Lentiviral Envelope Research Articles

Engineering lentivirus envelope VSV-G for liver targeted delivery of IDOL-shRNA to ameliorate hypercholesterolemia and atherosclerosis

Lentiviral vectors (LVs) have been widely used as a tool for gene therapies. However, tissue-selective transduction after systemic delivery remains a challenge. Inducible degrader of low-density lipoprotein receptor is an attractive target for treating hypercholesterolemia. Here, a liver-targeted lentiviral vector CS8-LV-shIDOL is developed by incorporating a hepatocyte-targeted peptide derived from circumsporozoite protein (CSP) into the lentivirus envelope for liver-targeted delivery of IDOL-shRNA to alleviate hypercholesterolemia. Tail-vein injection of CS8-LV-shIDOL results in extremely high accumulation in liver, and nearly undetectable in other organs in mice. Besides, it shows superior therapeutic efficacy in lowering serum LDL-C and reducing atherosclerotic lesions over unmodified LV-shIDOL in hyperlipidemic mice. Mechanically, the envelope-engineered CS8-LV-shIDOL can enter liver cells via low density lipoprotein receptor-related protein (LRP). Thus, this study provides a novel approach for liver-targeted delivery of IDOL-shRNA to treat hypercholesterolemia by using an envelope-engineered lentiviral vector, and this delivery system has great potential for liver-targeted transgene therapy.

One-step immunoassay of SARS-CoV-2 using screened Fv-antibodies and switching peptides

The Fv-antibodies were correponded to VH region of immunoglobulin G, which were composed of three complementarity determining regions (CDRs) for the specific binding of antigens. In this work, the Fv-antibodies against SARS-CoV-2 spike protein (SP) were screened from an autodisplayed Fv-antibody library which was expressed on E. coli outer membrane, and the receptor binding domain (RBD) of SP was used as a screening probe. The screened target clones were analyzed to have quantitative binding properties to the RBD, and the Fv-antibodies from the screened target clones were expressed as soluble proteins. The binding affinity (KD) of expressed Fv-antibodies to the RBD was estimated to be 70–85 nM using SPR biosensor. The specific binding properties of Fv-antibodies were analyzed for pseudo-virus particles with SARS-CoV-2 SP on the Lenti-virus envelope, such as wild type (Wuhan-1) and variants (Delta, Omicron BA.2, Omicron BA.4/5) using a SPR biosensor. The detection of real SARS-CoV-2 (Wild type, Wuhan-1) based on a SPR biosensor was also presented using the Fv-antibodies with the binding constant (KD) of cycle threshold value (Ct) = 33.8–32.9 (2.19–4.08 copies/μL) and LOD of 0.67–0.83 copies/μL (Ct = 35.5–35.2). Finally, one-step immunoassay based on switching peptide was demonstrated for the detection of the real SARS-CoV-2 (Wuhan-1) without any washing step. The binding constant (KD) was estimated to be Ct = 35.2–33.9 (0.83–2.04 copies/μL), and LOD was estimated to be 0.14–0.47 copies/μL (Ct = 37.8–36.0). Considering the LOD of the conventional RT-PCR (Ct = 35), the LOD of the one-step immunoassay based on the switching peptide was determined to be feasible for the medical diagnosis of COVID-19.

A CRISPR interference strategy for gene expression silencing in multiple myeloma cell lines

