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Lens epithelium-derived growth factor (LEDGF/p75) is an essential cofactor of HIV integration. Both stable overexpression of the C-terminal part of LEDGF/p75 (LEDGF(325-530)) containing the integrase (IN)-binding domain (IBD) and stable knockdown (KD) of LEDGF/p75 are known to inhibit HIV infection in laboratory cell lines. Here, primary human CD(4)(+) T-cells were transduced with lentiviral vectors encoding LEDGF(325-530), the interaction-deficient mutant LEDGF(325-530)D366N, or a hairpin depleting LEDGF/p75 and challenged with HIV. Maximal protection of primary T-cells from HIV infection was obtained after LEDGF(325-530) overexpression reducing HIV replication 40-fold without evidence of cellular toxicity. This strategy was subsequently evaluated in the NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NSG) mouse model. Threefold reduction in mean plasma viral load was obtained in mice engrafted with CD(4)(+) T-cells expressing LEDGF(325-530) in comparison with engraftment with LEDGF(325-530)D366N cells. Four weeks after transplantation with LEDGF(325-530)D366N cells, 70% of the CD(4)(+) cells were lost due to ongoing HIV replication. However, in mice transplanted with LEDGF(325-530) cells only a 20% decrease in CD(4)(+) cells was measured. Liver and spleen sections of LEDGF(325-530) mice contained less HIV than LEDGF(325-530)D366N mice as measured by p24 antigen detection. LEDGF(325-530) overexpression potently inhibits HIV replication in vivo and protects against HIV mediated cell killing of primary CD(4)(+) T-cells.

Drug toxicity and viral resistance limit the long-term efficacy of antiviral drug treatment for human immunodeficiency virus (HIV) infection. Thus, alternative therapies need to be explored. We tested the infusion of T lymphocytes transduced with a retroviral vector (M87o) that expresses an HIV entry-inhibitory peptide (maC46). Gene-modified autologous T cells were infused into ten HIV-infected patients with advanced disease and multidrug-resistant virus during anti-retroviral combination therapy. T-cell infusions were tolerated well, with no severe side effects. A significant increase of CD4 counts was observed after infusion. At the end of the 1-year follow-up, the CD4 counts of all patients were still around or above baseline. Gene-modified cells could be detected in peripheral blood, lymph nodes, and bone marrow throughout the 1-year follow-up, and marking levels correlated with the cell dose. No significant changes of viral load were observed during the first 4 months. Four of the seven patients who changed their antiviral drug regimen thereafter responded with a significant decline in plasma viral load. In conclusion, the transfer of gene-modified cells was safe, led to sustained levels of gene marking, and may improve immune competence in HIV-infected patients with advanced disease and multidrug-resistant virus.

Introduction: The generation of an HIV-1 vaccine able to induce long-lasting protective immunity remains a main challenge. Here, we aimed to modify nextgeneration soluble, prefusion-stabilized, close-to-native, glycan-engineered clade C gp140 envelope (Env) trimers (sC23v4 KIKO and ConCv5 KIKO) for optimal display on the cell surface following homologous or heterologous vector delivery. Methods: A combination of the following modifications scored best regarding the preservation of closed, native-like Env trimer conformation and antigenicity when using a panel of selected broadly neutralizing (bnAb) and non-neutralizing (nnAb) monoclonal antibodies for flow cytometry: i) replacing the natural cleavage site with a native flexible linker and introducing a single amino acid substitution to prevent CD4 binding (*), ii) fusing a heterologous VSV-G-derived transmembrane moiety to the gp140 C-terminus, and iii) deleting six residues proximal to the membrane. Results: When delivering membrane-tethered sC23v4 KIKO* and ConCv5 KIKO* via DNA, VSV-GP, and NYVAC vectors, the two native-like Env trimers provide differential antigenicity profiles. Whereas such patterns were largely consistent among the different vectors for either Env trimer, the membrane-tethered ConCv5 KIKO* trimer adopted a more closed and native-like structure than sC23v4 KIKO*. In immunized mice, VSV-GP and NYVAC vectors expressing the membranetethered ConCv5 KIKO* administered in prime/boost combination were the most effective regimens for the priming of Env-specific CD4 T cells among all tested combinations. The subsequent booster administration of trimeric ConCv5 KIKO* Env protein preserved the T cell activation levels between groups. The evaluation of the HIV-1-specific humoral responses induced in the different immunization groups after protein boosts showed that the various prime/boost protocols elicited broad and potent antibody responses, preferentially of a Th1-associated IgG2a subclass, and that the obtained antibody levels remained high at the memory phase. Discussion: In summary, we provide a feasible strategy to display multiple copies of native-like Env trimers on the cell surface, which translates into efficient priming of sustained CD4+ T cell responses after vector delivery as well as broad, potent, and sustained antibody responses following booster immunizations with the homologous, prefusion-stabilized, close-to-native ConCv5 KIKO* gp140 Env trimer. [ABSTRACT FROM AUTHOR]

