Engineering T-Cell Resistance to HIV-1 Infection via Knock-In of Peptides from the Heptad Repeat 2 Domain of gp41.

Previous studies suggest that short peptides from the heptad repeat 2 (HR2) domain of gp41 expressed on the cell surface are more potent inhibitors of HIV-1 entry than soluble analogs. However, their therapeutic potential has only been examined using lentiviral vectors. Here, we aimed to develop CRISPR/Cas9-based fusion inhibitory peptide knock-in (KI) technology for the generation and selection of HIV-1-resistant T cells. First, we embedded a series of HIV-1 fusion inhibitory peptides in CD52, the shortest glycosylphosphatidylinositol (GPI)-anchored protein, which efficiently delivers epitope tags to the cell surface and maintains a sufficient level of KI. Among the seven peptides tested, MT-C34, HP-23L, and 2P23 exhibited significant activity against both cell-free and cell-to-cell HIV-1 infection. The shed variant of MT-C34 provided insufficient protection against HIV-1 due to its low concentration in the culture medium. Using Cas9 plasmids or ribonucleoprotein electroporation and peptide-specific antibodies, we sorted CEM/R5 cells with biallelic KI of MT-C34 and 2P23 peptides at the CXCR4 locus. In combination, these peptides provided a higher level of protection than individual KI. By extending homology arms and cloning donor DNA into a plasmid containing signals for nuclear localization, we achieved KI of MT-C34 into the CXCR4 locus and HIV-1 proviral DNA at levels of up to 35% in the T-cell line and up to 4 to 5% in primary CD4 lymphocytes. Compared to lentiviral delivery, KI resulted in the higher MT-C34 surface expression and stronger protection of lymphocytes from HIV-1. Thus, we demonstrate that KI is a viable strategy for peptide-based therapy of HIV infection. IMPORTANCE HIV is a human lentivirus that infects CD4-positive immune cells and, when left untreated, manifests in the fatal disease known as AIDS. Antiretroviral therapy (ART) does not lead to viral clearance, and HIV persists in the organism as a latent provirus. One way to control infection is to increase the population of HIV-resistant CD4 lymphocytes via entry molecule knockout or expression of different antiviral genes. Peptides from the heptad repeat (HR) domain of gp41 are potent inhibitors of HIV-1 fusion, especially when designed to express on the cell surface. Individual gp41 peptides encoded by therapeutic lentiviral vectors have been evaluated and some have entered clinical trials. However, a CRISPR/Cas9-based gp41 peptide delivery platform that operates through concomitant target gene modification has not yet been developed due to low knock-in (KI) rates in primary cells. Here, we systematically evaluated the antiviral activity of different HR2 peptides cloned into the shortest carrier molecule, CD52. The resulting small-size transgene constructs encoding selected peptides, in combination with improvements to enhance donor vector nuclear import, helped to overcome precise editing restrictions in CD4 lymphocytes. Using KI into CXCR4, we demonstrated different options for target gene modification, effectively protecting edited cells against HIV-1.

"Cytoplasmic domain effects on exposure of co-receptor-binding sites of HIV-1 Env".

We defined the effects of the cytoplasmic domain (CT) of the Env glycoprotein on co-receptor usage of HIV-1 by reciprocal exchanges of regions containing V3-V5 loops between CD4-dependent and CD4-independent isolates. Primary HIV-1 isolate Env clones CD8 CXCR4-tropic 92UG046 CT84 with an 84-aa truncated CT domain, CD4 CXCR4-tropic 92UG046, and CD4 CCR5-tropic SF162 with full-length (FL) CT domains were used for comparison. The parental 92UG046 Env with CT84 was not fusogenic, but a chimeric SF162 V3-V5-CT84 with an 84-aa truncated CT domain, which demonstrated a switched co-receptor specificity, exhibited syncytium-formation activity with 3T3T4X4 cells. The wild-type (WT) SF162 Env with CT84 or full-length CT was fusogenic in 3T3T4R5 cells. By exchange of V3-V5 loops, we were able to alter WT SF162 to switch its co-receptor preference, which was not dependent on CT domain length. These results provide evidence that CT domains can induce conformational changes in functional regions of gp120 and determine receptor tropism but do not modulate HIV-1 co-receptor specificity.

Effects of modification of the HIV-1 Env cytoplasmic tail on immunogenicity of VLP vaccines.