BackgroundMultiple myeloma (MM) is the second most common hematologic neoplasm which is characterized by proliferation and infiltration of plasmatic cells in the bone marrow. Currently, MM is considered incurable due to resistance to treatment. The CRISPR/Cas9 system has emerged as a powerful tool for understanding the role of different genetic alterations in the pathogenesis of hematologic malignancies in both cell lines and mouse models. Despite current advances of gene editing tools, the use of CRISPR/Cas9 technology for gene editing of MM have not so far been extended. In this work, we want to repress Rnd3 expression, an atypical Rho GTPase involved in several cellular processes, in MM cell lines using a CRISPR interference strategy.ResultsWe have designed different guide RNAs and cloning them into a lentiviral plasmid, which contains all the machinery necessary for developing the CRISPR interference strategy. We co-transfected the HEK 293T cells with this lentiviral plasmid and 3rd generation lentiviral envelope and packaging plasmids to produce lentiviral particles. The lentiviral particles were used to transduce two different multiple myeloma cell lines, RPMI 8226 and JJN3, and downregulate Rnd3 expression. Additionally, the impact of Rnd3 expression absence was analyzed by a transcriptomic analysis consisting of 3’ UTR RNA sequencing. The Rnd3 knock-down cells showed a different transcriptomic profile in comparison to control cells.ConclusionsWe have developed a CRISPR interference strategy to generate stable Rnd3 knockdown MM cell lines by lentiviral transduction. We have evaluated this strategy in two MM cell lines, and we have demonstrated that Rnd3 silencing works both at transcriptional and protein level. Therefore, we propose CRISPR interference strategy as an alternative tool to silence gene expression in MM cell lines. Furthermore, Rnd3 silencing produces changes in the cellular transcriptomic profile.

SYNJ2BP Improves the Production of Lentiviral Envelope Protein by Facilitating the Formation of Mitochondrion-Associated Endoplasmic Reticulum Membrane.

Equine infectious anemia virus (EIAV) and HIV are both members of the Lentivirus genus and are similar in major virological characters. EIAV endangers the horse industry. In addition, EIAV can also be used as a model for HIV research. The maturation of the lentiviral Env protein, which is necessary for viral entry, requires Env to be folded in the endoplasmic reticulum (ER). It is currently unclear how this process is regulated. Mitochondrion-associated endoplasmic reticulum membrane (MAM) is a specialized part of the close connection between the ER and mitochondria, and one of the main functions of MAM is to promote oxidative protein production in the ER. SYNJ2BP is one of the key proteins that make up the MAM, and we found that SYNJ2BP is essential for EIAV replication. We therefore constructed a SYNJ2BP knockout HEK293T cell line in which the number of MAMs is significantly reduced. Moreover, overexpression of SYNJ2BP could increase the number of MAMs. Our study demonstrates that SYNJ2BP can improve the infectivity of the EIAV virus with elevated production of the viral Env protein through increased MAM formation. Interestingly, SYNJ2BP was able to improve the production of not only EIAV Env but also HIV. Further investigation showed that MAMs can provide more ATP and calcium ions, which are essential factors for Env production, to the ER and can also reduce ER stress induced by HIV or EIAV Envs to increase the Env production level in cells. These results may help us to understand the key production mechanisms of lentiviral Env. IMPORTANCE Lentiviral Env proteins, which are rich in disulfide bonds, need to be fully folded in the ER; otherwise, misfolded Env proteins will induce ER stress and be degraded by ER-associated protein degradation (ERAD). To date, it is still unclear about Env production mechanism in the ER. MAM is the structure of closely connection between the ER and mitochondria. MAMs play important roles in the calcium steady state and oxidative stress, especially in the production of oxidative protein. For the first time, we found that SYNJ2BP can promote the production of lentiviral Env proteins by providing the ATP and calcium ions required for oxidative protein production in the ER and by reducing ER stress through facilitating formation of MAMs. These studies shed light on how MAMs improve lentiviral Env production, which will lay the foundation for the study of replication mechanisms in other lentiviruses from the perspective of the cellular organelle microenvironment.

Domain Organization of Lentiviral and Betaretroviral Surface Envelope Glycoproteins Modeled with AlphaFold.

