines 2020 May 8;5(1):36. Epub 2020 May 8.

Department of Medicine, Duke University Medical Center, Durham, NC, USA.

Despite incredible scientific efforts, there is no cure for HIV infection. While antiretroviral treatment (ART) can help control the virus and prevent transmission, it cannot eradicate HIV from viral reservoirs established before the initiation of therapy. Further, HIV-infected individuals reliably exhibit viral rebound when ART is interrupted, suggesting that the host immune response fails to control viral replication in persistent reservoirs. Therapeutic vaccines are one current approach to improving antiviral host immune responses and enhance long term virus control. In the present study, we used an integrase defective lentiviral vector (IDLV) expressing SIV-Gag to boost anti-Gag specific immune responses in macaques chronically infected with the tier-2 SHIV-1157(QNE)Y173H. A single immunization with IDLV-SIV-Gag induced durable (>20 weeks) virus control in 55% of the vaccinated macaques, correlating with an increased magnitude of SIV-Gag specific CD8+ T-cell responses. IDLV-based therapeutic vaccines are therefore an effective approach to improve virus specific CD8+ T-cell responses and mediate virus control.
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http://dx.doi.org/10.1038/s41541-020-0186-5DOI Listing
May 2020

Prognostic Value of the Hemoglobin/Red Cell Distribution Width Ratio in Resected Lung Adenocarcinoma.

Cancers (Basel) 2021 Feb 9;13(4). Epub 2021 Feb 9.

Department of Thoracic Surgery, IEO European Institute of Oncology IRCCS, 20141 Milan, Italy.

Background: The ratio of hemoglobin to red cell distribution width (HRR) has been described as an effective prognostic factor in several types of cancer. The aim of this study was to investigate the prognostic role of preoperative HRR in resected-lung-adenocarcinoma patients.

Methods: We enrolled 342 consecutive patients. Age, sex, surgical resection, adjuvant treatments, pathological stage, preoperative hemoglobin, red cell distribution width, and their ratio were recorded for each patient.

Results: Mean age was 66 years (SD: 9.0). There were 163 females (47.1%); 169 patients (49.4%) had tumors at stage I, 71 (20.8%) at stage II, and 102 (29.8%) at stage III. In total, 318 patients (93.0%) underwent lobectomy, and 24 (7.0%) pneumonectomy. Disease-free survival multivariable analysis disclosed an increased hazard ratio (HR) of relapse for preoperative HRR lower than 1.01 (HR = 2.20, 95%CI: (1.30-3.72), = 0.004), as well as for N1 single-node (HR = 2.55, 95%CI: (1.33-4.90), = 0.005) and multiple-level lymph node involvement compared to N0 for both N1 (HR = 9.16, 95%CI:(3.65-23.0), < 0.001) and N2 (HR = 10.5, 95%CI:(3.44-32.2, < 0.001).

Conclusion: Pre-operative HRR is an effective prognostic factor of disease-free survival in resected-lung-adenocarcinoma patients, together with the level of pathologic node involvement.
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http://dx.doi.org/10.3390/cancers13040710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916257PMC
February 2021

Antibiotics in Bone Cements Used for Prosthesis Fixation: An Efficient Way to Prevent and Prosthetic Joint Infection.

Front Med (Lausanne) 2020 20;7:576231. Epub 2021 Jan 20.

Centre International de Recherche en Infectiologie, CIRI, Inserm U1111, CNRS UMR5308, ENS de Lyon, UCBL1, Lyon, France.

Prosthetic joint infections (PJIs) are one of the most frequent reasons for arthroplasty revision. These infections are mostly associated with the formation of biofilm, notably by staphylococci, such as and . To minimize the rates of PJIs following primary or revision total joint arthroplasty, antibiotic-loaded bone cements (ALBCs) can be used for prosthesis fixation. However, its use is still debated. Indeed, various studies reported opposite results. In this context, we aimed to compare the prophylactic anti-biofilm activity of ALBCs loaded with two antibiotics with ALBC loaded with only one antibiotic. We compared commercial ready-to-use cements containing gentamicin alone, gentamicin plus vancomycin, and gentamicin plus clindamycin to plain cement (no antibiotic), investigating staphylococcal biofilm formation for 10 strains of and with specific resistance to gentamicin, vancomycin, or clindamycin. Firstly, we performed disk diffusion assays with the elution solutions. We reported that only the cement containing gentamicin and clindamycin was able to inhibit bacterial growth at Day 9, whereas cements with gentamicin only or gentamicin and vancomycin lost their antibacterial activity at Day 3. Then, we observed that all the tested ALBCs can inhibit biofilm formation by methicillin-susceptible staphylococci without other antibiotic resistance ability. Similar results were observed when we tested vancomycin-resistant or clindamycin-resistant staphylococci, with some strain-dependent significant increase of efficacy for the two antibiotic ALBCs when compared with gentamicin-loaded cement. However, adding vancomycin or clindamycin to gentamicin allows a better inhibition of biofilm formation when gentamicin-resistant strains were used. Our results suggest that using commercially available bone cements loaded with gentamicin plus vancomycin or clindamycin for prosthesis fixation can help in preventing staphylococcal PJIs following primary arthroplasties, non-septic revisions or septic revisions, especially to prevent PJIs caused by gentamicin-resistant staphylococci.
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http://dx.doi.org/10.3389/fmed.2020.576231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856860PMC
January 2021

Robust neutralizing antibodies to SARS-CoV-2 develop and persist in subjects with diabetes and COVID-19 pneumonia.

J Clin Endocrinol Metab 2021 Jan 29. Epub 2021 Jan 29.

