Vesicular Stomatitis Virus Envelope Research Articles

Development of Robust Freeze-Drying Process for Long-Term Stability of rVSV-SARS-CoV-2 Vaccine.

The thermostability of vaccines, particularly enveloped viral vectored vaccines, remains a challenge to their delivery wherever needed. The freeze-drying of viral vectored vaccines is a promising approach but remains challenging due to the water removal process from the outer and inner parts of the virus. In the case of enveloped viruses, freeze-drying induces increased stress on the envelope, which often leads to the inactivation of the virus. In this study, we designed a method to freeze-dry a recombinant vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike glycoprotein. Since the envelope of VSV is composed of 50% lipids and 50% protein, the formulation study focused on both the protein and lipid portions of the vector. Formulations were prepared primarily using sucrose, trehalose, and sorbitol as cryoprotectants; mannitol as a lyoprotectant; and histidine as a buffer. Initially, the infectivity of rVSV-SARS-CoV-2 and the cake stability were investigated at different final moisture content levels. High recovery of the infectious viral titer (~0.5 to 1 log loss) was found at 3-6% moisture content, with no deterioration in the freeze-dried cakes. To further minimize infectious viral titer loss, the composition and concentration of the excipients were studied. An increase from 5 to 10% in both the cryoprotectants and lyoprotectant, together with the addition of 0.5% gelatin, resulted in the improved recovery of the infectious virus titer and stable cake formation. Moreover, the secondary drying temperature of the freeze-drying process showed a significant impact on the infectivity of rVSV-SARS-CoV-2. The infectivity of the vector declined drastically when the temperature was raised above 20 °C. Throughout a long-term stability study, formulations containing 10% sugar (sucrose/trehalose), 10% mannitol, 0.5% gelatin, and 10 mM histidine showed satisfactory stability for six months at 2-8 °C. The development of this freeze-drying process and the optimized formulation minimize the need for a costly cold chain distribution system.

Generation of stable suspension producer cell lines for serum-free lentivirus production.

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to4.64×107TU mL-1in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38×107TU mL-1in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of>1×107TUmL-1day-1for up to 29 consecutive days in a non-optimized 5L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.

Reverted HIV-1 Mutants in CD4+ T-Cells Reveal Critical Residues in the Polar Region of Viral Envelope Glycoprotein.

ABSTRACTHIV-1 envelope glycoprotein (Env) interacts with cell surface receptors and induces membrane fusion to enter cells and initiate infection. HIV-1 Env on virions comprises trimers of the gp120 and gp41 subunits. The polar region (PR) in the N-terminus of gp41 is composed of 17 conserved residues, including seven polar amino acids. We have reported that the PR is crucial for Env trimer stability and fusogenicity. Mutations of three highly conserved residues (S534P, T536A, or T538A) in the PR of HIV-1NL4-3 significantly decrease or eliminate viral infectivity due to defective fusion and increased gp120 shedding. To identify compensatory Env mutations that restore viral infectivity, we infected a CD4+ T-cell line with PR mutants pseudotyped with wild-type (WT) HIV-1 Env or vesicular stomatitis virus envelope glycoprotein (VSV-G). We found that PR mutant-infected CD4+ T-cells produced infectious viruses at 7 days postinfection (dpi). Sequencing of the env cDNA from cells infected with the recovered HIV-1 revealed that the S534P mutant reverted to serine or threonine at residue 534. Interestingly, the combined PR-mutant HIV-1 (S534P/T536A or S534P/T536A/T538A) recovered its infectivity and reverted to S534, but maintained the T536A or T538A mutation, suggesting that HIV-1 replication in CD4+ T-cells can tolerate T536A and T538A Env mutations, but not S534P. Moreover, VSV-G-pseudotyped HIV-1 mutants with a fusion-defective Env also recovered infectivity in CD4+ T-cells through reverted Env mutations. These new observations help define the Env residues critical for HIV-1 infection and demonstrate that Env-defective HIV-1 mutants can rapidly regain replication competency in CD4+ T-cells.IMPORTANCE Our previous mutagenesis study revealed that serine at position 534 of HIV-1 Env is critical for viral infectivity. We found that HIV-1 Env containing serine to proline mutation at position 534 (S534P) are incapable of supporting virus-cell and cell-cell fusion. To investigate whether these mutant viruses can recover infectivity and what amino acid changes account for recovered infectivity, we infected CD4+ T-cells with Env-mutant HIV-1 pseudotyped with WT HIV-1 Env or VSV-G and monitored cultures for the production of infectious viruses. Our results showed that most of the pseudotyped viruses recovered their infectivity within 1-week postinfection, and all the recovered viruses mutated proline at position 534. These observations help define the Env residues critical for HIV-1 replication. Because Env-defective HIV-1 mutants can rapidly regain replication competency in CD4+ T-cells, it is important to carefully monitor viral mutations for biosafety consideration when using HIV-1-derived lentivirus vectors pseudotyped with Env.

