Andrea Cara

Replication and compartmentalization of HIV-1 in kidney epithelium of patients with HIV-associated nephropathy

HIV-associated nephropathy is a clinicopathologic entity that includes proteinuria, focal segmental glomerulosclerosis often of the collapsing variant, and microcystic tubulointerstitial disease. Increasing evidence supports a role for HIV-1 infection of renal epithelium in the pathogenesis of HIV-associated nephropathy. Using in situ hybridization, we previously demonstrated HIV-1 gag and nef mRNA in renal epithelial cells of patients with HIV-associated nephropathy. Here, to investigate whether renal epithelial cells were productively infected by HIV-1, we examined renal tissue for the presence of HIV-1 DNA and mRNA by in situ hybridization and PCR, and we molecularly characterized the HIV-1 quasispecies in the renal compartment. Infected renal epithelial cells were removed by laser-capture microdissection from biopsies of two patients, DNA was extracted, and HIV-1 V3-loop or gp120-envelope sequences were amplified from individually dissected cells by nested PCR. Phylogenetic analysis of kidney-derived sequences as well as corresponding sequences from peripheral blood mononuclear cells of the same patients revealed evidence of tissue-specific viral evolution. In phylogenetic trees constructed from V3 and gp120 sequences, kidney-derived sequences formed tissue-specific subclusters within the radiation of blood mononuclear cell-derived viral sequences from both patients. These data, along with the detection of HIV-1-specific proviral DNA and mRNA in tubular epithelium cells, argue strongly for localized replication of HIV-1 in the kidney and the existence of a renal viral reservoir.

HIV-1 Nef Induces Proliferation and Anchorage-Independent Growth in Podocytes

HIV-associated nephropathy (HIVAN) is now the third leading cause of end-stage renal disease in the African American population. HIV-1 infects renal tubular and glomerular epithelial cells or podocytes, cells that are a critical part of the filtration barrier. HIV-1 infection induces the loss of podocyte differentiation markers and increases podocyte proliferation. It has been previously shown that HIV-infection induces loss of contact inhibition. Here, the HIV-1 gene responsible for proliferative changes is identified by using cultured podocytes in vitro. The HIV-1 proviral construct, pNL4-3 was rendered noninfectious by replacing the HIV-1 gag/pol sequences with an EGFP reporter gene (pNL4-3: DeltaG/P-EGFP). This construct was then pseudotyped with VSV.G envelope to infect podocytes that were conditionally immortalized with SV-40 T antigen. In addition, mutated constructs were engineered with premature stop codons in the HIV-1 env, vif, vpr, vpu, nef, or rev genes. The parental construct and all the other mutated constructs, with the exception of nef, induced proliferation under nonpermissive conditions and anchorage-independent growth (colony formation in soft agar) under permissive conditions. In contrast, deletion of nef markedly reduced proliferation and colony formation. Although tat alone, or tat plus rev induced marginal levels of anchorage-independent growth, coexpression with nef significantly increased colony formation. Finally, stable expression of Nef in a retroviral vector, pBabe-puro, was sufficient to induce increased proliferation and colony formation. Moreover, nef induced saturation density and loss of contact inhibition. These data indicate that Nef induces multiple proliferative effects in podocytes in culture and that nef may therefore be an important gene in the pathogenesis of HIVAN in vivo.

Novel integrase-defective lentiviral episomal vectors for gene transfer.

High levels of circular viral extrachromosomal DNA (E-DNA) are normally produced after infection with integration-competent and -incompetent lentiviruses. Although E-DNA has been shown to be transcriptionally active, it lasts for only a short time in replicating cells. Here, we report an integrase (IN)-defective lentiviral episomal vector in which insertion of the simian virus 40 (SV40) promoter, containing the origin of replication (ori), is associated with long-term expression and persistence of E-DNA in the presence of SV40 large T antigen (TAg) from 293T cells. 293 and 293T cell lines transduced with IN-competent lentiviral vectors expressing green fluorescent protein (GFP) or luciferase from the cytomegalovirus (CMV) or SV40 promoter gave similar levels of transduction and expression. In contrast, only transient reporter expression occurred when using the CMV IN-defective control vector in both 293 and 293T cells. However, reporter gene expression was maintained for more than 8 weeks in 293T, but not 293, cells transduced with the IN-defective lentiviral vector containing the SV40-ori promoter. Polymerase chain reaction for two-long terminal repeat (2LTR) extrachromosomal circular forms, a marker of lentiviral E-DNA, and fluorescence in situ hybridization analysis confirmed the persistence and episomal nature of circular E-DNA up to 60 days after transduction. Taken together, these results indicate that insertion of the SV40-ori promoter in a lentiviral vector contributes to long-term expression by promoting episomal replication when TAg is provided in trans. Lentiviral episomal vectors may serve as specific tools for therapeutic approaches to diseases, particularly those associated with episomal replication of DNA viruses including papillomaviruses, polyomaviruses, and herpesviruses.

Intracellular expression of antibody fragments directed against HIV reverse transcriptase prevents HIV infection in vitro

We have tested a novel strategy of intracellular immunization to block human immunodeficiency virus (HIV) infection. The expression of a specific antibody within a cell was achieved by transduction of genes that encode for immunoglobulin chains with specificity to viral reverse transcriptase. We demonstrated that inhibition of this enzyme makes cells resistant to HIV infection by blocking an early stage of viral replication. If high efficiency transduction with a stable vector into lymphohaematopoietic stem cells or mature lymphocytes can be achieved, gene transfer-mediated intracellular immunization might be a feasible treatment strategy in AIDS.