Diabetes Research Institute, IRCCS Ospedale San Raffaele, Milan, Italy.

Purpose: The aim of this study was to characterise the kinetics and durability of neutralizing antibody (Nab) response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the presence of hyperglycemia.

Methods: Using a lentiviral-vector-based SARS-CoV-2 neutralization assay to measure Nabs, we characterised 150 patients randomly selected from a cohort of 509 patients with confirmed COVID-19 pneumonia. We analysed Nab response according to the presence of diabetes or hyperglycaemia, at the time of hospitalisation and during the post discharge follow-up, i.e., 1, 3 and 6 months outpatient visits.

Results: Among 150 randomly selected patients 40 (26.6%) had diabetes. Diabetes (HR 8.9, p < 0.001), glucose levels (HR 1.25 x 1.1 mmol/L, p < 0.001) and glucose variability (HR 1.17 x 0.6 mmol/L, p < 0.001) were independently associated with an increased risk of mortality. The neutralizing activity of SARS-CoV-2 antibodies in patients with diabetes was superimposable, as for kinetics and extent, to that of patients without diabetes. It was similar across glucose levels and correlated with the humoral response against the SARS-CoV-2 spike protein. Positivity for Nabs at the time of hospital admission conferred protection on mortality, both in the presence (HR 0.28, p=0.046) or absence of diabetes (HR 0.26, p=0.030). The longevity of the Nab response was not affected by diabetes.

Conclusions: Diabetes and hyperglycaemia do not affect the kinetics and durability of the neutralizing antibody response to SARS-CoV-2. These findings provide the rational to include patients with diabetes in the early phase of the vaccination campaign against SARS-CoV-2.
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http://dx.doi.org/10.1210/clinem/dgab055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928901PMC
January 2021

Integrase-Defective Lentiviral Vectors for Delivery of Monoclonal Antibodies against Influenza.

Viruses 2020 12 17;12(12). Epub 2020 Dec 17.

Department of Medicine, Weill Cornell Medical College, New York, NY 10021, USA.

Delivering rapid protection against infectious agents to non-immune populations is a formidable public health challenge. Although passive immunotherapy is a fast and effective method of protection, large-scale production and administration of monoclonal antibodies (mAbs) is expensive and unpractical. Viral vector-mediated delivery of mAbs offers an attractive alternative to their direct injection. Integrase-defective lentiviral vectors (IDLV) are advantageous for this purpose due to the absence of pre-existing anti-vector immunity and the safety features of non-integration and non-replication. We engineered IDLV to produce the humanized mAb VN04-2 (IDLV-VN04-2), which is broadly neutralizing against H5 influenza A virus (IAV), and tested the vectors' ability to produce antibodies and protect from IAV in vivo. We found that IDLV-transduced cells produced functional VN04-2 mAbs in a time- and dose-dependent fashion. These mAbs specifically bind the hemagglutinin (HA), but not the nucleoprotein (NP) of IAV. VN04-2 mAbs were detected in the serum of mice at different times after intranasal (i.n.) or intramuscular (i.m.) administration of IDLV-VN04-2. Administration of IDLV-VN04-2 by the i.n. route provided rapid protection against lethal IAV challenge, although the protection did not persist at later time points. Our data suggest that administration of mAb-expressing IDLV may represent an effective strategy for rapid protection against infectious diseases.
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http://dx.doi.org/10.3390/v12121460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7767071PMC
December 2020

Mild SARS-CoV-2 Infection After Gene Therapy in a Child With Wiskott-Aldrich Syndrome: A Case Report.

Front Immunol 2020 24;11:603428. Epub 2020 Nov 24.

Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy.

In this work we present the case of SARS-CoV-2 infection in a 1.5-year-old boy affected by severe Wiskott-Aldrich Syndrome with previous history of autoinflammatory disease, occurring 5 months after treatment with gene therapy. Before SARS-CoV-2 infection, the patient had obtained engraftment of gene corrected cells, resulting in WASP expression restoration and early immune reconstitution. The patient produced specific immunoglobulins to SARS-CoV-2 at high titer with neutralizing capacity and experienced a mild course of infection, with limited inflammatory complications, despite pre-gene therapy clinical phenotype.
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http://dx.doi.org/10.3389/fimmu.2020.603428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732473PMC
December 2020

Immunogenicity, safety, and efficacy of sequential immunizations with an SIV-based IDLV expressing CH505 Envs.

NPJ Vaccines 2020 Nov 18;5(1):107. Epub 2020 Nov 18.

Department of Medicine, Duke University School of Medicine, Durham, NC, USA.

A preventative HIV-1 vaccine is an essential intervention needed to halt the HIV-1 pandemic. Neutralizing antibodies protect against HIV-1 infection in animal models, and thus an approach toward a protective HIV-1 vaccine is to induce broadly cross-reactive neutralizing antibodies (bnAbs). One strategy to achieve this goal is to define envelope (Env) evolution that drives bnAb development in infection and to recreate those events by vaccination. In this study, we report the immunogenicity, safety, and efficacy in rhesus macaques of an SIV-based integrase defective lentiviral vector (IDLV) expressing sequential gp140 Env immunogens derived from the CH505 HIV-1-infected individual who made the CH103 and CH235 bnAb lineages. Immunization with IDLV expressing sequential CH505 Envs induced higher magnitude and more durable binding and neutralizing antibody responses compared to protein or DNA +/- protein immunizations using the same sequential envelopes. Compared to monkeys immunized with a vector expressing Envs alone, those immunized with the combination of IDLV expressing Env and CH505 Env protein demonstrated improved durability of antibody responses at six months after the last immunization as well as lower peak viremia and better virus control following autologous SHIV-CH505 challenge. There was no evidence of vector mobilization or recombination in the immunized and challenged monkeys. Although the tested vaccines failed to induce bnAbs and to mediate significant protection following SHIV-challenge, our results show that IDLV proved safe and successful at inducing higher titer and more durable immune responses compared to other vaccine platforms.
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http://dx.doi.org/10.1038/s41541-020-00252-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674457PMC
November 2020

Lung Recruitability of COVID-19 Pneumonia in Patients Undergoing Helmet CPAP.