Inclusion of cGAMP within virus-like particle vaccines enhances their immunogenicity

Cyclic GMP-AMP (cGAMP) is an immunostimulatory molecule produced by cGAS that activates STING. cGAMP is an adjuvant when administered alongside antigens. cGAMP is also incorporated into enveloped virus particles during budding. Here, we investigate whether inclusion of cGAMP within viral vaccine vectors enhances their immunogenicity. We immunise mice with virus-like particles (VLPs) containing HIV-1 Gag and the vesicular stomatitis virus envelope glycoprotein G (VSV-G). cGAMP loading of VLPs augments CD4 and CD8 T-cell responses. It also increases VLP- and VSV-G-specific antibody titres in a STING-dependent manner and enhances virus neutralisation, accompanied by increased numbers of T follicular helper cells. Vaccination with cGAMP-loaded VLPs containing haemagglutinin induces high titres of influenza A virus neutralising antibodies and confers protection upon virus challenge. This requires cGAMP inclusion within VLPs and is achieved at markedly reduced cGAMP doses. Similarly, cGAMP loading of VLPs containing the SARS-CoV-2 Spike protein enhances Spike-specific antibody titres. cGAMP-loaded VLPs are thus an attractive platform for vaccination.

Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization.

The human complement system is an important part of the innate immune system. Its effector pathways largely mediate virus neutralization. Vesicular stomatitis virus (VSV) activates the classical pathway of the complement, leading to virus neutralization by lysis. Two host-derived membrane-associated regulators of complement activation (RCA), CD55 and CD46, which are incorporated into the VSV envelope during egress, confer protection by delaying/resisting complement-mediated neutralization. We showed previously that CD55 is more effective than CD46 in the inhibition of neutralization. In this study, we identified that, at the protein level, VSV infection resulted in the down-regulation of CD46 but not CD55. The mRNA of both the RCAs was significantly down-regulated by VSV, but it was delayed in the case of CD55. The immunoblot analysis of the levels of RCAs in the progeny virion harvested at three specific time intervals, points to an equal ratio of its distribution relative to viral proteins. Besides reconfirming the dominant role of CD55 over CD46 in shielding VSV from complement, our results also highlight the importance of the subtle modulation in the expression pattern of RCAs in a system naturally expressing them.

Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and Neutralization Assays: A 1-Week Protocol.

The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus requires reliable assays for studying viral entry mechanisms which remains poorly understood. This knowledge is important for the development of therapeutic approaches to control SARS-CoV-2 infection by permitting the screening for neutralizing antibodies and other agents that can block infection. This is particularly important for patients who are at high risk for severe outcomes related to COVID-19. The production of pseudotyped viral particles may seem like a daunting task for a non-virology laboratory without experience in the two most commonly used pseudotyping systems, namely retro/lentiviruses and vesicular stomatitis virus (VSV) which lacks the VSV envelope glycoprotein (VSVΔG). By incorporating the most up-to-date knowledge, we have developed a detailed, easy-to-follow novel protocol for producing SARS-CoV-2 spike-bearing pseudovirus using the VSV-ΔG system. We describe the infection assay which uses GFP fluorescence as a measure of infection in a 24-well live imaging system. We present results of our optimization of the system to enhance viral infection levels through the over-expression of human ACE2 receptor and the overexpression of at least one of two proteases - TMPRSS2 or Furin, as well as, supplementation with Poloxamer 407 (P407) and Prostaglandin E2 (PGE2) as adjuvants. We show that the system works efficiently in three unrelated, clinically relevant cell lines: human 293T (renal epithelial) cells, human Calu-3 (lung epithelial) cells, and the non-human primate (African Green Monkey) cell line, Vero-E6 (renal epithelial) cells. In addition, we have used this system to show infection of human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs). This system is efficient (virus generation, titration, and infection assays can be performed in 1 week), quantitative, inexpensive, and readily scalable for application in drug development and therapeutic screening approaches.