The surface envelope glycoproteins of nonprimate lentiviruses and betaretroviruses share sequence similarity with the inner proximal domain β-sandwich of the human immunodeficiency virus type 1 (HIV-1) gp120 glycoprotein that faces the transmembrane glycoprotein as well as patterns of cysteine and glycosylation site distribution that points to a similar two-domain organization in at least some lentiviruses. Here, high-reliability models of the surface glycoproteins obtained with the AlphaFold algorithm are presented for the gp135 glycoprotein of the small ruminant caprine arthritis-encephalitis (CAEV) and visna lentiviruses and the betaretroviruses Jaagsiekte sheep retrovirus (JSRV), mouse mammary tumor virus (MMTV), and consensus human endogenous retrovirus type K (HERV-K). The models confirm and extend the inner domain structural conservation in these viruses and identify two outer domains with a putative receptor binding site in the CAEV and visna virus gp135. The location of that site is consistent with patterns of sequence conservation and glycosylation site distribution in gp135. In contrast, a single domain is modeled for the JSRV, MMTV, and HERV-K betaretrovirus envelope proteins that is highly conserved structurally in the proximal region and structurally diverse in apical regions likely to interact with cell receptors. The models presented here identify sites in small ruminant lentivirus and betaretrovirus envelope glycoproteins likely to be critical for virus entry and virus neutralization by antibodies and will facilitate their functional and structural characterization. IMPORTANCE Structural information on the surface envelope proteins of lentiviruses and related betaretroviruses is critical to understand mechanisms of virus-host interactions. However, experimental determination of these structures has been challenging, and only the structure of the human immunodeficiency virus type 1 gp120 has been determined. The advent of the AlphaFold artificial intelligence method for structure prediction allows high-quality modeling of the structures of small ruminant lentiviral and betaretroviral surface envelope proteins. The models are consistent with much of the previously described experimental data, show regions likely to interact with receptors, and identify domains that may be involved in mechanisms of antibody neutralization resistance in the small ruminant lentiviruses. The models will allow more precise design of mutants to further determine mechanisms of viral entry and immune evasion in this group of viruses and constructs for structural determination of these surface envelope proteins.

Truncation of the Cytoplasmic Tail of Equine Infectious Anemia Virus Increases Virion Production by Improving Env Cleavage and Plasma Membrane Localization.

ABSTRACTEnvelope glycoproteins (Envs) of lentiviruses harbor unusually long cytoplasmic tails (CTs). Natural CT truncations always occur in vitro and are accompanied by attenuated virulence, but their effects on viral replication have not been fully elucidated. The Env in equine infectious anemia virus (EIAV) harbors the longest CT in the lentiviral family, and a truncated CT was observed in a live attenuated vaccine. This study demonstrates that CT truncation significantly increased EIAV production, as determined by comparing the virion yields from EIAV infectious clones in the presence and absence of the CT. A significant increase in a cleaved product from the CT-truncated Env precursor, but not the full-length Env, was observed. We further confirmed that the presence of the CT inhibited the cleavage of the Env precursor and found that a functional domain located at the C terminus was responsible for this function. Moreover, CT-truncated Env was mainly localized at the plasma membrane (PM), while full-length Env was mainly localized in the cytoplasm. The CT truncation caused a dramatic reduction in the endocytosis of Env. These results suggest that the CT can modulate the processing and trafficking of EIAV Env and thus regulate EIAV replication.IMPORTANCE The mature lentivirus envelope glycoprotein (Env) is composed of a surface unit (SU) and a transmembrane unit (TM), which are cleaved products of the Env precursor. After mature Env is heterodimerically formed from the cleavage of the Env precursor, it is trafficked to the plasma membrane (PM) for incorporation and virion assembly. Env harbors a long cytoplasmic tail (CT), which has been increasingly found to play multiple roles in the Env biological cycle. Here, we revealed for the first time that the CT of equine infectious anemia virus (EIAV) Env inhibits cleavage of the Env precursor. Simultaneously, the CT promoted Env endocytosis, resulting in weakened Env localization at the PM. We also validated that the CT could significantly decrease EIAV production. These findings suggest that the CT regulates the processing and trafficking of EIAV Env to balance virion production.