The impact of telomere erosion on memory CD8+ T cells in patients with X-linked lymphoproliferative syndrome.

Patients with X-linked lymphoproliferative syndrome (XLP) experience excessive T cell proliferation after primary Epstein-Barr virus (EBV) infection, due to mutations in the signalling lymphocyte activation molecule (SLAM) associated protein (SAP) molecule. We examined the impact of dysfunctional proliferative control on the extent of CD8+ T cell differentiation in XLP patients who recovered from primary EBV infection. Although these young patients have normal numbers of lytic and latent EBV-epitope-specific CD8+ T cells, they were extremely differentiated as defined by loss of CCR7 and CD27, low telomerase activity and very short telomeres. This was not a direct effect arising from the loss of SAP, but was due to excessive T cell stimulation due to this defect. Thus, transduction of XLP CD8+ T cells with the catalytic component of telomerase (hTERT), but not SAP, prevented telomere loss and considerably extended proliferative lifespan in vitro. These results indicate that excessive proliferation in CD8+ T cells in XLP patients may lead to end-stage differentiation and loss of functional EBV-specific CD8+ T cells through replicative senescence. This may contribute to the defective immunity found in XLP patients who survive acute EBV infection who develop EBV-related B cell lymphomas before the fourth decade of life.

HIV-1 Tat-Based Vaccines: From Basic Science to Clinical Trials

Vaccination against human immunodeficiency virus (HIV)-1 infection requires candidate antigen(s) (Ag) capable of inducing an effective, broad, and long-lasting immune response against HIV-1 despite mutation events leading to differences in virus clades. The HIV-1 Tat protein is more conserved than envelope proteins, is essential in the virus life cycle and is expressed very early upon virus entry. In addition, both humoral and cellular responses to Tat have been reported to correlate with a delayed progression to disease in both humans and monkeys. This suggested that Tat is an optimal target for vaccine development aimed at controlling virus replication and blocking disease onset. Here are reviewed the results of our studies including the effects of the Tat protein on monocyte-derived dendritic cells (MDDCs) that are key antigen-presenting cells (APCs), and the results from vaccination trials with both the Tat protein or tat DNA in monkeys. We provide evidence that the HIV-1 Tat protein is very efficiently taken up by MDDCs and promotes T helper (Th)-1 type immune responses against itself as well as other Ag. In addition, a Tat-based vaccine elicits an immune response capable of controlling primary infection of monkeys with the pathogenic SHIV89.6P at its early stages allowing the containment of virus spread. Based on these results and on data of Tat conservation and immune cross-recognition in field isolates from different clades, phase I clinical trials are being initiated in Italy for both preventive and therapeutic vaccination.

Macaca mulatta, fascicularis and nemestrina in AIDS vaccine development

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.

Retroviral E-DNA: persistence and gene expression in nondividing immune cells

Following retroviral infection of cells, not only is the proviral DNA integrated into the host genome, but there is also an accumulation of unintegrated extrachromosomal DNA (E‐DNA), both linear and circular. Although the integrated DNA is responsible for the production of viral proteins and new viral progeny, the role of E‐DNA has remained uncertain. Several reports have shown that E‐DNA is transcriptionally active producing both RNA, as well as viral proteins and that circular E‐DNA can persist in nondividing cells, raising questions regarding the potential consequences of this reservoir. Furthermore, integrase inhibitors, presently in clinical trials, shifts the balance of proviral DNA to the E‐DNA form. This review is focused on recent work in this field with an emphasis on exploring the potential role of E‐DNA in both pathogenesis of retroviral infections, especially HIV‐1, and as a tool to deliver and express genes.

Development and use of SIV-based Integrase defective lentiviral vector for immunization

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.

Effect of Tissue Processing on the Ability to Recover Nucleic Acid from Specific Renal Tissue Compartments by Laser Capture Microdissection

The anatomic heterogeneity of the nephron poses obstacles to microdissection of individual renal compartments for analysis of gene expression. We have systematically analyzed the effects of fixation time and nuclear staining on the ability to recover nucleic acid from individual renal compartments by laser capture microdissection (LCM). Formalin-fixed kidney sections from Wistar rats and archival human renal biopsies were used for DNA analysis. From 1 to 10 individual glomeruli and from 1 to 10 individual proximal tubules were captured sequentially onto polymer films. DNA for β-globin could be amplified by PCR from even a single glomerulus or tubule. Optimal conditions for DNA amplification were brief (1- or 2-day) formalin fixation. Use of nuclear counterstains, including Weigert’s hematoxylin, Harris’s hematoxylin, Mayer’s hematoxylin, or methyl green, did not adversely affect the ability to extract and amplify DNA. For RNA extraction, glomeruli and tubules were microdissected from renal cryostat sections stored for up to 6 months. By RT-PCR, mRNA expression of the glomerulus-specific gene, Wilms’ tumor-1, was identified in as few as 5 microdissected glomeruli and of the tubule-specific gene, aminopeptidase N, in as few as 5 microdissected tubules, with no cross-contamination between renal compartments. Our findings indicate that the LCM method can successfully microdissect pure glomerular and tubular tissue compartments and that the optimal fixation and staining conditions are those employed routinely for renal biopsies, namely overnight formalin fixation and hematoxylin counterstain for DNA extraction, and cryostat sectioning with hematoxylin counterstain for RNA extraction. The specificity of LCM coupled with the sensitivity of RT-PCR should prove a powerful tool for the analysis of gene expression in specific renal compartments from archival human renal biopsies.