We investigated the effects on assembly and antigenic properties of specific modifications of the transmembrane spanning (TMS) and cytoplasmic tail (CT) domains of HIV-1 Env from a transmitted/founder (T/F) ZM53 Env glycoprotein. A construct containing a short version of the TMS domain derived from the mouse mammary tumor virus (MMTV) Env with or without a GCN4 trimerization sequence in the CT exhibited the highest levels of incorporation into VLPs and induced the highest titers of anti-Env IgG immune responses in a VLP context. Sera from guinea pigs immunized by VLPs with high Env content, and containing the CT trimerization sequence, had increased neutralization activity and antibody avidity. A cross-clade prime-boost regimen with clade B SF162 or clade C ZM53 Env DNA priming and boosting with VLPs containing modified ZM53 Env further enhanced these immune responses. The modified VLPs demonstrate improved potential as HIV-1 vaccine antigens.

Effects of stabilization of the gp41 cytoplasmic domain on fusion activity and infectivity of SIVmac239.

We investigated the effects of introducing specific sequences that are predicted to affect trimer stability into the CT domain of the SIV Env protein. Two constructs, 3HBai and 3HBaa, with additional GCN4-related sequences in the CT domain (45 aa) had enhanced infectivity, and differed in their fusion activity and trimer stability. Another construct, 3HBii, exhibited a very stable trimeric structure. Pseudotyped virions containing 3HBii retained infectivity despite the lack of syncytia formation. In contrast, 3HBai and 3HBaa, which caused extensive syncytia formation, had a less stable trimeric structure. We observed an inverse correlation between trimer stability and fusion activity but no correlation between syncytia formation activity and infectivity. Quantitative cell-cell fusion assays, analysis of Env incorporation, measurement of ectodomain conformation by CD4 binding, and CCR5 blocking assays indicated differential effects on fusion activity and infectivity of the viruses with Env CT modifications. Differences in interaction with CD4 were not affected by trimer stability and were not related to fusion activity or infectivity. The results indicate that changes in the stability of the CT domain can have significant effects on functional activities of the Env external domain and can impact viral biological properties.

Enhanced mucosal immune responses to HIV virus-like particles containing a membrane-anchored adjuvant.

Previously, a modified HIV Env protein with a heterologous membrane anchor was found to be incorporated into HIV virus-like particles (VLPs) at 10-fold-higher levels than those of unmodified Env. To further improve the immunogenicity of such VLPs, membrane-anchored forms of bacterial flagellin (FliC) or a flagellin with a truncated variable region (tFliC) were constructed to be incorporated into the VLPs as adjuvants. HIV-specific immune responses induced by the resulting VLPs were determined in a guinea pig model. The VLPs induce enhanced systemic antibody responses by either systemic or mucosal vaccination and enhanced mucosal immunity by a mucosal immunization route, as demonstrated by high levels of HIV-specific serum IgG and mucosal IgG and IgA. The quality of the antibody responses was also improved, as shown by enhanced neutralization capacity. VLPs incorporating FliC were more effective in inducing systemic responses, while VLPs containing tFliC were more effective in inducing mucosal IgA responses. The IgG titers in sera were found to last for at least 5 months without a significant drop. These results indicate that HIV VLPs incorporating high levels of Env and a molecular adjuvant have excellent potential for further development as a prophylactic HIV vaccine.

Role of the long cytoplasmic domain of the SIV Env glycoprotein in early and late stages of infection.

BACKGROUND: The Env glycoproteins of retroviruses play an important role in the initial steps of infection involving the binding to cell surface receptors and entry by membrane fusion. The Env glycoprotein also plays an important role in viral assembly at a late step of infection. Although the Env glycoprotein interacts with viral matrix proteins and cellular proteins associated with lipid rafts, its possible role during the early replication events remains unclear. Truncation of the cytoplasmic tail (CT) of the Env glycoprotein is acquired by SIV in the course of adaptation to human cells, and is known to be a determinant of SIV pathogenicity. RESULTS: We compared SIV viruses with full length or truncated (T) Env glycoproteins to analyze possible differences in entry and post-entry events, and assembly of virions. We observed that early steps in replication of SIV with full length or T Env were similar in dividing and non-dividing cells. However, the proviral DNA of the pathogenic virus clone SIVmac239 with full length Env was imported to the nucleus about 20-fold more efficiently than proviral DNA of SIVmac239T with T Env, and 100-fold more efficiently than an SIVmac18T variant with a single mutation A239T in the SU subunit and with a truncated cytoplasmic tail (CT). In contrast, proviral DNA of SIVmac18 with a full length CT and with a single mutation A239T in the SU subunit was imported to the nucleus about 50-fold more efficiently than SIVmac18T. SIV particles with full length Env were released from rhesus monkey PBMC, whereas a restriction of release of virus particles was observed from human 293T, CEMx174, HUT78 or macrophages. In contrast, SIV with T Envs were able to overcome the inhibition of release in human HUT78, CEMx174, 293T or growth-arrested CEMx174 cells and macrophages resulting in production of infectious particles. We found that the long CT of the Env glycoprotein was required for association of Env with lipid rafts. An Env mutant C787S which eliminated palmitoylation did not abolish Env incorporation into lipid rafts, but prevented virus assembly. CONCLUSION: The results indicate that the long cytoplasmic tail of the SIV Env glycoprotein may govern post-entry replication events and plays a role in the assembly process.