Arch Bronconeumol 2021 Jan 22;57 Suppl 1:92-94. Epub 2020 Oct 22.

Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Respiratory Unit and Cystic Fibrosis Adult Center, Milan, Italy; Università degli Studi di Milano, Department of Pathophysiology and Transplantation, Milan, Italy.

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http://dx.doi.org/10.1016/j.arbres.2020.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7577873PMC
January 2021

Therapeutic vaccination with IDLV-SIV-Gag results in durable viremia control in chronically SHIV-infected macaques.

NPJ Vaccines 2020 8;5:36. Epub 2020 May 8.

1Department of Medicine, Duke University Medical Center, Durham, NC USA.

Despite incredible scientific efforts, there is no cure for HIV infection. While antiretroviral treatment (ART) can help control the virus and prevent transmission, it cannot eradicate HIV from viral reservoirs established before the initiation of therapy. Further, HIV-infected individuals reliably exhibit viral rebound when ART is interrupted, suggesting that the host immune response fails to control viral replication in persistent reservoirs. Therapeutic vaccines are one current approach to improving antiviral host immune responses and enhance long term virus control. In the present study, we used an integrase defective lentiviral vector (IDLV) expressing SIV-Gag to boost anti-Gag specific immune responses in macaques chronically infected with the tier-2 SHIV-1157(QNE)Y173H. A single immunization with IDLV-SIV-Gag induced durable (>20 weeks) virus control in 55% of the vaccinated macaques, correlating with an increased magnitude of SIV-Gag specific CD8+ T-cell responses. IDLV-based therapeutic vaccines are therefore an effective approach to improve virus specific CD8+ T-cell responses and mediate virus control.
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http://dx.doi.org/10.1038/s41541-020-0186-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7210278PMC
May 2020

Skeletal Muscle Is an Antigen Reservoir in Integrase-Defective Lentiviral Vector-Induced Long-Term Immunity.

Mol Ther Methods Clin Dev 2020 Jun 13;17:532-544. Epub 2020 Mar 13.

Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.

We previously developed integrase-defective lentiviral vectors (IDLVs) as an antigen delivery system for inducing strong and prolonged immunity in animal models. Here, we examined the association between persistence of antigen expression and durability of immune response. Following a single intramuscular (i.m.) or subcutaneous (s.c.) injection of IDLV delivering GFP in mice, we evaluated antigen expression and inflammation at the site of injection and persistence of antigen-specific T cells at early and late time points. Durable antigen expression was detected up to 90 days only after i.m. immunization. Mononuclear inflammation was evident soon after IDLV injection in both i.m. and s.c. immunized mice, but remained detectable up to 30 days postinjection only in i.m. immunized mice. Similarly, GFP-specific T cells were more persistent in the i.m. immunized mice. Interestingly, GFP muscle fibers were co-expressing major histocompatibility complex (MHC) class I, suggesting that muscle cells are competent for presenting antigens to T cells . In experiments, we demonstrated that although both primary myoblasts and myocytes present the antigen to GFP-specific T cells through MHC class I, myoblasts are more resistant to Fas-dependent cytotoxic T lymphocyte (CTL) killing activity. Overall, these data indicate that muscle cells may serve as an antigen reservoir that contributes to the long-term immunity induced by IDLV vaccination.
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http://dx.doi.org/10.1016/j.omtm.2020.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7114633PMC
June 2020

Development and Preclinical Evaluation of an Integrase Defective Lentiviral Vector Vaccine Expressing the HIVACAT T Cell Immunogen in Mice.

Mol Ther Methods Clin Dev 2020 Jun 4;17:418-428. Epub 2020 Feb 4.

National Center for Global Health, Istituto Superiore di Sanità, Rome, Italy.

Cellular immune responses play a fundamental role in controlling viral replication and AIDS progression in human immunodeficiency virus (HIV)-infected subjects and in simian immunodeficiency virus (SIV)-infected macaques. Integrase defective lentiviral vector (IDLV) represents a promising vaccine candidate, inducing functional and durable immune responses in mice and non-human primates. Here, we designed HIV- and SIV-based IDLVs to express the HIVACAT T cell immunogen (HTI), a mosaic antigen designed to cover vulnerable sites in HIV-1 Gag, Pol, Vif, and Nef. We observed that HTI expression during lentiviral vector production interfered profoundly with IDLV particles release because of sequestration of both HIV- and SIV-Gag proteins in the cytoplasm of the vector-producing cells. However, modifications in IDLV design and vector production procedures greatly improved recovery of both HIV- and SIV-based IDLV-HTI. Immunization experiments in BALB/c mice showed that both IDLVs elicited HTI-specific T cell responses. However, immunization with HIV-based IDLV elicited also a T cell response toward exogenous HIV proteins in IDLV particles, suggesting that SIV-based IDLV may be a preferable platform to assess the induction of transgene-specific immune responses against rationally designed HIV structural antigens. These data support the further evaluation of IDLV as an effective platform of T cell immunogens for the development of an effective HIV vaccine.
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http://dx.doi.org/10.1016/j.omtm.2020.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056611PMC
June 2020

Persistence of Integrase-Deficient Lentiviral Vectors Correlates with the Induction of STING-Independent CD8 T Cell Responses.