A chimeric vesicular stomatitis virus with the glycoprotein of the lymphocytic choriomeningitis virus, VSV-GP, is a potent viral vaccine vector that overcomes several of the limitations of wild-type VSV. Here, we evaluated the potential of VSV-GP as an HIV vaccine vector. We introduced genes for different variants of the HIV-1 envelope protein Env, i.e., secreted or membrane-anchored, intact or mutated furin cleavage site or different C-termini, into the genome of VSV-GP. We found that the addition of the Env antigen did not attenuate VSV-GP replication. All HIV-1 Env variants were expressed in VSV-GP infected cells and some were incorporated very efficiently into VSV-GP particles. Crucial epitopes for binding of broadly neutralizing antibodies against HIV-1 such as MPER (membrane-proximal external region), CD4 binding site, V1V2 and V3 loop were present on the surface of VSV-GP-Env particles. Binding of quaternary antibodies indicated a trimeric structure of VSV-GP incorporated Env. We detected high HIV-1 antibody titers in mice and showed that vectors expressing membrane-anchored Env elicited higher antibody titers than vectors that secreted Envs. In rabbits, Tier 1A HIV-1 neutralizing antibodies were detectable after prime immunization and titers further increased after boosting with a second immunization. Taken together, VSV-GP-Env is a promising vector vaccine against HIV-1 infection since this vector permits incorporation of native monomeric and/or trimeric HIV-1 Env into a viral membrane. [ABSTRACT FROM AUTHOR]

Despite continued progress in the development of novel antiretroviral therapies, it has liecome increasingly evident that drug-based treatments will not lead to a functional or sterilizing cure for HIV+ patients. In 2009, an HIV+ patient was effectively cured of HIV following allogeneic transplantation of hematopoietic stem cells (HSCs) from a CCR5-/-donor. The utility of this approach, however, is severely limited because of the difficulty in finding matched donors. Hence, we studied the potential of HIV-resistant stem cells in the autologous setting in a nonhuman primate AIDS model and incorporated a fusion inhibitor (mC46) as the means for developing infection-resistant cells. Pigtail macaques underwent identical transplants and Simian-Human Immunodeficiency Virus (SHIV) challenge procedures with the only variation between control and mC46 macaques being the inclusion of a fusion-inhibitor expression cassette. Following SHIV challenge, mC46 macaques, but not control macaques, showed a positive selection of gene-modified CD4+ T cells in peripheral blood, gastrointestinal tract, and lymph nodes, accounting for >90% of the total CD4+ T-cell population. mC46 macaques also maintained high frequencies of SHIV-specific, gene-modified CD4+ T cells, an increase in nonmodified CD4+ T cells, enhanced cytotoxic. T lymphocyte function, and antibody responses. These data suggest that HSC protection may be a potential alternative to conventional antiretroviral therapy in patients with HIV/AIDS. [ABSTRACT FROM AUTHOR]

Gene therapeutic strategies for human immunodeficiency virus type 1 (HIV-1) infection could potentially overcome the limitations of standard antiretroviral drug therapy (ART). However, in none of the clinical gene therapy trials published to date, therapeutic levels of genetic protection have been achieved in the target cell population for HIV-1. To improve systemic antiviral efficacy, C peptides, which are efficient inhibitors of HIV-1 entry, were engineered for high-level secretion by genetically modified cells. The size restrictions for efficient peptide export through the secretory pathway were overcome by expressing the C peptides as concatemers, which were processed into monomers by furin protease cleavage. These secreted antiviral entry inhibitory (SAVE) peptides mediated a substantial protective bystander effect on neighboring nonmodified cells, thus suppressing virus replication even if only a small fraction of cells was genetically modified. Accordingly, these SAVE peptides may provide a strong benefit to AIDS patients in future, and, if applied by direct in vivo gene delivery, could present an effective alternative to antiretroviral drug regimen. [ABSTRACT FROM AUTHOR]

Soluble peptides derived from the C-terminal heptad repeat domain of human immunodeficiency virus type 1 (HIV-1) gp41 are potent inhibitors of HIV-1 entry and gp41-induced fusion. Target membrane-anchored variants of these peptides have been shown to retain inhibitory activity. Both soluble and membrane-anchored C peptides (MACs) are thought to block fusion by binding to the N-terminal coiled coil domain of gp41 and preventing formation of the final six-helix bundle structure. However, interactions of target MACs with gp41 must be restricted to a subset of trimers that have their hydrophobic fusion peptides inserted into the target membrane. This unique feature of MACs was used to identify the intermediate step of fusion at which gp41 engaged the target membrane. Fusion between HIV envelope-expressing effector cells and target cells was measured by fluorescence microscopy. Expression of MACs in target cells led to less than twofold reduction in the extent of fusion. However, when reaction was first arrested by adding lysolipids that diHIvored membrane merger, and the lipids were subsequently removed by washing, control cells supported fusion, whereas those that expressed MACs did not. The drastically improved potency of MACs implies that, at lipid-arrested stage, gp41 bridges the viral and target cell membranes and therefore more optimally binds the membrane-anchored peptides. Experimental demonstration of this intermediate shows that, similar to fusion induced by many other viral glycoproteins, engaging the target membrane by HIV-1 gp41 permits coupling between six-helix bundle formation and membrane merger. [ABSTRACT FROM AUTHOR]