Immunogenicity of virus-like particles containing modified human immunodeficiency virus envelope proteins.

Extensive glycosylation and variable loops of the HIV envelope protein (Env) are reported to shield some neutralizing epitopes. Here, we investigated the immunogenicity of mutated HIV Envs presented in virus-like particles (VLPs). We immunized mice with simian human immunodeficiency virus (SHIV) VLPs containing mutant HIV Env with reduced glycosylation (3G), variable loop-deleted mutations (dV1V2), or combinations of both types of mutations (3G-dV2-1G), and evaluated immune responses. Immune sera from mice that received VLPs with modified HIV Envs (3G or dV1V2) showed higher neutralizing activities against the homologous HIV 89.6 virus as well as heterologous viruses when compared with wild type SHIV VLP-immunized mice. Lymphocytes from immunized mice produced HIV Env-specific cytokines, with the 3G-dV2-1G mutant producing high levels of cytokines. Interestingly, both dendritic cells and B cells were found to interact with VLPs suggesting that VLPs are effective immunogens. Therefore, this study suggests that VLPs containing modified HIV Env have the potential to be developed as candidate vaccines capable of inducing cellular and humoral immune responses including neutralizing activities.

Parameters of inhibition of HIV-1 infection by small anionic microbicides.

Sulfonated porphyrins and phthalocyanines have been shown to have anti-HIV activity and are under consideration as microbicides. Both categories of compounds are small negatively charged molecules and both were previously shown to inhibit cell fusion induced by the HIV Env protein and to block binding of gp120 to the CD4 receptor. In the present study we show that these compounds inhibit transmission of cell-associated HIV, inactivate a broad range of HIV-1 primary isolates and are active against DS polyanion-resistant virus. The compounds tested are active over a range of pH values, and possess no detectable activity against normal bacterial flora. These results support the conclusion that anionic tetrapyrroles are promising candidates as microbicides for HIV prevention.

Incorporation of glycosylphosphatidylinositol-anchored granulocyte- macrophage colony-stimulating factor or CD40 ligand enhances immunogenicity of chimeric simian immunodeficiency virus-like particles.

The rapid worldwide spread of human immunodeficiency virus (HIV) mandates the development of successful vaccination strategies. Since live attenuated HIV is not accepted as a vaccine due to safety concerns, virus-like particles (VLPs) offer an attractive safe alternative because they lack the viral genome yet they are perceived by the immune system as a virus particle. We hypothesized that adding immunostimulatory signals to VLPs would enhance their efficacy. To accomplish this we generated chimeric simian immunodeficiency virus (SIV) VLPs containing either glycosylphosphatidylinositol (GPI)-anchored granulocyte-macrophage colony-stimulating factor (GM-CSF) or CD40 ligand (CD40L) and investigated their biological activity and ability to enhance immune responses in vivo. Immunization of mice with chimeric SIV VLPs containing GM-CSF induced SIV Env-specific antibodies as well as neutralizing activity at significantly higher levels than those induced by standard SIV VLPs, SIV VLPs containing CD40L, or standard VLPs mixed with soluble GM-CSF. In addition, mice immunized with chimeric SIV VLPs containing either GM-CSF or CD40L showed significantly increased CD4(+)- and CD8(+)-T-cell responses to SIV Env, compared to standard SIV VLPs. Taken together, these results demonstrate that the incorporation of immunostimulatory molecules enhances humoral and cellular immune responses. We propose that anchoring immunostimulatory molecules into SIV VLPs can be a promising approach to augmenting the efficacy of VLP antigens.

Sulfonated naphthyl porphyrins as agents against HIV-1.