Cell Rep 2019 01;26(5):1242-1257.e7

Institut Pasteur, Unité de Régulation Immunitaire et Vaccinologie, Equipe Labellisée Ligue Contre le Cancer, 75015 Paris, France; INSERM U1041, 75015 Paris, France. Electronic address:

Lentiviruses are among the most promising viral vectors for in vivo gene delivery. To overcome the risk of insertional mutagenesis, integrase-deficient lentiviral vectors (IDLVs) have been developed. We show here that strong and persistent specific cytotoxic T cell (CTL) responses are induced by IDLVs, which persist several months after a single injection. These responses were associated with the induction of mild and transient maturation of dendritic cells (DCs) and with the production of low levels of inflammatory cytokines and chemokines. They were independent of the IFN-I, TLR/MyD88, interferon regulatory factor (IRF), retinoic acid induced gene I (RIG-I), and stimulator of interferon genes (STING) pathways but require NF-κB signaling in CD11c DCs. Despite the lack of integration of IDLVs, the transgene persists for 3 months in the spleen and liver of IDLV-injected mice. These results demonstrate that the capacity of IDLVs to trigger persistent adaptive responses is mediated by a weak and transient innate response, along with the persistence of the vector in tissues.
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http://dx.doi.org/10.1016/j.celrep.2019.01.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679900PMC
January 2019

IDLV-HIV-1 Env vaccination in non-human primates induces affinity maturation of antigen-specific memory B cells.

Commun Biol 2018 5;1:134. Epub 2018 Sep 5.

Department of Medicine, Duke University Medical Center, Durham, 27710, NC, USA.

HIV continues to be a major global health issue. In spite of successful prevention interventions and treatment methods, the development of an HIV vaccine remains a major priority for the field and would be the optimal strategy to prevent new infections. We showed previously that a single immunization with a SIV-based integrase-defective lentiviral vector (IDLV) expressing the 1086.C HIV-1-envelope induced durable, high-magnitude immune responses in non-human primates (NHPs). In this study, we have further characterized the humoral responses by assessing antibody affinity maturation and antigen-specific memory B-cell persistence in two vaccinated macaques. These animals were also boosted with IDLV expressing the heterologous 1176.C HIV-1-Env to determine if neutralization breadth could be increased, followed by evaluation of the injection sites to assess IDLV persistence. IDLV-Env immunization was associated with persistence of the vector DNA for up to 6 months post immunization and affinity maturation of antigen-specific memory B cells.
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http://dx.doi.org/10.1038/s42003-018-0131-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125466PMC
September 2018

A steam-based method to investigate biofilm.

Sci Rep 2018 08 29;8(1):13040. Epub 2018 Aug 29.

CIRI - Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Univ Lyon, Lyon, France.

Biofilm has become a major topic of interest in medical, food, industrial, and environmental bacteriology. To be relevant, investigation of biofilm behavior requires effective and reliable techniques. We present herein a simple and robust method, adapted from the microplate technique, in which steam is used as a soft washing method to preserve biofilm integrity and to improve reproducibility of biofilm quantification. The kinetics of steam washing indicated that the method is adapted to remove both planktonic bacteria and excess crystal violet (CV) staining for S. aureus, S. epidermidis, S. carnosus, P. aeruginosa, and E. coli biofilm. Confocal laser scanning microscopy confirmed that steam washing preserved the integrity of the biofilm better than pipette-based washing. We also investigated the measurement of the turbidity of biofilm resuspended in phosphate-buffered saline (PBS) as an alternative to staining with CV. This approach allows the discrimination of biofilm producer strains from non-biofilm producer strains in a way similar to CV staining, and subsequently permits quantification of viable bacteria present in biofilm by culture enumeration from the same well. Biofilm quantification using steam washing and PBS turbidity reduced the technical time needed, and data were highly reproducible.
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http://dx.doi.org/10.1038/s41598-018-31437-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115380PMC
August 2018

Integrase Defective Lentiviral Vector as a Vaccine Platform for Delivering Influenza Antigens.

Front Immunol 2018 5;9:171. Epub 2018 Feb 5.

Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy.

Viral vectors represent an attractive technology for vaccine delivery. We exploited the integrase defective lentiviral vector (IDLV) as a platform for delivering relevant antigens within the context of the ADITEC collaborative research program. In particular, Influenza virus hemagglutinin (HA) and nucleoprotein (NP) were delivered by IDLVs while H1N1 A/California/7/2009 subunit vaccine (HAp) with or without adjuvant was used to compare the immune response in a murine model of immunization. In order to maximize the antibody response against HA, both IDLVs were also pseudotyped with HA (IDLV-HA/HA and IDLV-NP/HA, respectively). Groups of CB6F1 mice were immunized intramuscularly with a single dose of IDLV-NP/HA, IDLV-HA/HA, HAp alone, or with HAp together with the systemic adjuvant MF59. Six months after the vaccine prime all groups were boosted with HAp alone. Cellular and antibody responses to influenza antigens were measured at different time points after the immunizations. Mice immunized with HA-pseudotyped IDLVs showed similar levels of anti-H1N1 IgG over time, evaluated by ELISA, which were comparable to those induced by HAp + MF59 vaccination, but significantly higher than those induced by HAp alone. The boost with HAp alone induced an increase of antibodies in all groups, and the responses were maintained at higher levels up to 18 weeks post-boost. The antibody response was functional and persistent overtime, capable of neutralizing virus infectivity, as evaluated by hemagglutination inhibition and microneutralization assays. Moreover, since neuraminidase (NA)-expressing plasmid was included during IDLV preparation, immunization with IDLV-NP/HA and IDLV-HA/HA also induced functional anti-NA antibodies, evaluated by enzyme-linked lectin assay. IFNγ-ELISPOT showed evidence of HA-specific response in IDLV-HA/HA immunized animals and persistent NP-specific CD8+ T cell response in IDLV-NP/HA immunized mice. Taken together our results indicate that IDLV can be harnessed for producing a vaccine able to induce a comprehensive immune response, including functional antibodies directed toward HA and NA proteins present on the vector particles in addition to a functional T cell response directed to the protein transcribed from the vector.
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http://dx.doi.org/10.3389/fimmu.2018.00171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5807328PMC
April 2019