Lentiviral shRNA Construction in Gene Therapy for Knock Down CYPIA1, and YWHAZ gene in Lung Cancer

Lung cancer is a disease that is in the top 10 causes of death globally. It is caused by CYP1A1 and YWHAZ genes expressed in the lungs. One of the solutions offered for lung cancer sufferers is gene therapy. Gene therapy for lung cancer is focused on the knock-down of expressed CYP1A1 and YWHAZ genes. It can be using shRNA transfected into lung cells by lentivirus. The lentivirus envelope is modified using glycoprotein filovirus, one of which is ebolavirus. Lentivirus pseudotyping can recognize certain cells. Especially, Ebolavirus can recognize lung cells. The lentiviral gene is designed to carry the gene of interest (shRNA) but no the infectious gene. The shRNA gene transfused into lung cancer cells will recognize and complement the specific target mRNA sites of the CYP1A1 and YWHAZ genes. After that, shRNA will cut the mRNA which should be expressed into a cancer protein. Unexpressed mRNA into protein causes cancer cell growth inhibited. Thus, this gene therapy solution is expected to be able to reduce the rate of death caused by lung cancer. Keywords: Lung cancer, CYP1A1 and YWHAZ genes, gene therapy, lentiviral, shRNA

Integrase-derived peptides together with CD24-targeted lentiviral particles inhibit the growth of CD24 expressing cancer cells

The integration of viral DNA into the host genome is mediated by viral integrase, resulting in the accumulation of double-strand breaks. Integrase-derived peptides (INS and INR) increase the number of integration events, leading to escalated genomic instability that induces apoptosis. CD24 is a surface protein expressed mostly in cancer cells and is very rarely found in normal cells. Here, we propose a novel targeted cancer therapeutic platform based on the lentiviral integrase, stimulated by integrase-derived peptides, that are specifically delivered to cancerous cells via CD24 antigen-antibody targeting. INS and INR were synthesized and humanized and anti-CD24 antibodies were fused to the lentivirus envelope. The activity, permeability, stability, solubility, and toxicity of these components were analyzed. Cell death was measured by fluorescent microscopy and enzymatic assays and potency were tested in vitro and in vivo. Lentivirus particles, containing non-functional DNA led to massive cell death (40–70%). Raltegravir, an antiretroviral drug, inhibited the induction of apoptosis. In vivo, single and repeated administrations of INS/INR were well tolerated without any adverse effects. Tumor development in nude mice was significantly inhibited (by 50%) as compared to the vehicle arm. In summary, a novel and generic therapeutic platform for selective cancer cell eradication with excellent efficacy and safety are presented.

Efficient and Highly Specific Gene Transfer Using Mutated Lentiviral Vectors Redirected with Bispecific Antibodies.

Despite their exceptional potencies, the broad tropism of most commonly used lentivirus (LV) vectors limits their use for targeted gene delivery in vivo We hypothesized that we could improve the specificity of LV targeting by coupling (i) reduction of their binding to off-target cells with (ii) redirection of the vectors with a bispecific antibody (bsAb) that binds both LV and receptors on target cells. As a proof of concept, we pseudotyped nonreplicating LV using a mutated Sindbis envelope (mSindbis) with ablated binding to native receptors, while retaining the capacity to facilitate efficient fusion and endosomal escape. We then evaluated the transduction potencies of the mSindbis LV for HER2-positive (HER2+) (SKBR3) breast and HER2-negative (HER2-) (A2780) cells when redirected with different bsAbs. mSindbis LV alone failed to induce appreciable green fluorescent protein (GFP) expression in either cell. When mixed with HER2-targeting bsAb, mSindbis LV was exceptionally potent, transducing 12% to 16% of the SKBR3 cells at a multiplicity of infection (MOI [ratio of viral genome copies to target cells]) of 3. Transduction was highly specific, resulting in ∼50-fold-greater selectivity toward SKBR3 cells versus A2780 cells. Redirecting mSindbis LV led to a 10-fold improvement in cell-specific targeting compared to redirecting wild-type Sindbis LV with the same bsAb, underscoring the importance of ablating native virus tropism in order to maximize targeting specificity. The redirection of mutated LV using bsAb represents a potent and highly versatile platform for targeted gene therapy.IMPORTANCE The goal of gene therapy is specific delivery and expression of therapeutic genes to target cells and tissues. Common lentivirus (LV) vectors are efficient gene delivery vehicles but offer little specificity. Here, we report an effective and versatile strategy to redirect LV to target cells using bispecific antibodies (bsAbs) that bind both cell receptors and LV envelope domains. Importantly, we ablated the native receptor binding of LV to minimize off-target transduction. Coupling bsAb specificity and ablated native LV tropism synergistically enhanced the selectivity of our targeted gene delivery system. The modular nature of our bsAb-based redirection enables facile targeting of the same LV to diverse tissues/cells. By abrogating the native broad tropism of LV, our bsAb-LV redirection strategy may enable lentivirus-based gene delivery in vivo, expanding the current use of LV beyond ex vivo applications.