In Vivo Gene Therapy for Canine SCID-X1 Using Cocal-Pseudotyped Lentiviral Vector.

Hematopoietic stem and progenitor cell (HSPC)-based ex vivo gene therapy has demonstrated clinical success for X-linked severe combined immunodeficiency (SCID-X1) patients who lack a suitable donor for HSPC transplantation. Nevertheless, this form of treatment is associated with an increased risk of infectious disease complications and genotoxicity mainly due to the conditioning regimen. In addition, ex vivo gene therapy approaches require sophisticated facilities to manufacture gene-modified cells and to care for the patients after chemotherapy. Considering these impediments, we have developed an in vivo gene therapy approach to treat canine SCID-X1 after HSPC mobilization and systemic delivery of the therapeutic vector. Here, we investigated the use of the cocal envelope to pseudotype a lentiviral (LV) vector expressing a functional gammaC gene. The cocal envelope is resistant to serum inactivation compared with the commonly used vesicular stomatitis virus envelope glycoprotein (VSV-G) envelope and thus well suited for systemic delivery. Two SCID-X1 neonatal canines treated with this approach achieved long-term therapeutic immune reconstitution with no prior conditioning. Therapeutic levels of gene-corrected CD3+ T cells were demonstrated for at least 16 months, and all other correlates of T cell functionality were within normal range. Retroviral integration-site analysis demonstrated polyclonal T cell reconstitution. Comparative analysis of integration profiles of foamy viral (FV) vector and cocal LV vector after in vivo gene therapy found distinct integration-site patterns. These data demonstrate that clinically relevant and durable correction of canine SCID-X1 can be achieved with in vivo delivery of cocal LV. Since manufacturing of cocal LV is similar to VSV-G LV, this approach is easily translatable to a clinical setting, thus providing for a highly portable and accessible gene therapy platform for SCID-X1.

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

Genetic labeling of extracellular vesicles for studying biogenesis and uptake in living mammalian cells.

Molecular imaging methods are powerful tools for gaining insight into the cellular organization of living cells. To understand the biogenesis and uptake of extracellular vesicles (EVs) as well as to engineer cell-derived vesicles for targeted drug delivery and therapy, genetic labeling with fluorescent proteins has increasingly been used to determine the structures, locations, and dynamics of EVs in vitro and in vivo. Here, we report a genetic method for the stable labeling of EVs to study their biogenesis and uptake in living human cells. Fusing a green fluorescent protein (GFP) with either the endogenous CD63 (CD63-GFP) or a vesicular stomatitis virus envelope glycoprotein, VSVG (VSVG-GFP), we successfully obtained distinct fluorescence signals in the cytoplasm, revealing the biogenesis of EVs in post-transfected cells. We describe experimental procedures in detail for EV isolation, imaging, and cellular uptake using both confocal microscopy and flow cytometry. We also provide a perspective on how genetic labeling methods can be used to study EV biology, characterization of engineered EVs, and development of EV-based nano-medicine.

Molecular Dynamics Simulation Reveals Exposed Residues in the Ligand-Binding Domain of the Low-Density Lipoprotein Receptor that Interacts with Vesicular Stomatitis Virus-G Envelope.