A high susceptibility to redox imbalance of the transmissible stages of Plasmodium falciparum revealed with a luciferase-based mature gametocyte assay.

Mol Microbiol 2017 04 21;104(2):306-318. Epub 2017 Feb 21.

Dipartimento di Malattie Infettive, Parassitarie ed Immunomediate, Istituto Superiore di Sanità, Rome, Italy.

The goal to prevent Plasmodium falciparum transmission from humans to mosquitoes requires the identification of targetable metabolic processes in the mature (stage V) gametocytes, the sexual stages circulating in the bloodstream. This task is complicated by the apparently low metabolism of these cells, which renders them refractory to most antimalarial inhibitors and constrains the development of specific and sensitive cell-based assays. Here, we identify and functionally characterize the regulatory regions of the P. falciparum gene PF3D7_1234700, encoding a CPW-WPC protein and named here Upregulated in Late Gametocytes (ULG8), which we have leveraged to express reporter genes in mature male and female gametocytes. Using transgenic parasites containing a pfULG8-luciferase cassette, we investigated the susceptibility of stage V gametocytes to compounds specifically affecting redox metabolism. Our results reveal a high sensitivity of mature gametocytes to the glutathione reductase inhibitor and redox cycler drug methylene blue (MB). Using isobologram analysis, we find that a concomitant inhibition of the parasite enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase, a key component of NADPH synthesis, potently synergizes MB activity. These data suggest that redox metabolism and detoxification activity play an unsuspected yet vital role in stage V gametocytes, rendering these cells exquisitely sensitive to decreases in NADPH concentration.
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http://dx.doi.org/10.1111/mmi.13626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5380559PMC
April 2017

Immunization with an SIV-based IDLV Expressing HIV-1 Env 1086 Clade C Elicits Durable Humoral and Cellular Responses in Rhesus Macaques.

Mol Ther 2016 Nov 21;24(11):2021-2032. Epub 2016 Jun 21.

Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA; Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Rome, Italy. Electronic address:

The design of an effective HIV-1 vaccine remains a major challenge. Several vaccine strategies based on viral vectors have been evaluated in preclinical and clinical trials, with largely disappointing results. Integrase defective lentiviral vectors (IDLV) represent a promising vaccine candidate given their ability to induce durable and protective immune responses in mice after a single immunization. Here, we evaluated the immunogenicity of a SIV-based IDLV in nonhuman primates. Six rhesus monkeys were primed intramuscularly with IDLV-Env and boosted with the same vector after 1 year. A single immunization with IDLV-Env induced broad humoral and cellular immune responses that waned over time but were still detectable at 1 year postprime. The boost with IDLV-Env performed at 1 year from the prime induced a remarkable increase in both antibodies and T-cell responses. Antibody binding specificity showed a predominant cross-clade gp120-directed response. Monkeys' sera efficiently blocked anti-V2 and anti-CD4 binding site antibodies, neutralized the tier 1 MW965.26 pseudovirus and mediated antibody-dependent cellular cytotoxicity (ADCC). Durable polyfunctional Env-specific T-cell responses were also elicited. Our study demonstrates that an IDLV-Env-based vaccine induces functional, comprehensive, and durable immune responses in Rhesus macaques. These results support further evaluation of IDLV as a new HIV-1 vaccine delivery platform.
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http://dx.doi.org/10.1038/mt.2016.123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154473PMC
November 2016

HIV-1 DNA dynamics and variations in HIV-1 DNA protease and reverse transcriptase sequences in multidrug-resistant patients during successful raltegravir-based therapy.

J Med Virol 2016 12 30;88(12):2115-2124. Epub 2016 May 30.

Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Rome, Italy.

There is limited information on the variations of HIV-1 DNA mutation profile in reverse transcriptase (RT) and protease (PR) genes during suppressive antiretroviral treatment (plasma HIV-1 RNA continuously <50 copies/ml) with raltegravir (RAL)-based regimens in patients with baseline RT/PR resistant HIV. Twelve multidrug resistant (RT: 12/12, PR: 8/12) HIV-infected patients were followed during effectively suppressive RAL-based therapy. Total and integrated HIV-1 DNA were assessed by real time PCR at baseline and every 6 months. Ultrasensitive (threshold: 2.5 copies/ml) plasma HIV-1 RNA and genotypic analysis of RT and PR in proviral DNA were performed at baseline and at 24 months. Half of the patients had full viral suppression (plasma HIV-RNA < 2.5 copies/ml) at month 12. Total HIV-1 DNA declined significantly after 12 months of therapy (from 249.2 to 145.7 copies/10 cells, P = 0.023), and remained stable until 24 months, when total HIV-1 DNA levels raised, concomitantly with a less stringent suppression of HIV-1 RNA (81.8% of patients with >2.5 copies/ml). Integrated HIV-1 DNA did not show fluctuations during the study period. Sequencing of the PR and RT regions from HIV-1 DNA revealed changes in the resistance mutation profile in five patients. Total HIV-1 DNA declined after the introduction of RAL-based therapy, with a rebound after 2 years. No changes were observed in levels of integrated DNA, suggesting limited effect on archived HIV. The RT and PR sequence changes in archived HIV-1 DNA suggest that variation of the mutation profile can occur even in the absence of detectable HIV-1 RNA. J. Med. Virol. 88:2115-2124, 2016. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jmv.24581DOI Listing
December 2016

Identification of HIV-1 genitourinary tract compartmentalization by analyzing the env gene sequences in urine.