Generation and identification of a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes.

Coiled-coil domain containing 67 (CCDC67) gene is a tumor suppressor gene that exhibits a significant inhibitory effect on a variety of tumors. Our previous study demonstrated that the upregulation of CCDC67 gene in TPC-1 cells inhibited cell proliferation, migration and invasion, and promoted apoptosis in vitro. However, due to the lack of a suitable cell tool, these results were not validated in vivo. In the present study, a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes was generated and identified. Firstly, cDNA clones of the CCDC67 gene were obtained by reverse transcription using a custom-designed primer. The results of subsequent electrophoresis analysis and sequencing revealed that the cDNA clones of CCDC67 gene were obtained successfully, with a length of 1,862 bp. The lentiviral vectors, containing the CCDC67, luciferase reporter and puromycin acetyltransferase genes, were co-transfected with two plasmids that encode lentiviral structural proteins and envelope proteins into 293T cells. Following ultracentrifugation, the titer of lentivirus was determined by ELISA to be 5.0×108 TU/ml. The constructed lentiviral vector was used to transfect TPC-1 thyroid cancer cells, and stabilization was achieved by puromycin screening. The expression of CCDC67 gene, luciferase activity and tumorigenic ability of the generated cell line were detected. Reverse transcription-qPCR results demonstrated that the expression levels of CCDC67 gene in TPC-1 cells following transfection were increased 194,46.782-fold compared with those in the negative control group (P<0.01). A higher fluorescence intensity was detected in the generated cell line, while no detectable fluorescence was observed in untransfected TPC-1 cells. The tumorigenic ability of TPC-1-Luc-Puromycin-CCDC67 cells was verified by bioluminescence imaging and histopathological analysis using a pulmonary metastasis model. These results demonstrated that a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes was generated successfully. The TPC-1-Luc-Puromycin-CCDC67 cell line may be a helpful tool for further research on CCDC67 in vivo.

Generation of a caged lentiviral vector through an unnatural amino acid for photo-switchable transduction.

Application of viral vectors in gene delivery is attracting widespread attention but is hampered by the absence of control over transduction, which may lead to non-selective transduction with adverse side effects. To overcome some of these limitations, we proposed an unnatural amino acid aided caging–uncaging strategy for controlling the transduction capability of a viral vector. In this proof-of-principle study, we first expanded the genetic code of the lentiviral vector to incorporate an azido-containing unnatural amino acid (Nϵ-2-azidoethyloxycarbonyl-l-lysine, NAEK) site specifically within a lentiviral envelope protein. Screening of the resultant vectors indicated that NAEK incorporation at Y77 and Y116 was capable of inactivating viral transduction upon click conjugation with a photo-cleavable chemical molecule (T1). Exposure of the chimeric viral vector (Y77-T1) to UVA light subsequently removed the photo-caging group and restored the transduction capability of lentiviral vector both in vitro and in vivo. Our results indicate that the use of the photo-uncage activation procedure can reverse deactivated lentiviral vectors and thus enable regulation of viral transduction in a switchable manner. The methods presented here may be a general approach for generating various switchable vectors that respond to different stimulations and adapt to different viral vectors.

Meeting Review: 2018 International Workshop on Structure and Function of the Lentiviral gp41 Cytoplasmic Tail.