Sulfonated 5,10,15,20-tetra(1-naphthyl)porphyrin (T1NapS) and 5,10,15,20-tetra(2-naphthyl)porphyrin (T2NapS) and their copper and iron chelates show activity against the human immunodeficiency virus (HIV-1). The porphyrins were prepared by sulfonation of the parent structures with sulfuric acid. More highly sulfonated structures were prepared by sulfonation for longer times. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry showed species with as many as eight sulfonates. Some of the mass spectral peaks for the copper chelates were consistent with loss of water, apparently from intramolecular sulfone formation between two adjacent naphthalene rings that took place during copper insertion. The compounds could be separated using capillary electrophoresis; addition of beta- or gamma-cyclodextrin gave substantially better separation of the components. Activity against HIV was evaluated using an epithelial HeLa-CD4-CCR5 cell line; EC50 values for HIV-1 IIIB and HIV-1 JR-FL ranged from 1 to 15 microg/ml. The compounds exhibit low toxicity for human epithelial cells and have potential as microbicides which might be used to provide protection against sexual transmission of HIV.

Multiple domains of the SIV Env protein determine virus replication efficiency and neutralization sensitivity.

SIVmac239 and SIVmac1A11 are wild-type viruses encoding Env proteins with full-length or truncated cytoplasmic tails (CTs), respectively. A mutant designated SIVmac239T has a site-specific mutation which introduces a stop codon in the env gene resulting a truncated protein of similar length to SIVmac1A11 Env. To investigate the role of specific sequence differences in these Env proteins, we constructed SIV mutants encoding 1A11 or 239 Env proteins with reciprocal exchanges of the CT or exchanges of both the surface unit (SU) and CT sequences. A truncated CT in the context of the 1A11 SU subunit was found to significantly enhance replication in CEMx174 (human T-cell line) and rhesus PBMCs. However, similar Env CT truncation did not enhance replication of SIVmac239 in human or monkey cells. SIVmac1A11 with a full-length SIVmac239 CT did not replicate in human T-cell lines, but truncation of the CT by a stop codon resulted in replication. We also observed that these viruses differed significantly in sensitivity to neutralization by antibody. Taken together, the results indicated that the length of the CT domain as well as specific sequence differences in the SU domain affect viral replication capacity as well as sensitivity to neutralization.

Surface stability and immunogenicity of the human immunodeficiency virus envelope glycoprotein: role of the cytoplasmic domain.

The effects of two functional domains, the membrane-proximal YXXPhi motif and the membrane-distal inhibitory sequence in the long cytoplasmic tail of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env), on immunogenicity of the envelope protein were investigated. Genes with codons optimized for mammalian expression were synthesized for the HIV 89.6 Env and a truncated Env with 50 amino acids in the cytoplasmic domain to delete the membrane distal inhibitory sequence for surface expression. Additional genes were generated in which the tyrosine residue in the YXXPhi motif was changed into a serine. Pulse-chase radioactive labeling and immunoprecipitation studies indicated that both domains can mediate endocytosis of the HIV Env, and removal of both domains is required to enhance HIV Env protein surface stability. Analysis of immune responses induced by DNA immunization of mice showed that the DNA construct for the mutant Env exhibiting enhanced surface stability induced significantly higher levels of antibody responses against the HIV Env protein. Our results suggest that the HIV Env cytoplasmic domain may play important roles in virus infection and pathogenesis by modulating its immunogenicity.

Enhancement of immunogenicity of an HIV Env DNA vaccine by mutation of the Tyr-based endocytosis motif in the cytoplasmic domain.

We investigated the effect of the conserved tyrosine-based endocytosis motif (YXXPhi) in the cytoplasmic domain of the human immunodeficiency viruses (HIV) envelope protein (Env) on its immunogenicity. Genes with codons optimized for mammalian expression were synthesized for the HIV 89.6 Env with a truncated cytoplasmic domain and a mutant Env in which the tyrosine residue in the YXXPhi motif was changed into a serine. Mutation of the Tyr residue enhanced surface expression of the Env protein. Analysis of immune responses induced by DNA immunization of mice showed that the DNA construct for the Tyr mutant Env induced moderately higher levels of T cell responses. More interestingly, the DNA construct for the mutant Env induced significantly higher levels of antibody responses against the Env protein in comparison to the construct for the wild type Env. Our results suggest that the YXXPhi motif in the HIV Env cytoplasmic domain may play a role in virus evasion of host immune responses through affecting its immunogenicity.

Prevention of HIV-1 infection by phthalocyanines.