AIDS 2015 Aug;29(13):1651-7

aDuke University Medical Center, Durham, North Carolina, USA bIstituto Superiore di Sanità, Rome, Italy.

Objective: HIV-1 persists indefinitely in memory CD4 T cells and other long-lived cellular reservoirs despite antiretroviral therapy. Our group had previously demonstrated that HIV-1 can establish a productive infection in renal epithelial cells and that the kidney represents a separate compartment for HIV-1 replication. Here, to better understand the viruses in this unique site, we genetically characterized and compared the viruses in blood and urine specimens from 24 HIV-1 infected patients with detectable viremia.

Design And Methods: Blood and urine samples were obtained from 35 HIV-1 positive patients. Single-genome amplification was performed on HIV-1 env RNA and DNA isolated from urine supernatants and urine-derived cell pellets, respectively, as well as from plasma and peripheral blood mononuclear cell from the same individuals. Neighbor-joining trees were constructed under the Kimura 2-parameter model.

Results: We amplified and sequenced the full-length HIV-1 envelope (env) gene from 12 of the 24 individuals, indicating that 50% of the viremic HIV-1-positive patients had viral RNA in their urine. Phylogenetic analysis of the env sequences from four individuals with more than 15 urine-derived env sequences showed that the majority of the sequences from urine formed distinct cluster(s) independent of those peripheral blood mononuclear cell and plasma-derived sequences, consistent with viral compartmentalization in the urine.

Conclusion: Our results suggest the presence of a distinct HIV compartment in the genitourinary tract.
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http://dx.doi.org/10.1097/QAD.0000000000000757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4710139PMC
August 2015

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

Retrovirology 2015 Jan 22;12. Epub 2015 Jan 22.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

Background: 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. Moreover, 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.

Results: CCL2 neutralization potently reduced the number of p24 Gag+ cells during the course of either productive or single cycle infection with HIV-1. 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. Notably, the accumulation of viral DNA, both total, integrated and 2-LTR circles, was strongly impaired by neutralization of CCL2. Looking for correlates of HIV-1 DNA accumulation inhibition, we found that the antiviral effect of CCL2 neutralization was independent of the modulation 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 replication mediated by CCL2 blocking. Interestingly, the CCL2 neutralization mediated increase of APOBEC3A expression was type I IFN independent. Moreover, the transcriptome analysis of the effect of CCL2 blocking on global gene expression revealed that the neutralization of this chemokine resulted in the upmodulation of additional genes involved in the defence response to viruses.

Conclusions: Neutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primary MDM affecting post-entry steps of the viral life cycle with a mechanism independent of SAMHD1. In addition, CCL2 blocking is associated with induction of APOBEC3A expression, thus unravelling a novel mechanism which might 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 defences against HIV-1 and protecting macrophages from infection.
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http://dx.doi.org/10.1186/s12977-014-0132-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4314729PMC
January 2015

Murine granulocyte-macrophage colony-stimulating factor expressed from a bicistronic simian immunodeficiency virus-based integrase-defective lentiviral vector does not enhance T-cell responses in mice.

Viral Immunol 2014 Dec;27(10):512-20

1 Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità , Rome, Italy .

As a prelude to immunization studies in nonhuman primates, we compared in mice the immunogenicity of a simian immunodeficiency virus (SIV)-based integrase (IN)-defective lentiviral vector (IDLV) encoding the model antigen-enhanced green fluorescence protein (eGFP) in the presence or absence of the murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) expressed from an internal ribosomal entry site (IRES) sequence. BALB/c mice were immunized once intramuscularly with IDLV expressing eGFP alone or eGFP and mGM-CSF and immune responses were evaluated up to 90 days from the single intramuscular immunization. Results indicated that the mGM-CSF was unable to improve the magnitude and quality of the immune response against the eGFP transgene in the context of the SIV-based IDLV, as evaluated by enzyme-linked immunosorbent spot (ELISPOT) assays for interferon-γ (IFN-γ) and by intracellular cytokine staining for IFN-γ, interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-α). These findings suggest that for vaccination purposes, the presence of mGM-CSF expressed after the IRES in a SIV-based IDLV system does not favor the improvement of the immunological response against the transgene of interest. Further studies should investigate whether the selection of a different cytokine gene might improve the immune response against the transgene.
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http://dx.doi.org/10.1089/vim.2014.0062DOI Listing
December 2014

Optimization of mucosal responses after intramuscular immunization with integrase defective lentiviral vector.

PLoS One 2014 11;9(9):e107377. Epub 2014 Sep 11.

Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.