Familial hypercholesterolemia (FH) is an autosomal dominant disease most often caused by mutations in the low-density lipoprotein receptor (LDLR) gene, which consists of 18 exons spanning 45 kb and codes for a precursor protein of 860 amino acids. Mutations in the LDLR gene lead to a reduced hepatic clearance of LDL as well as a high risk of coronary artery disease (CAD) and sudden cardiac death (SCD). Recently, LDLR transgenes have generated interest as potential therapeutic agents. However, LDLR packaging using a lentiviral vector (LVV) system pseudotyped with a vesicular stomatitis virus (VSV)-G envelope is not efficient. In this study, we modified the LVV system to improve transduction efficiency and investigated the LDLR regions responsible for transduction inhibition. Transduction efficiency of 293T cells with a 5′-LDLReGFP-3′ fusion construct was only 1.55% compared to 42.32% for the eGFP construct. Moreover, co-expression of LDLR affected eGFP packaging. To determine the specific region of the LDLR protein responsible for packaging inhibition, we designed constructs with mutations or sequential deletions at the 3′ and 5′ ends of LDLR cDNA. All constructs except one without the ligand-binding domain (LBD) (pWoLBD–eGFP) resulted in low transduction efficiency, despite successful packaging of viral RNA in the VSV envelope, as confirmed through RT-PCR. When we evaluated a direct interaction between LDLR and the VSV envelope glycoprotein using MD simulation and protein–protein interactions, we uncovered Val119, Thr120, Thr67, and Thr118 as exposed residues in the LDLR receptor that interact with the VSV protein. Together, our results suggest that the LBD of LDLR interacts with the VSV-G protein during viral packaging, which significantly reduces transduction efficiency.

Bacterial flagellin promotes viral entry via an NF-kB and Toll Like Receptor 5 dependent pathway

Viruses and bacteria colonize hosts by invading epithelial barriers. Recent studies have shown that interactions between the microbiota, pathogens and the host can potentiate infection through poorly understood mechanisms. Here, we investigated whether diverse bacterial species could modulate virus internalization into host cells, often a rate-limiting step in establishing infections. Lentiviral pseudoviruses expressing influenza, measles, Ebola, Lassa or vesicular stomatitis virus envelope glycoproteins enabled us to study entry of viruses that exploit diverse internalization pathways. Salmonella Typhimurium, Escherichia coli and Pseudomonas aeruginosa significantly increased viral uptake, even at low bacterial frequencies. This did not require bacterial contact with or invasion of host cells. Studies determined that the bacterial antigen responsible for this pro-viral activity was the Toll-Like Receptor 5 (TLR5) agonist flagellin. Exposure to flagellin increased virus attachment to epithelial cells in a temperature-dependent manner via TLR5-dependent activation of NF-ΚB. Importantly, this phenotype was both long lasting and detectable at low multiplicities of infection. Flagellin is shed from bacteria and our studies uncover a new bystander role for this protein in regulating virus entry. This highlights a new aspect of viral-bacterial interplay with significant implications for our understanding of polymicrobial-associated pathogenesis.

Role of Basal Cells in Producing Persistent Lentivirus-Mediated Airway Gene Expression.

Cystic fibrosis (CF) lung disease is an ideal candidate for a genetic therapy. It has been shown previously that preconditioning with lysophosphatidylcholine (LPC) prior to lentiviral (LV) vector delivery results in long-term in vivo gene expression in the airway epithelium of CF mice. It was hypothesized that this outcome is largely due to transduction of airway basal cells that in turn pass the transgene onto their progeny. The aim of these studies was to confirm if the in vivo delivery of a human immunodeficiency virus type 1 (HIV-1) vesicular stomatitis virus envelope glycoprotein (VSV-G) pseudotyped LV vector following LPC airway conditioning results in transduction of mouse airway basal cells in situ and if the transgene is passed onto their progeny. Additionally, the study sought to determine the efficiency of in vitro transduction of human airway basal cells. First, normal mouse nasal airways were pretreated with LPC prior to delivery of a HIV-1 VSV-G pseudotyped LV vector carrying a LacZ marker gene (LV-LacZ). An epithelial ablation model utilizing polidocanol was then used to demonstrate that clonal outgrowth of linear and spotted clusters of transgene expressing ciliated, basal, and goblet cells occurs following transduction of basal cells. Second, human basal cells were cultured from primary bronchial epithelial cells, with identity confirmed by keratin 5 staining. High levels of transgene expression were found following LV-LacZ transduction. This study demonstrates the ability of the vector delivery protocol to transduce mouse airway basal cells, the LV vector to transduce human basal cells, and the likely role of these cells in maintaining long-term gene expression. These findings support and further develop the potential of LV gene transfer for persistent correction of CF airway disease.

Lentiviral Transduction of Primary Human Glioblastoma Cultures.