Gene therapy has the potential to control human immunodeficiency virus (HIV) in patients who do not respond to traditional antiviral therapy. In this study, we tested foamy virus (FV) vectors expressing three anti-HIV transgenes, both individually and in a combination vector. The transgenes tested in this study are RevM10, a dominant negative version of the viral rev protein, Sh1, a short hairpin RNA directed against a conserved overlapping sequence of tat and rev, and membrane-associated C46 (maC46), a membrane-attached peptide that blocks HIV cell entry. FV vectors efficiently transduce hematopoietic stem cells and, unlike lentivirus (LV) vectors, do not share viral proteins with HIV. The titers of the FV vectors described in this study were not affected by anti-HIV transgenes. On a direct comparison of FV vectors expressing the individual transgenes, entry inhibition using the maC46 transgene was found to be the most effective at blocking HIV replication. A clinically relevant FV vector expressing three anti-HIV transgenes effectively blocked HIV infection in primary macrophages derived from transduced, peripheral blood CD34-selected cells and in a cell line used for propagating HIV, CEM×174. These results suggest that there are potential benefits of using FV vectors in HIV gene therapy.Molecular Therapy (2007) 16 1, 46–51. doi:10.1038/sj.mt.6300335 [ABSTRACT FROM AUTHOR]

The expression of a membrane-anchored gp41-derived peptide (M87) has been shown to confer protection from infection through human immunodeficiency virus type 1 (HIV-1) (Hildinger el al., J. Virol. 75:3038–3042, 2001). In an effort to characterize the mechanism of action of this membrane-anchored peptide in comparison to the soluble peptide T-20, we selected resistant variants of HIV-1NL4-3 and HIV-1BaL by serial virus passage using PM1 cells stably expressing peptide M87. Sequence analysis of the resistant isolates showed different patterns of selected point mutations in heptad repeat regions 1 and 2 (HR1 and HR2, respectively) for the two viruses analyzed. For HIV-1NL4-3 a single amino acid change at position 33 in HR1 (L33S) was selected, whereas for HIV-1BaL the majority of the sequences obtained showed two amino acid changes, one in HR1 and one in HR2 (I48V/N126K). In both selections the most important contiguous 3-amino-acid sequence, GIV, within HR1, associated with resistance to soluble T-20, was not changed. Site-directed mutagenesis studies confirmed the importance of the characterized point mutations to confer resistance to M87 as well as to soluble T-20 and T-649. Replication capacity and dual-color competition assays revealed that the double mutation I48V/N126K in HIV-1BaL results in a strong reduction of viral fitness, whereas the L33S mutation in HIV-1NL4-3 did enhance viral fitness compared to the respective parental viruses. However, the selected point mutations did not confer resistance to the more recently described optimized membrane-anchored fusion inhibitor M870 (Egelhofer et al., J. Virol. 78:568–575, 2004), strengthening the importance of this novel antiviral concept for gene therapy approaches. [ABSTRACT FROM AUTHOR]

Binding of the human immunodeficiency virus (HIV) envelope glycoprotein (Env) to the cellular CD4 receptor and a chemokine coreceptor initiates a series of conformational changes in the Env subunits gp120 and gp41. Eventually, the trimeric gp41 folds into a six-helix bundle, thereby inducing fusion of the viral and cellular membranes. C peptides derived from the C-terminal heptad repeat (CHR) of gp41 are efficient entry inhibitors as they block the six-helix bundle formation. Previously, we developed a membrane-anchored C peptide (maC46) expressed from a retroviral vector that also shows high activity against virus strains resistant to enfuvirtide (T-20), an antiviral C peptide approved for clinical use. Here, we present a systematic analysis of mutations in Env that confer resistance of HIV type 1 (HIV-1) to maC46. We selected an HIV-1 BaL strain with 10-fold reduced sensitivity to maC46 (BaL_C46) by passaging virus for nearly 200 days in the presence of gradually increasing concentrations of maC46. In comparison to wild-type BaL, BaL_C46 had five mutations at highly conserved positions in Env, three in gp120, one in the N-terminal heptad-repeat (NHR), and one in the CHR of gp41. No mutations were found in the NHR domain around the GIV motif that are known to cause resistance to enfuvirtide. Instead, maC46 resistance was found to depend on complementary mutations in the NHR and CHR that considerably favor binding of the mutated NHR to the mutated CHR over binding to maC46. In addition, resistance was highly dependent on mutations in gp120 that accelerated entry. Taken together, resistance to maC46 did not develop readily and required multiple cooperating mutations at conserved positions of the viral envelope glycoproteins gp120 and gp41. [ABSTRACT FROM AUTHOR]