Many infectious agents infiltrate the host at the mucosal surfaces and then spread systemically. This implies that an ideal vaccine should induce protective immune responses both at systemic and mucosal sites to counteract invasive mucosal pathogens. We evaluated the in vivo systemic and mucosal antigen-specific immune response induced in mice by intramuscular administration of an integrase defective lentiviral vector (IDLV) carrying the ovalbumin (OVA) transgene as a model antigen (IDLV-OVA), either alone or in combination with sublingual adjuvanted OVA protein. Mice immunized intramuscularly with OVA and adjuvant were compared with IDLV-OVA immunization. Mice sublingually immunized only with OVA and adjuvant were used as a positive control of mucosal responses. A single intramuscular dose of IDLV-OVA induced functional antigen-specific CD8+ T cell responses in spleen, draining and distal lymph nodes and, importantly, in the lamina propria of the large intestine. These results were similar to those obtained in a prime-boost regimen including one IDLV immunization and two mucosal boosts with adjuvanted OVA or vice versa. Remarkably, only in groups vaccinated with IDLV-OVA, either alone or in prime-boost regimens, the mucosal CD8+ T cell response persisted up to several months from immunization. Importantly, following IDLV-OVA immunization, the mucosal boost with protein greatly increased the plasma IgG response and induced mucosal antigen-specific IgA in saliva and vaginal washes. Overall, intramuscular administration of IDLV followed by protein boosts using the sublingual route induced strong, persistent and complementary systemic and mucosal immune responses, and represents an appealing prime-boost strategy for immunization including IDLV as a delivery system.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0107377PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161417PMC
October 2015

Multicolor bioluminescence boosts malaria research: quantitative dual-color assay and single-cell imaging in Plasmodium falciparum parasites.

Anal Chem 2014 Sep 15;86(17):8814-21. Epub 2014 Aug 15.

INBB, Istituto Nazionale di Biostrutture e Biosistemi , 00136 Rome, Italy.

New reliable and cost-effective antimalarial drug screening assays are urgently needed to identify drugs acting on different stages of the parasite Plasmodium falciparum, and particularly those responsible for human-to-mosquito transmission, that is, the P. falciparum gametocytes. Low Z' factors, narrow dynamic ranges, and/or extended assay times are commonly reported in current gametocyte assays measuring gametocyte-expressed fluorescent or luciferase reporters, endogenous ATP levels, activity of gametocyte enzymes, or redox-dependent dye fluorescence. We hereby report on a dual-luciferase gametocyte assay with immature and mature P. falciparum gametocyte stages expressing red and green-emitting luciferases from Pyrophorus plagiophthalamus under the control of the parasite sexual stage-specific pfs16 gene promoter. The assay was validated with reference antimalarial drugs and allowed to quantitatively and simultaneously measure stage-specific drug effects on parasites at different developmental stages. The optimized assay, requiring only 48 h incubation with drugs and using a cost-effective luminogenic substrate, significantly reduces assay cost and time in comparison to state-of-the-art analogous assays. The assay had a Z' factor of 0.71 ± 0.03, and it is suitable for implementation in 96- and 384-well microplate formats. Moreover, the use of a nonlysing D-luciferin substrate significantly improved the reliability of the assay and allowed one to perform, for the first time, P. falciparum bioluminescence imaging at single-cell level.
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http://dx.doi.org/10.1021/ac502098wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4151787PMC
September 2014

Renal epithelial cells produce and spread HIV-1 via T-cell contact.

AIDS 2014 Oct;28(16):2345-53

aDuke University Medical Center, Durham, North Carolina bIcahn School of Medicine at Mount Sinai, New York, USA cIstituto Superiore di Sanità, Rome, Italy.

Objectives: Increasing evidence supports the role of the kidney as a reservoir for HIV-1. In-vitro co-cultivation of HIV-infected T cells with renal tubule epithelial (RTE) cells results in virus transfer to the latter, whereas cell-free virus infection is inefficient. We further characterized the fate of HIV-1 after it is internalized in renal epithelial cells.

Methods: Primary or immortalized CD4 cells were infected with a green fluorescent protein (GFP)-expressing replication competent HIV-1. HIV-1 transfer from T cells to RTE cells was carried out in a co-culture system and evaluated by fluorescence-activated cell sorting analysis. HIV-1 integration in renal cells was evaluated by Alu-PCR and the production of infectious particles was assessed by p24-ELISA and TZM-bl assay. HIV-infected renal cells were used as donor cells in a co-culture system to evaluate their ability to transfer the virus back to T cells.

Results: Renal cells become productively infected by HIV-1 and multiple copies of HIV-1 can be transferred from infected T cells to renal cells. Two separate cell populations were identified among infected renal cells based on reporter gene GFP expression level (low vs. high), only the high showing sensitivity to azidothymidine and ritonavir. Co-cultivation of HIV-1-infected renal cells with noninfected T cells resulted in HIV-1 transmission to T cells, supporting bidirectional exchange of virus between T cells and kidney-derived cells. Persistent expression and generation of infectious virus in renal cells required HIV integration.

Conclusion: These results support the kidney as a potential reservoir where virus is exchanged between interstitial T cells and RTE cells.
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http://dx.doi.org/10.1097/QAD.0000000000000398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4721518PMC
October 2014

Mucosal immunization with integrase-defective lentiviral vectors protects against influenza virus challenge in mice.

PLoS One 2014 13;9(5):e97270. Epub 2014 May 13.

Department of Public Health, Weill Medical College of Cornell University, New York, New York, United States of America; Department of Medicine, Weill Medical College of Cornell University, New York, New York, United States of America.