This chapter provides detailed step-by-step instructions for the production of lentiviral particles and the transduction of primary human glioblastoma cultures. Lentiviruses stably transduce both dividing and non-dividing cells, such as quiescent cancer stem cell populations. The viral envelope is pseudotyped with the vesicular stomatitis virus envelope glycoprotein G (VSV-G), which renders the lentiviral particles pantropic, so that they can infect theoretically all cell types. The third generation packaging system used in this protocol produces lentiviruses with important safety features, including replication incompetence and self-inactivation (SIN). The protocol we describe here leads to transduction of primary human glioblastoma cultures with efficiencies of up to 90%.

Genome editing for scalable production of alloantigen‐free lentiviral vectors for in vivo gene therapy

Lentiviral vectors (LV) are powerful and versatile vehicles for gene therapy. However, their complex biological composition challenges large‐scale manufacturing and raises concerns for in vivo applications, because particle components and contaminants may trigger immune responses. Here, we show that producer cell‐derived polymorphic class‐I major histocompatibility complexes (MHC‐I) are incorporated into the LV surface and trigger allogeneic T‐cell responses. By disrupting the beta‐2 microglobulin gene in producer cells, we obtained MHC‐free LV with substantially reduced immunogenicity. We introduce this targeted editing into a novel stable LV packaging cell line, carrying single‐copy inducible vector components, which can be reproducibly converted into high‐yield LV producers upon site‐specific integration of the LV genome of interest. These LV efficiently transfer genes into relevant targets and are more resistant to complement‐mediated inactivation, because of reduced content of the vesicular stomatitis virus envelope glycoprotein G compared to vectors produced by transient transfection. Altogether, these advances support scalable manufacturing of alloantigen‐free LV with higher purity and increased complement resistance that are better suited for in vivo gene therapy.

288. Development of Cocal Glycoprotein Envelope Producer Cell Lines for Robust Lentiviral Gene Transfer into Hematopoietic Stem Cells and T Cells

Lentiviral vectors (LVs) are routinely used for stable gene transfer and have demonstrated great promise in hematopoietic stem cell gene therapy and also immunotherapy using genetically modified T cells. LVs are commonly pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G), which confers broad tropism to the vector and allows for vector concentration by centrifugation. However, the use of VSV-G has several limitations, such as susceptibility to inactivation by human serum complement making it unsuitable for in vivo delivery. Furthermore, VSV-G is toxic when constitutively expressed, which has impeded efforts to generate stable producer cell lines. In this study, we first validate the use of cocal vesiculovirus envelope to pseudotype LVs by demonstrating that cocal LVs transduce hematopoietic stem cells and CD4+ T cells more efficiently than VSV-G LVs. We also provide evidence that cocal and VSV-G envelopes use the same receptor for cell entry. We then describe the development of two high-titer, cocal-pseudotyped, LV producer cell lines for a GFP reporter and for a WT1 tumor-specific T cell receptor (TCR). The different 3rd generation lentiviral helper genes were sequentially introduced in HEK293T cells by co-transfection with plasmids encoding antibiotic resistance genes followed by selection to allow for stable protein expression. Cells expressing the cocal envelope produced over 10-times more infectious LV particles as compared to VSV-G expressing cells. High-titer cocal producer cells were isolated by screening for best single clones, which were capable of generating concentrated titers above 108 infectious units per mL. We found that these producer cells were stable after serial passages for over 3 months, with no drop in titer detected over time. The resulting GFP and WT1-TCR vectors performed at least as well as identical vectors made with our standard transient transfection protocol for the transduction of CD34+ and CD4+ T cells, respectively. Cocal LV producer cells were also adapted for growth in suspension, serum-free culture, which will facilitate efforts for the scaling up of vector production. In summary, we have successfully developed two independent LV producer cells lines with clinically usable titers. The broad applicability of our cocal packaging cell line offers a promising tool toward the generation of large-scale, clinical grade LV.

Development of Third-generation Cocal Envelope Producer Cell Lines for Robust Lentiviral Gene Transfer into Hematopoietic Stem Cells and T-cells.