The ability of selected phthalocyanines and metallophthalocyanines to block HIV infection has been evaluated in an epithelial HeLa-CD4 cell line with an integrated LTR-beta-galactosidase gene. Sulfonated phthalocyanine itself (PcS), as well as its copper, nickel, and vanadyl chelates, were the most effective in blocking viral infection. These compounds were also very effective in blocking the fusion activity of the viral Env proteins. All of these compounds are expected to bind axial ligands weakly or not at all. In contrast, sulfonated phthalocyanines bearing metals expected to bind axial ligands more tightly (aluminum, cobalt, chromium, iron, silicon, and zinc) were less effective in blocking HIV infection and also less effective at inhibiting fusion. A number of active compounds were found to block binding of gp120 to CD4. Selected cationic and carboxy phthalocyanines, as well as porphyrazines, were also evaluated. Our results indicate that at least some of the compounds render the virus noninfectious, i.e. that they are virucidal. These compounds have potential as microbicides that might be used to provide protection against sexually transmitted HIV.

Prevention of poxvirus infection by tetrapyrroles.

BACKGROUND: Prevention of poxvirus infection is a topic of great current interest. We report inhibition of vaccinia virus in cell culture by porphyrins and phthalocyanines. Most previous work on the inhibition of viruses with tetrapyrroles has involved photodynamic mechanisms. The current study, however, investigates light-independent inhibition activity. METHODS: The Western Reserve (WR) and International Health Department-J (IHD-J) strains of vaccinia virus were used. Virucidal and antiviral activities as well as the cytotoxicity of test compounds were determined. RESULTS: Examples of active compounds include zinc protoporphyrin, copper hematoporphyrin, meso(2,6-dihydroxyphenyl)porphyrin, the sulfonated tetra-1-naphthyl and tetra-1-anthracenylporphyrins, selected sulfonated derivatives of halogenated tetraphenyl porphyrins and the copper chelate of tetrasulfonated phthalocyanine. EC50 values for the most active compounds are as low as 0.05 microg/mL (40 nM). One of the most active compounds was the neutral meso(2,6-dihydroxyphenyl)porphyrin, indicating that the compounds do not have to be negatively charged to be active. CONCLUSIONS: Porphyrins and phthalocyanines have been found to be potent inhibitors of infection by vaccinia virus in cell culture. These tetrapyrroles were found to be active against two different virus strains, and against both enveloped and non-enveloped forms of the virus, indicating that these compounds may be broadly effective in their ability to inhibit poxvirus infection.

Virus-like particle and DNA-based candidate AIDS vaccines.

Both humoral and cellular immune responses are critical for the control of HIV infection and replication. We have established systems for production of HIV and SIV virus-like particles containing high levels of viral Env proteins using the baculovirus expression system. Evaluation of immunogenicity showed that immunization with virus-like particles induced both cellular and neutralizing antibody responses. Furthermore, mucosal administration of virus-like particles effectively induced both mucosal and systemic immune responses. These results indicate that virus-like particles consisting of HIV structural proteins are an attractive vaccine platform for eliciting anti-viral immune responses, especially neutralizing antibody responses. We have also synthesized codon-optimized genes for HIV Env proteins and evaluated their immunogenicity. Combinations of virus-like particle and DNA-based vaccination are promising for inducing strong cellular and neutralizing antibody responses against HIV.

Inactivation of human immunodeficiency virus type 1 by porphyrins.

We have evaluated the anti-human immunodeficiency virus (HIV) activity of a series of natural and synthetic porphyrins to identify compounds that could potentially be used as microbicides to provide a defense against infection by sexually transmitted virus. For assays we used an epithelial HeLa-CD4 cell line with an integrated long terminal repeat-beta-galactosidase gene. For structure-activity analysis, we divided the porphyrins tested into three classes: (i) natural porphyrins, (ii) metallo-tetraphenylporphyrin tetrasulfonate (metallo-TPPS4) derivatives, and (iii) sulfonated tetra-arylporphyrin derivatives. None of the natural porphyrins studied reduced infection by more than 80% at a concentration of 5 micro g/ml in these assays. Some metal chelates of TPPS4 were more active, and a number of sulfonated tetra-aryl derivatives showed significantly higher activity. Some of the most active compounds were the sulfonated tetranaphthyl porphyrin (TNapPS), sulfonated tetra-anthracenyl porphyrin (TAnthPS), and sulfonated 2,6-difluoro-meso-tetraphenylporphine [TPP(2,6-F2)S] and its copper chelate [TPP(2,6-F2)S,Cu], which reduced infection by 99, 96, 94, and 96%, respectively. Our observations indicate that at least some of these compounds are virucidal, i.e., that they render the virus noninfectious. The active compounds were found to inhibit binding of the HIV type 1 gp120 to CD4 and also to completely inhibit the ability of Env proteins expressed from recombinant vectors to induce cell fusion with receptor-bearing target cells. These results support the conclusion that modified porphyrins exhibit substantial activity against HIV and that their target is the HIV Env protein.