Recent reports highlight the potential for integrase-defective lentiviral vectors (IDLV) to be developed as vaccines due to their ability to elicit cell-mediated and humoral immune responses after intramuscular administration. Differently from their integrase-competent counterpart, whose utility for vaccine development is limited by the potential for insertional mutagenesis, IDLV possess a mutation in their integrase gene that prevents genomic integration. Instead, they are maintained as episomal DNA circles that retain the ability to stably express functional proteins. Despite their favorable profile, it is unknown whether IDLV elicit immune responses after intranasal administration, a route that could be advantageous in the case of infection with a respiratory agent. Using influenza as a model, we constructed IDLV expressing the influenza virus nucleoprotein (IDLV-NP), and tested their ability to generate NP-specific immune responses and protect from challenge in vivo. We found that administration of IDLV-NP elicited NP-specific T cell and antibody responses in BALB/c mice. Importantly, IDLV-NP was protective against homologous and heterosubtypic influenza virus challenge only when given by the intranasal route. This is the first report demonstrating that IDLV can induce protective immunity after intranasal administration, and suggests that IDLV may represent a promising vaccine platform against infectious agents.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0097270PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4019533PMC
December 2014

Effects of raltegravir on 2-long terminal repeat circle junctions in HIV type 1 viremic and aviremic patients.

AIDS Res Hum Retroviruses 2013 Oct 24;29(10):1365-9. Epub 2013 Jul 24.

1 Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità , Rome, Italy .

Although 2-long terminal repeat (2-LTR) circles are only a fraction of the total viral DNA in infected cells, sequence analysis of 2-LTR circles reveals critical information regarding viral DNA synthesis and the nature of actively replicating virus. It was observed that a large proportion of the 2-LTR circular molecules in the peripheral blood mononuclear cell (PBMC) DNA of infected individuals are mutated at the circle junction. The integrase inhibitor raltegravir (RAL) blocks the strand transfer step of the integration of HIV-1; as a consequence of abortive integration a significant increase of episomal 2-LTR circles is observed. Moreover, it was demonstrated that in patients treated with highly active retroviral therapy (HAART) changes in 2-LTR concentration did not affect junction sequences and flanking regions of 2-LTR. Here we evaluated whether RAL therapy could have a differential impact on the 2-LTR circle junctional sequences in patients with different virological profiles at the time of starting RAL therapy. Sequence analysis indicates that RAL acts differently in the two populations.
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http://dx.doi.org/10.1089/AID.2013.0047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3785805PMC
October 2013

No evidence of autoimmune disorders in antiretroviral-experienced HIV-1-infected individuals after long-term treatment with raltegravir.

Antivir Ther 2013 9;18(3):321-7. Epub 2012 Oct 9.

Istituto Superiore di Sanità, Rome, Italy.

Background: The HIV integrase inhibitor raltegravir (RAL) can exacerbate autoimmune diseases in genetically predisposed mice. To evaluate whether this may occur in clinical practice, we clinically monitored HIV-positive patients treated with RAL and measured a panel of autoantibodies (auto-Abs) during the first year of RAL treatment.

Methods: This was a longitudinal study in 109 antiretroviral-experienced patients who started a RAL-based regimen and were followed up for more than 2 years. A total of 45 patients were tested at baseline (before starting RAL) and after 12 months for the presence of the following auto-Abs: anti-nuclear antibodies, anti-double-stranded DNA, anti-smooth-muscle antibodies, anti-thyreoglobulin and anti-thyroid peroxidase antibodies, anti-cardiolipin immunoglobulin G and immunoglobulin M and anti-nuclear extractable antigens, including anti-SM ribonucleoprotein antigen, anti-Ro antigen and anti-La antigen.

Results: A low rate of clinically relevant autoimmune diseases was observed at study entry (3/109; 2.8%; 95% CI 0.004, 0.059). No exacerbations were observed during follow-up. During the second year of RAL-based therapy a previously healthy patient developed psoriasis. At baseline, 17/45 (37.8%) patients tested for the presence of auto-Abs were positive. Most patients (n=13) were positive for anti-cardiolipin. After 12 months of RAL exposure, 9/45 patients were positive (20%; P=0.063). A positive correlation was found between HIV-1 RNA and anti-cardiolipin antibody concentration (P=0.010).

Conclusions: According to these results, RAL does not promote antibody-mediated immune disorders, at least not in the mid-term. A prolonged follow-up and an extension of the panel of auto-Abs are recommended to support these results.
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http://dx.doi.org/10.3851/IMP2433DOI Listing
September 2013

Simian immunodeficiency virus-Vpx for improving integrase defective lentiviral vector-based vaccines.

Retrovirology 2012 Aug 22;9:69. Epub 2012 Aug 22.

Department of Infectious, Parasitic and Immune-mediated Diseases, Viale Regina Elena 299, Rome 00161, Italy.

Background: Integrase defective lentiviral vectors (IDLV) represent a promising delivery system for immunization purposes. Human dendritic cells (DC) are the main cell types mediating the immune response and are readily transduced by IDLV, allowing effective triggering of in vitro expansion of antigen-specific primed CD8+ T cells. However, IDLV expression in transduced DC is at lower levels than those of the integrase (IN) competent counterpart, thus requiring further improvement of IDLV for future use in the clinic.

Results: In this paper we show that the addition of simian immunodeficiency (SIV)-Vpx protein in the vector preparation greatly improves transduction of human and simian DC, but not of murine DC, thus increasing the ability of transduced DC to act as functional antigen presenting cells, in the absence of integrated vector sequences. Importantly, the presence of SIV-Vpx allows for using lower dose of input IDLV during in vitro transduction, thus further improving the IDLV safety profile.

Conclusions: These results have significant implications for the development of IDLV-based vaccines.
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http://dx.doi.org/10.1186/1742-4690-9-69DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3468359PMC
August 2012