Lentiviral vectors (LVs) pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G) have demonstrated great promise in gene therapy trials employing hematopoietic stem cell and T-cells. The VSV-G envelope confers broad tropism and stability to the vector but is toxic when constitutively expressed, which has impeded efforts to generate stable producer cell lines. We previously showed that cocal pseudotyped LVs offer an excellent alternative to VSV-G vectors because of their broad tropism and resistance to human serum inactivation. In this study, we demonstrate that cocal LVs transduce CD34(+) and CD4(+) T-cells more efficiently than VSV-G LVs and share the same receptor(s) for cell entry. 293T-cells stably expressing the cocal envelope produced significantly higher LV titers than VSV-G expressing cells. We developed cocal pseudotyped, third-generation, self-inactivating LV producer cell lines for a GFP reporter and for a WT1 tumor-specific T-cell receptor, which achieved concentrated titers above 10(8) IU/ml and were successfully adapted for growth in suspension, serum-free culture. The resulting LVs were at least as effective as standard LVs in transducing CD34(+) and CD4(+) T-cells. Our stable cocal LV producer cell lines should facilitate the production of large-scale, high titer clinical grade vectors.

A Novel Role for the Receptor of the Complement Cleavage Fragment C5a, C5aR1, in CCR5-Mediated Entry of HIV into Macrophages.

The complement system is an ancient pattern recognition system that becomes activated during all stages of HIV infection. Previous studies have shown that C5a can enhance the infection of monocyte-derived macrophages and T cells indirectly through the production of interleukin (IL)-6 and tumor necrosis factor (TNF)-α and the attraction of dendritic cells. C5a exerts its multiple biologic functions mainly through activation of C5a receptor 1 (C5aR1). Here, we assessed the role of C5aR1 as an enhancer of CCR5-mediated HIV infection. We determined CCR5 and C5aR1 heterodimer formation in myeloid cells and the impact of C5aR1 blockade on HIV entry and genomic integration. C5aR1/CCR5 heterodimer formation was identified by immunoprecipitation and western blotting. THP-1 cells and monocyte-derived macrophages (MDM) were infected by R5 laboratory strains or HIV pseudotyped for the vesicular stomatitis virus (VSV) envelope. Levels of integrated HIV were measured by quantitative PCR after targeting of C5aR1 by a C5aR antagonist, neutralizing C5aR1 monoclonal antibody (mAb) or hC5a. C5aR1 was also silenced by specific siRNA prior to viral entry. We found that C5aR1 forms heterodimers with the HIV coreceptor CCR5 in myeloid cells. Targeting C5aR1 significantly decreased integration by R5 viruses but not by VSV-pseudotyped viruses, suggesting that C5aR1 is critical for viral entry. The level of inhibition achieved with C5aR1-blocking reagents was comparable to that of CCR5 antagonists. Mechanistically, C5aR1 targeting decreased CCR5 expression. MDM from CCR5Δ32 homozygous subjects expressed levels of C5aR1 similar to CCR5 WT individuals, suggesting that mere C5aR1 expression is not sufficient for HIV infection. HIV appeared to preferentially enter THP-1 cells expressing high levels of both C5aR1 and CCR5. Targeted reduction of C5aR1 expression in such cells reduced HIV infection by ~50%. Our data thus suggest that C5aR1 acts as an enhancer of CCR5-mediated HIV entry into macrophages, the targeting of which may prove useful to reduce HIV infection by R5 strains.

528. Generation of a Cocal Envelope Packaging Cell Line for Robust Lentiviral Gene Transfer Into Hematopoietic Stem Cells and T Cells