Recent developments in defining the role of the lentiviral envelope glycoprotein (Env) cytoplasmic tail (CT) in Env trafficking and incorporation into virus particles have advanced our understanding of viral replication and transmission. To stimulate additional progress in this field, the two-day International Workshop on Structure and Function of the Lentiviral gp41 Cytoplasmic Tail, co-organized by Eric Freed and James Hoxie, was held at the National Cancer Institute in Frederick, MD (26–27 April 2018). The meeting served to bring together experts focused on the role of gp41 in HIV replication and to discuss the emerging mechanisms of CT-dependent trafficking, Env conformation and structure, host protein interaction, incorporation, and viral transmission. The conference was organized around the following three main hot topics in gp41 research: the role of host factors in CT-dependent Env incorporation, Env structure, and CT-mediated trafficking and transmission. This review highlights important topics and the advances in gp41 research that were discussed during the conference.

Abstract 5922: "Gammora": Selective killing of cancer cells bearing CD24 by viral integrase

Abstract The HIV integrase is important in the integration of viral DNA to the host genome resulting in double-stranded breaks (DSB). Only 1-2 copies are integrated, due to inhibition of HIV-1 Rev protein, making the cells invisible to the immune system. CD24 is mostly expressed on cancer and not normal cells. We hypothesize that specific targeting of the viral particles using mAb to CD24 together with stimulating the enzyme activity and increasing the frequency of integration events, would increase the occurrence of DSB, that will lead to genomic instability, and ultimately to apoptosis of the target cells. Aim To develop a therapeutic platform, for selective eradication of cancer cells, which consists of targeted lentivirus particles and IN-derived peptides. Methods Integrase derived peptides (designated as INR2 and INS) were synthesized. Humanized anti-CD24 antibody (scFv) was engineered and fused to the lentivirus envelope. Stability, solubility and toxicity of the peptides were analyzed by HPLC-MS and MTD, respectively, as well as their activity and permeability into cells. Cell death was measured qualitatively by fluorescent microscopy and quantified by the enzymatic MTT assay. Colorectal, pancreatic and lung cancer cells were used for testing the potency of the lentiviral-based system. The efficacy of the suggested system was assessed in vivo, in nude mice bearing xenografts of lung cancer. The mice were treated systemically twice weekly. Results INS and INR2 stimulate viral integrase in vitro. A single IV administration of INR2 and INS was well tolerated without adverse effects. These lentivirus particles contain nonfunctional DNA with flanked LTRs allowing their integration into the target cells DNA and formation of DSB due to the action of the integrase whose activity was stimulated by the IN-derived peptides. Massive cell death (40-70%) in a dose- dependent manner was achieved. Raltegravir, an antiretroviral FDA-approved drug, was able to inhibit cell death (2-3 times). In vivo, the INR2 and INS combination led to a significant reduction (50%) in tumor volume. Live imaging (IVIS device) of the tumors and selected organs confirmed that the viruses indeed reach the tumor after systemic injection and that the anti-CD24 enhanced the selectivity of infection. Conclusions The use of IN-derived peptides together with the CD24-targeted lenti-therapy approach suggests a novel strategy that specifically promotes killing and removal of a wide range of cancer cells. Citation Format: Shiran Shapira, Eynat Finkelshtein, Abraham Loyter, Nadir Arber. "Gammora": Selective killing of cancer cells bearing CD24 by viral integrase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5922.

Identification of the membrane-spanning domain of glycoprotein 45 in bovine immunodeficiency virus.

The membrane-spanning domain (MSD) of the transmembrane subunit (TM) anchors the envelope glycoprotein (Env) on the lipid bilayer of the host cell membrane and virions. Its functions include membrane fusion efficiency and intracellular trafficking of the lentivirus envelope protein. Our study aimed to determine the MSD of bovine immunodeficiency virus (BIV) glycoprotein 45 (gp45) and reveal structural characteristics of the BIV Env protein. We have predicted the region of the BIV MSD and obtained the sequence using bioinformatics software. Various kinds of assays, including analogy analysis, fluorescence microscopy, and dye-transfer-based assays, were carried out to validate the prediction. The results, for the first time, show that the BIV MSD is located at the D170 to M191 amino acids of gp45, and the identified MSD divides gp45 into the extracellular domain (ED), MSD and cytoplasmic domain (CT). We further found that the BIV MSD had a similar structure and function as the HIV MSD using amino acid sequence alignment and fluorescence microscopy. Additionally, the dye-transfer-based assay demonstrates that deletion of the BIV MSD efficiently decreases cell-cell fusion. Based on the identification of the MSD, a "snorkeling" model, in which the flanking charged amino acid residues are buried in the lipid bilayer while their side chains interact with polar head groups, was proposed for the BIV MSD. Ultimately, we further improved the primary structure of the BIV envelope glycoprotein.

