[Show abstract][Hide abstract] ABSTRACT: New reliable and cost-effective anti-malarial 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, i.e. 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 48h 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 non-lysing D-luciferin substrate significantly improved the reliability of the assay and allowed to perform, for the first time, P. falciparum bioluminescence imaging at single-cell level.
[Show abstract][Hide abstract] ABSTRACT: 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.
AIDS (London, England) 07/2014; · 4.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 01/2014; 9(5):e97270. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 01/2014; 9(9):e107377. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although 2-LTR circle are only a fraction of total viral DNA in the infected cells, sequence analysis of 2-LTR circle contains 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 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 HAART treated patients changes in 2-LTR concentration did not affect junction sequences and flanking regions of 2-LTR. Here we evaluated if RAL therapy could differentially impact on the 2-LTR circles junctional sequences in patients with different virological profiles at time of starting RAL therapy. Sequence analysis indicate that RAL acts differently in the two populations.
AIDS research and human retroviruses 06/2013; · 2.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 two years. Forty-five of them were tested at baseline (before starting RAL) and after 12 months for the presence of the following auto-Abs: anti-nuclear antibodies (ANA), anti-double-stranded (ds)DNA, anti-smooth-muscle antibodies (ASMA), anti-thyreoglobulin (anti-TG) and anti-thyroid peroxidase (anti-TPO) antibodies, anti-cardiolipin (anti-CL) IgG and IgM, anti-nuclear extractable antigens (ENA) including anti-SM RNP antigen, anti-Ro (SSA) antigen and anti-La (SSB) 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 subjects (13) were positive for anti-CL. After 12 months of RAL exposure 9/45 subjects were positive (20%, p = 0063). A positive correlation was found between HIV-1 RNA and anti-CL antibody concentration (p = 0.010). CONCLUSIONS: According to these results, RAL does not promote antibody-mediated immune disorders at least in the mid-term. A prolonged follow up and an extension of autoAbs' panel are recommended to support these results.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
These results have significant implications for the development of IDLV-based vaccines.
[Show abstract][Hide abstract] ABSTRACT: Persistent infection with high risk genotypes of human papillomavirus (HPV) is the cause of cervical cancer, one of most common cancer among woman worldwide, and represents an important risk factor associated with other anogenital and oropharyngeal cancers in men and women. Here, we designed a therapeutic vaccine based on integrase defective lentiviral vector (IDLV) to deliver a mutated nononcogenic form of HPV16 E7 protein, considered as a tumor specific antigen for immunotherapy of HPV-associated cervical cancer, fused to calreticulin (CRT), a protein able to enhance major histocompatibility complex class I antigen presentation (IDLV-CRT/E7). Vaccination with IDLV-CRT/E7 induced a potent and persistent E7-specific T cell response up to 1 year after a single immunization. Importantly, a single immunization with IDLV-CRT/E7 was able to prevent growth of E7-expressing TC-1 tumor cells and to eradicate established tumors in mice. The strong therapeutic effect induced by the IDLV-based vaccine in this preclinical model suggests that this strategy may be further exploited as a safe and attractive anticancer immunotherapeutic vaccine in humans.
International Journal of Cancer 06/2012; · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION: The development of new strategies for the induction of potent and broad immune responses is of high priority in the vaccine field. In this setting, integrase-defective lentiviral vectors (IDLV) represent a new and promising delivery system for immunization purposes. AREAS COVERED: In this review we describe the development and application of IDLV for vaccination. IDLV are turning out to be a new class of vectors endowed with peculiar characteristics, setting them apart from the parental integration-competent lentiviral vectors. Recent data suggest that IDLV are able to induce strong antigen-specific immune responses in terms of quantity, persistence and quality of CD8(+) T cell response following a single immunization in mice. EXPERT OPINION: IDLV are a recent acquisition in the field of genetic immunization, thus allowing for the opportunity of further upgrading, including increasing antigen expression and potency of immune response. Based on recent reports showing the potential of IDLV for immunization in mouse models, further development and validation of IDLV, including comparison with other vaccine protocols and use in non-human primate models, are warranted.