Lentiviral vectors (LVs) are routinely used for stable gene transfer and have demonstrated great promise in clinical hematopoietic stem cell and immune cell gene therapy. LVs are commonly pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G), which confers broad tropism to the vector and allows for vector concentration by centrifugation. However, the use of VSV-G has several limitations, such as susceptibility to inactivation by human serum complement making it unsuitable for in vivo delivery, and toxicity when constitutively expressed, which has impeded efforts to generate stable producer cell lines. With the goal to generate a self-inactivating LV packaging cell line, we stably expressed either the VSV-G or cocal envelope in HEK293T cells along with other required helper genes, 3rd generation gagpol and rev. Genes were sequentially introduced in cells by co-transfection with plasmids containing an antibiotic resistance gene. After selection, best producer clones were isolated by limiting dilution and were treated with the histone deacetylase inhibitor sodium butyrate to enhance vector titer. The resulting cocal packaging cell line produces 20 to 50-fold more infectious particles as compared to VSV-G expressing cells, reaching titers averaging 108 infectious units/mL upon concentration. We also extend on our previous studies (Trobridge, 2010) by showing more robust gene transfer for cocal-pseudotyped LVs as compared to VSV-G pseudotyped LVs in human (81% cocal vs. 45% VSV-G, MOI 5) and non-human primate (Macaca nemestrina) CD34+ HSPCs (45% cocal vs. 18% VSV-G, MOI 2×5) while preserving similar differentiation potential as determined by CFC assays. Cocal vectors also transduced human CD3+ blood cells more efficiently than VSV-G vectors (65% cocal vs. 42% VSV-G vs., MOI 3) and showed comparable transduction efficiency in non-human primate CD3+ cells. Using a competitive repopulation approach in the non-human primate model, we find increased gene marking in repopulating cells transduced with a cocal pseudotyped LVs compared to repopulating cells transduced with the same lentivirus backbone pseudotyped with VSV-G. We observed a dramatic difference in the granulocyte fraction where marking with the cocal vector reaches ~90% at 70 days post transplantation. Retroviral integration site analysis is currently underway to characterize the integration profile of cocal vs. VSV-G modified clones. Overall, our study suggests that the cocal envelope outperforms the VSV-G envelope in a stable lentivirus packaging cell line, which may prove useful in current gene therapy efforts to generate large-scale clinical grade vectors.

Evaluation of cre recombinase delivery in mammalian cells using baculovirus infection

In vivo conditional knock-out of a protein is a method of choice to decipher its biological function. It can be achieved by encoding the cre-recombinase on a recombinant virus to exert spatio-temporal control of its expression and enzymatic activity and, subsequently, of the target gene deletion. Recombinant baculoviruses have been successfully used to express a wide range of proteins in insect cells. More recently, their potential to infect mammalian cells has been addressed but, so far, their ability to yield a conditional knock-out as a result of efficient in vivo cre-recombinase gene delivery has not been examined. Cre-recombinase fused to the green fluorescent protein was cloned under the control of the CAG promoter in a recombinant Autographa californica baculovirus expressing the vesicular stomatitis virus envelope G protein for increased mammalian cell infection. Gene delivery was evaluated in vitro in mammalian cells, neuroblastoma and mouse primary neuronal cultures as well as in vivo in the mouse brain. Infection with adeno-associated viruses encoding the cre-recombinase fused to the green fluorescent protein was performed as a positive control. Our results indicate that baculovirus infection leads to functional cre-recombinase expression in non-neuronal and neuroblastoma cell lines but not in mouse primary neuronal cultures or brain.

Efficacy of DNA Vaccines Forming E7 Recombinant Retroviral Virus-Like Particles for the Treatment of Human Papillomavirus-Induced Cancers

Human papillomavirus (HPV) is involved in the development of anogenital tumors and also in the development of oropharyngeal head and neck carcinomas, where HPV-16, expressing the E6 and E7 oncoproteins, is the most frequent serotype. Although vaccines encoding L1 and L2 capsid HPV proteins are efficient for the prevention of HPV infection, they are inadequate for treating established tumors. Hence, development of innovative vaccine therapies targeting E6/E7 is important for controlling HPV-induced cancers. We have engineered a nononcogenic mutated E7-specific plasmo-retroVLP vaccine (pVLP-E7), consisting of plasmid DNA, that is able to form recombinant retrovirus-based virus-like particles (VLPs) that display E7 antigen into murine leukemia virus Gag proteins pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G). pVLP-E7 vaccinations were studied for their ability to generate specific immune responses and for induction of protective immunity against tumor cell challenge in preventive and therapeutic models. The produced VLPs induce the maturation of human dendritic cells in vitro and mount specific E7 T cell responses. Intradermic vaccinations of mice with pVLP-E7 show their efficacy to generate antigen-specific T cell responses, to prevent and protect animals from early TC-1 tumor development compared with standard DNA or VLP immunizations. The vaccine efficacy was also evaluated for advanced tumors in mice vaccinated at various time after the injection of TC-1 cells. Data show that pVLP-E7 vaccination can cure mice with already established tumors only when combined with Toll-like receptor-7 (TLR7) and TLR9 agonists. Our findings provide evidence that pVLPs, combining the advantages of DNA and VLP vaccines, appear to be a promising strategy for the treatment of HPV-induced cancers.