Selective Targeting of CD133-Expressing Glioblastoma Stem Cells Using Lentiviral Vectors.

Several lines of evidence suggest a cellular hierarchy in glioblastoma (GBM). In this hierarchy, GBM stem-like cells (GSCs) play critical roles in tumor progression and recurrence, by virtue of their robust tumor-propagating potential and resistance to conventional chemoradiotherapy. Therefore, targeting GSCs holds significant therapeutic promise. Expression of CD133 (PROM1), a cell surface glycoprotein, has been associated with the GSC phenotype and used as a GSC marker. Here, we describe a protocol that allows the selective lentiviral transduction of CD133-expressing GBM cells. This selectivity is conferred by pseudotyping the lentiviral envelope with a single-chain antibody against an extracellular epitope on CD133. We previously demonstrated the efficacy and specificity of this lentiviral vector using patient-derived GBM cultures. This chapter outlines the preparation of the vector and the transduction of human GBM cells.

Utilizing Lentiviral Gene Transfer in Primary Endothelial Cells to Assess Lymphocyte-Endothelial Interactions.

A major impediment when studying primary human endothelial cell function is the resistance of these cells to gene transfer. Here we describe methods for transferring genes into primary endothelial cells prior to incorporation into a static adhesion assay to analyze the adhesion and migration of isolated lymphocytes. Human embryonic kidney (HEK)-293T (HEK-293 cells expressing the large T-antigen of simian virus 40) cells are initially transfected with plasmids containing the lentiviral packaging and envelope genes and the target sequence, such as a gene of interest or short hairpin loop RNA (shRNA). These cells produce lentivirus packaged with this target sequence and are used to transduce primary human umbilical vein endothelial cells (HUVECs). Human peripheral blood lymphocytes (PBLs) isolated from venous blood are co-incubated with lentivirally transduced cytokine-stimulated endothelial cells to assess lymphocyte adhesion in a static adhesion assay. Direct observations of lymphocyte adhesion and migration over a time course can also be made. In general, lentiviral transduction of primary endothelial cells provides an invaluable system to manipulate gene expression levels when studying the cellular adhesion dynamics that regulate leukocyte adhesion and extravasation.

Broadening the versatility of lentiviral vectors as a tool in nucleic acid research via genetic code expansion.

With the aim of broadening the versatility of lentiviral vectors as a tool in nucleic acid research, we expanded the genetic code in the propagation of lentiviral vectors for site-specific incorporation of chemical moieties with unique properties. Through systematic exploration of the structure–function relationship of lentiviral VSVg envelope by site-specific mutagenesis and incorporation of residues displaying azide- and diazirine-moieties, the modifiable sites on the vector surface were identified, with most at the PH domain that neither affects the expression of envelope protein nor propagation or infectivity of the progeny virus. Furthermore, via the incorporation of such chemical moieties, a variety of fluorescence probes, ligands, PEG and other functional molecules are conjugated, orthogonally and stoichiometrically, to the lentiviral vector. Using this methodology, a facile platform is established that is useful for tracking virus movement, targeting gene delivery and detecting virus–host interactions. This study may provide a new direction for rational design of lentiviral vectors, with significant impact on both basic research and therapeutic applications.

Protective efficacy of centralized and polyvalent envelope immunogens in an attenuated equine lentivirus vaccine.