[Show abstract][Hide abstract] ABSTRACT: Virus-like particles (VLPs) are excellent tools for vaccines against pathogens and tumors. They can accommodate foreign polypeptides whose incorporation efficiency and immunogenicity however decrease strongly with the increase of their size. We recently described the CD8(+) T cell immune response against a small foreign antigen (i.e., the 98 amino acid long human papilloma virus E7 protein) incorporated in human immunodeficiency virus (HIV)-1 based VLPs as product of fusion with an HIV-1 Nef mutant (Nef(mut)). Here, we extended our previous investigations by testing the antigenic/immunogenic properties of Nef(mut)-based VLPs incorporating much larger heterologous products, i.e., human hepatitis C virus (HCV) NS3 and influenza virus NP proteins, which are composed of 630 and 498 amino acids, respectively. We observed a remarkable cross-presentation of HCV NS3 in dendritic cells challenged with Nef(mut)-NS3 VLPs, as detected using a NS3 specific CD8(+) T cell clone as well as PBMCs from HCV infected patients. On the other hand, when injected in mice, Nef(mut)-NP VLPs elicited strong anti-NP CD8(+) T cell and CTL immune responses. In addition, we revealed the ability of Nef(mut) incorporated in VLPs to activate and mature primary human immature dendritic cells (iDCs). This phenomenon correlated with the activation of Src tyrosine kinase-related intracellular signaling, and can be transmitted from VLP-challenged to bystander iDCs. Overall, these results prove that Nef(mut)-based VLPs represent a rather flexible platform for the design of innovative CD8(+) T cell vaccines.
[Show abstract][Hide abstract] ABSTRACT: Macrophages represent an important site for productive infection of HIV-1 and the evaluation of integrase (IN) inhibitors on this cell subset is of fundamental importance. In this report, preclinical evaluation of IN inhibitors on primary human macrophages was attempted successfully using a 96-well microtiter phenotypic assay developed recently for the evaluation of IN inhibitors in a cell-based system by taking advantage of HIV-derived lentiviral vectors expressing luciferase. IN inhibitors were also tested using a lentiviral vector containing an IN with introduced T66I/S153Y mutations, known to affect the activity of azido-group-containing diketo acid (DKA) IN inhibitors. Utilizing different classes of HIV integrase inhibitors against the wild-type IN and the mutant mentioned above, some of the IN inhibitors used were also active on this particular mutant, suggesting that should HIV-1 develop additional or different mutations to become resistant to such anti-IN drugs, new drugs can be developed with a better resistance profile. This assay provides a standardized method for the preclinical evaluation of the efficacy of IN inhibitors on wild-type and mutated IN that can be adapted easily for the evaluation of anti-IN activity on IN sequences derived from patients.
Journal of virological methods 09/2010; 168(1-2):272-6. · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nonintegrating lentiviral vectors are being developed as a efficient and safe delivery system for both gene therapy and vaccine purposes. Several reports have demonstrated that a single immunization with integration-defective lentiviral vectors (IDLVs) delivering viral or tumor model antigens in mice was able to elicit broad and long-lasting specific immune responses in the absence of vector integration. At present, no evidence has been reported showing that IDLVs are able to expand preexisting immune responses in the human context. In the present study, we demonstrate that infection of human antigen-presenting cells (APCs), such as monocyte-derived dendritic cells (DCs) and macrophages with IDLVs expressing influenza matrix M1 protein resulted in effective induction of in vitro expansion of M1-primed CD8(+) T cells, as evaluated by both pentamer staining and cytokine production. This is the first demonstration that IDLVs represent an efficient delivery system for gene transfer and expression in human APCs, useful for immunotherapeutic applications.
Human gene therapy 03/2010; 21(8):1029-35. · 4.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genetic immunization with lentiviral vectors is under evaluation as a means for induction of sustained immune response. Lentiviral vectors showed reduced antivector immune responses and efficiently transduce post-mitotic cells in vivo, including antigen presenting cells, such as dendritic cells and macrophages, thus providing a significant benefit over other vector-based antigen delivery platforms. Several evidences indicate that a single immunization with lentiviral vectors induces strong and sustained effector and memory T-cell immune responses, as well as antibody production. New generation of lentiviral vectors with improved biosafety profile are also under development. In particular, integration defective lentiviral vectors have been generated and used as an efficient and safe delivery system for both gene therapy and immunization purposes. Taken together, these evidences support the ongoing development of lentiviral vector-based genetic immunization strategies for safe applications in the clinic.