Lentiviral Envelope (Env) antigenic variation and related immune evasion present major hurdles to effective vaccine development. Centralized Env immunogens that minimize the genetic distance between vaccine proteins and circulating viral isolates are an area of increasing study in HIV vaccinology. To date, the efficacy of centralized immunogens has not been evaluated in the context of an animal model that could provide both immunogenicity and protective efficacy data. We previously reported on a live-attenuated (attenuated) equine infectious anemia (EIAV) virus vaccine, which provides 100% protection from disease after virulent, homologous, virus challenge. Further, protective efficacy demonstrated a significant, inverse, linear relationship between EIAV Env divergence and protection from disease when vaccinates were challenged with viral strains of increasing Env divergence from the vaccine strain Env. Here, we sought to comprehensively examine the protective efficacy of centralized immunogens in our attenuated vaccine platform. We developed, constructed, and extensively tested a consensus Env, which in a virulent proviral backbone generated a fully replication-competent pathogenic virus, and compared this consensus Env to an ancestral Env in our attenuated proviral backbone. A polyvalent attenuated vaccine was established for comparison to the centralized vaccines. Additionally, an engineered quasispecies challenge model was created for rigorous assessment of protective efficacy. Twenty-four EIAV-naïve animals were vaccinated and challenged along with six-control animals six months post-second inoculation. Pre-challenge data indicated the consensus Env was more broadly immunogenic than the Env of the other attenuated vaccines. However, challenge data demonstrated a significant increase in protective efficacy of the polyvalent vaccine. These findings reveal, for the first time, a consensus Env immunogen that generated a fully-functional, replication-competent lentivirus, which when experimentally evaluated, demonstrated broader immunogenicity that does not equate to higher protective efficacy.

Understanding the Process of Envelope Glycoprotein Incorporation into Virions in Simian and Feline Immunodeficiency Viruses

The lentiviral envelope glycoproteins (Env) mediate virus entry by interacting with specific receptors present at the cell surface, thereby determining viral tropism and pathogenesis. Therefore, Env incorporation into the virions formed by assembly of the viral Gag polyprotein at the plasma membrane of the infected cells is a key step in the replication cycle of lentiviruses. Besides being useful models of human immunodeficiency virus (HIV) infections in humans and valuable tools for developing AIDS therapies and vaccines, simian and feline immunodeficiency viruses (SIV and FIV, respectively) are relevant animal retroviruses; the study of which provides important information on how lentiviral replication strategies have evolved. In this review, we discuss the molecular mechanisms underlying the incorporation of the SIV and FIV Env glycoproteins into viral particles.

Envelope Determinants of Equine Lentiviral Vaccine Protection

Lentiviral envelope (Env) antigenic variation and associated immune evasion present major obstacles to vaccine development. The concept that Env is a critical determinant for vaccine efficacy is well accepted, however defined correlates of protection associated with Env variation have yet to be determined. We reported an attenuated equine infectious anemia virus (EIAV) vaccine study that directly examined the effect of lentiviral Env sequence variation on vaccine efficacy. The study identified a significant, inverse, linear correlation between vaccine efficacy and increasing divergence of the challenge virus Env gp90 protein compared to the vaccine virus gp90. The report demonstrated approximately 100% protection of immunized ponies from disease after challenge by virus with a homologous gp90 (EV0), and roughly 40% protection against challenge by virus (EV13) with a gp90 13% divergent from the vaccine strain. In the current study we examine whether the protection observed when challenging with the EV0 strain could be conferred to animals via chimeric challenge viruses between the EV0 and EV13 strains, allowing for mapping of protection to specific Env sequences. Viruses containing the EV13 proviral backbone and selected domains of the EV0 gp90 were constructed and in vitro and in vivo infectivity examined. Vaccine efficacy studies indicated that homology between the vaccine strain gp90 and the N-terminus of the challenge strain gp90 was capable of inducing immunity that resulted in significantly lower levels of post-challenge virus and significantly delayed the onset of disease. However, a homologous N-terminal region alone inserted in the EV13 backbone could not impart the 100% protection observed with the EV0 strain. Data presented here denote the complicated and potentially contradictory relationship between in vitro virulence and in vivo pathogenicity. The study highlights the importance of structural conformation for immunogens and emphasizes the need for antibody binding, not neutralizing, assays that correlate with vaccine protection.