Current HIV research 03/2010; 8(4):274-81. · 1.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lentiviral vectors are a powerful tool for gene transfer into target cells in vitro and in vivo. However, there are concerns about safety with regard to their use in gene transfer protocols because of insertional mutagenesis following viral infection. Once in the target cells, and in addition to the integrated proviral DNA, lentiviral vectors produce episomal forms of DNA (E-DNA), which are transcriptionally active. Therefore, one strategy to improve safety would envision the block integration of the lentiviral vector while allowing production of E-DNA. Such nonintegrating lentiviral vectors can be produced by introducing mutations in the Integrase (IN) protein of the parental packaging vector. These vectors are fundamentally different from the parental IN competent counterpart, thus opening new avenues for this class of lentiviral vectors as a new gene delivery system for gene therapy strategies, vaccination protocols and as a tool for anti-Integrase drug discovery.
[Show abstract][Hide abstract] ABSTRACT: CD8+ T cells are an essential component of an effective host immune response to tumors and viral infections. Genetic immunization is particularly suitable for inducing CTL responses, because the encoded proteins enter the MHC class I processing pathway through either transgene expression or cross-presentation. In order to compare the efficiency and persistence of immune response induced by genetic vaccines, BALB/c mice were immunized either twice intramuscularly with DNA plasmid expressing a codon-optimized HIV-1 gp120 Envelope sequence together with murine GM-CSF sequence or with a single immunization using an integrase defective lentiviral vector (IDLV) expressing the same proteins. Results strongly indicated that the schedule based on IDLV vaccine was more efficient in inducing specific immune response, as evaluated three months after the last immunization by IFNgamma ELISPOT in both splenocytes and bone marrow- (BM-) derived cells, chromium release assay in splenocytes, and antibody detection in sera. In addition, IDLV immunization induced high frequency of polyfunctional CD8+ T cells able to simultaneously produce IFNgamma, TNFalpha, and IL2.
BioMed Research International 01/2010; 2010:534501. · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Integrase (IN) defective lentiviral vectors have a high safety profile and might prove useful as immunizing agents especially against HIV-1. However, IN defective SIV-based vectors must be developed in order to test their potential in the non-human primate models (NHP) of AIDS. To this aim we tested a novel SIV-based IN defective lentiviral vector for its ability to induce sustained immune responses in mice. BALB/c mice were immunized once intramuscularly with a SIV-based IN defective lentiviral vector expressing the model antigen enhanced green fluorescence protein (eGFP). Immune responses were evaluated 90 days after the injection and compared with those elicited with the IN competent counterpart. The IN defective vector was able to efficiently elicit specific and long-lasting polyfunctional immune responses as evaluated by enzyme-linked immunospot (ELISPOT) assays for interferon-gamma (IFN-gamma) in spleens, bone marrow (BM) and draining lymph nodes, and by intracellular staining (ICS) for IFN-gamma, Interleukin-2 (IL-2) and tumor necrosis factor (TNF-alpha) in both splenocytes and BM cells without integration of the vector into the host genome. This is the first demonstration that an IN defective SIV-based lentiviral vector provides effective immunization, thus paving the way for the construction of IN defective vectors expressing SIV antigen(s) and test their efficacy against a SIV virus challenge in the NHP model of AIDS.
[Show abstract][Hide abstract] ABSTRACT: Over the past 20 years, many efforts have been made to develop a vaccine against AIDS. The lack of an animal model that can be productively infected with HIV-1 has been partially replaced by macaque species infected with SIV or chimeric SHIV. Natural SIV and chimeric SHIV cause an infection resembling human AIDS, and Asian monkeys of genus Macaca (species mulatta, fascicularis and nemestrina) should be considered a useful surrogate in vaccine trials. A multitude of vaccines and immunization approaches have been evaluated, including live-attenuated viruses, DNA vaccines, subunit proteins and viral and bacterial vectors. The results of all these studies are often difficult to interpret due to lack of standardizations, choice of challenging virus and differences in the macaque species used. This article aims at summarizing the main characteristics of the three macaque species used in vaccine trials.
Expert Review of Vaccines 12/2008; 7(9):1419-34. · 4.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lentiviral vectors have been shown to be good candidates for gene transfer protocols; however, prevention of insertional mutagenesis remains problematic. Here we report on the design of a conditionally replicating integrase (IN)-defective lentiviral-hybrid episomal vector in which the insertion of the SV40 promoter/origin of replication provides long-term persistence of the extrachromosomal DNA in the presence of the corresponding trans-acting T antigen (Tag) for targeted suicide gene therapy. SV40-driven GFP expression from the IN-defective lentiviral-hybrid vector was sustained only in the Tag positive 293T cell line, while expression was transient in the parental Tag deficient cell line 293. Quantitative PCR for the 2-LTR circular forms indicated that the unintegrated forms remained stable in 293T for up to 56 days post-transduction, while they were undetectable in the cell line 293 after day 14. Transduction of 293T cells with the IN-defective lentiviral-hybrid episomal vector containing the thymidine kinase (TK) gene rendered the Tag expressing cells highly susceptible to ganciclovir (GCV) treatment, as opposed to the cells infected with the control vector or in Tag negative cells. These data suggest that conditionally replicating IN-defective lentiviral-hybrid episomal vectors could prove useful as vehicles for suicide gene therapy, in particular in cells transformed by SV40.
Antiviral research 12/2008; 80(3):288-94. · 3.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Conceptually, blocking human immunodeficiency virus type 1 (HIV-1) integration is the last possibility for preventing irreversible cellular infection. Using cocultures of monocyte-derived dendritic cells and CD4(+) T cells, which represent primary targets in sexual transmission, we demonstrated that blocking integration with integrase strand transfer inhibitors (InSTIs), particularly L-870812, could consistently block cell-free and cell-associated HIV-1 infection. In a pretreatment setting in which the compound was present before and during infection and was afterwards gradually diluted during the culture period, the naphthyridine carboxamide L-870812 blocked infection with the cell-free and cell-associated HIV-1 Ba-L strain at concentrations of, respectively, 1,000 and 10,000 nM. The potency of L-870812 was similar to that of the nucleotide reverse transcriptase inhibitor R-9-(2-phosphonylmethoxypropyl) adenine (PMPA) but one or two orders of magnitude lower than those of the nonnucleoside reverse transcriptase inhibitors UC781 and TMC120. In contrast, the diketo acid RDS derivative InSTIs showed clear-cut but weaker antiviral activity than L-870812. Moreover, L-870812 completely blocked subtype C and CRFO2_AG primary isolates, which are prevalent in the African heterosexual epidemic. Furthermore, the addition of micromolar concentrations of L-870812 even 24 h after infection could still block both cell-free and cell-associated Ba-L, opening the prospect of postexposure prophylaxis. Finally, an evaluation of the combined activity of L-870812 with either T20, zidovudine, PMPA, UC781, or TMC120 against replication-deficient HIV-1 Ba-L (env) pseudovirus suggested synergistic activity for all combinations. Importantly, compounds selected for the study by using the coculture model were devoid of acute or delayed cytotoxic effects at HIV-blocking concentrations. Therefore, these findings provide evidence supporting consideration of HIV-1 integration as a target for microbicide development.
Antimicrobial Agents and Chemotherapy 08/2008; 52(7):2544-54. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An RNA-based, non-cytopathic replicon vector system, based on the flavivirus Kunjin, has shown considerable promise as a new vaccine delivery system. Here we describe the testing in mice of four different SIVmac239 gag vaccines delivered by Kunjin replicon virus-like-particles. The four vaccines encoded the wild type gag gene, an RNA-optimised gag gene, a codon-optimised gag gene and a modified gag-pol gene construct. The vaccines behaved quite differently for induction of effector memory and central memory responses, for mediation of protection, and with respect to insert stability, with the SIV gag-pol vaccine providing the optimal performance. These results illustrate that for an RNA-based vector the RNA sequence of the antigen can have profound and unforeseen consequences on vaccine behaviour.