A mathematical model of HIV dynamics treated with a population of gene-edited haematopoietic progenitor cells exhibiting threshold phenomenon.

The use of gene-editing technology has the potential to excise the CCR5 gene from haematopoietic progenitor cells, rendering their differentiated CD4-positive (CD4+) T cell descendants HIV resistant. In this manuscript, we describe the development of a mathematical model to mimic the therapeutic potential of gene editing of haematopoietic progenitor cells to produce a class of HIV-resistant CD4+ T cells. We define the requirements for the permanent suppression of viral infection using gene editing as a novel therapeutic approach. We develop non-linear ordinary differential equation models to replicate HIV production in an infected host, incorporating the most appropriate aspects found in the many existing clinical models of HIV infection, and extend this model to include compartments representing HIV-resistant immune cells. Through an analysis of model equilibria and stability and computation of $R_0$ for both treated and untreated infections, we show that the proposed therapy has the potential to suppress HIV infection indefinitely and return CD4+ T cell counts to normal levels. A computational study for this treatment shows the potential for a successful 'functional cure' of HIV. A sensitivity analysis illustrates the consistency of numerical results with theoretical results and highlights the parameters requiring better biological justification. Simulations of varying level production of HIV-resistant CD4+ T cells and varying immune enhancements as the result of these indicate a clear threshold response of the model and a range of treatment parameters resulting in a return to normal CD4+ T cell counts.

Analysis of CRISPR/Cas9 Guide RNA Efficiency and Specificity Against Genetically Diverse HIV-1 Isolates.

Gene editing approaches using CRISPR/Cas9 are being developed as a means for targeting the integrated HIV-1 provirus. Enthusiasm for the use of gene editing as an anti-HIV-1 therapeutic has been tempered by concerns about the specificity and efficacy of this approach. Guide RNAs (gRNAs) that target conserved sequences across a wide range of genetically diverse HIV-1 isolates will have greater clinical utility. However, on-target efficacy should be considered in the context of off-target cleavage events as these may comprise an essential safety parameter for CRISPR-based therapeutics. We analyzed a panel of Streptococcus pyogenes Cas9 (SpCas9) gRNAs directed to the 5' and 3' long terminal repeat (LTR) regions of HIV-1. We used in vitro cleavage assays with genetically diverse HIV-1 LTR sequences to determine gRNA activity across HIV-1 clades. Lipid-based transfection of gRNA/Cas9 ribonucleoproteins was used to assess targeting of the integrated HIV-1 proviral sequence in cells (in vivo). For both the in vitro and in vivo experiments, we observed increased efficiency of sequence disruption through the simultaneous use of two distinct gRNAs. Next, CIRCLE-Seq was utilized to identify off-target cleavage events using genomic DNA from cells with integrated HIV-1 proviral DNA. We identified a gRNA targeting the U3 region of the LTR (termed SpCas9-127HBX2) with broad cleavage efficiency against sequences from genetically diverse HIV-1 strains. Based on these results, we propose a workflow for identification and development of anti-HIV CRISPR therapeutics.

Induction of cross-reactive HIV-1 specific antibody responses by engineered V1V2 immunogens with reduced conformational plasticity.

Antibodies against the HIV-1 V1V2 loops were the only correlate of reduced infection risk in the RV144 vaccine trial, highlighting the V1V2 loops as promising targets for vaccine design. The V1V2 loops are structurally plastic, exhibiting either an α-helix-coil or ß-strand conformation. V1V2-specific antibodies may thus recognize distinct conformations, and an antibody's conformational specificity can be an important determinant of breadth and function. Restricting V1V2 conformational plasticity in an immunogen may thus provide control over the conformational specificity and quality of a vaccine-elicited antibody response. Previously, we identified a V1V2 sequence variant (K155M) that results in enhanced recognition by cross-reactive antibodies recognizing the ß-strand conformation. Here, we relate V1V2 antigenicity to immunogenicity by comparing the immunogenicity profiles of wildtype and K155M immunogens in two mouse models. In one model, immunization with gp70 V1V2 K155M but not wildtype elicited antibody responses that were cross-reactive to a panel of heterologous gp120 and gp140 antigens. In a second model, we compared the effect of K155M on immunogenicity in the context of gp70 V1V2, gD V1V2 and gp120, examining the effects of scaffold, epitope-focusing and immunization regimen. K155M variants, especially in the context of a gp120 immunogen, resulted in more robust, durable and cross-reactive antibody responses than wildtype immunogens. Restriction of the ß-stranded V1V2 conformation in K155M immunogens may thus be associated with the induction of cross-reactive antibody responses thought to be required of a protective HIV-1 vaccine.

Transcriptional interference by independently regulated genes occurs in any relative arrangement of the genes and is influenced by chromosomal integration position.

Transcriptional interference is the influence, generally suppressive, of one active transcriptional unit on another unit linked in cis. Its wide occurrence in experimental systems suggests that it may also influence transcription in many loci, but little is known about its precise nature or underlying mechanisms. Here we report a study of the interaction of two nearly identical transcription units juxtaposed in various arrangements. Each reporter gene in the constructs has its own promoter and enhancer and a strong polyadenylation signal. We used recombinase-mediated cassette exchange (RMCE) to insert the constructs into previously tagged genomic sites in cultured cells. This strategy also allows the constructs to be assessed in both orientations with respect to flanking chromatin. In each of the possible arrangements (tandem, divergent, and convergent), the presence of two genes strongly suppresses expression of both genes compared to that of an identical single gene at the same integration site. The suppression is most severe with the convergent arrangement and least severe in total with the divergent arrangement, while the tandem arrangement is most strongly influenced by the integration site and the genes' orientation within the site. These results suggest that transcriptional interference could underlie some position effects and contribute to the regulation of genes in complex loci.

VISTA deficiency attenuates antibody-induced arthritis and alters macrophage gene expression in response to simulated immune complexes.

BACKGROUND: In addition to activated T cells, the immune checkpoint inhibitor "V domain-containing Ig suppressor of T-cell activation" (VISTA) is expressed by myeloid cell types, including macrophages and neutrophils. The importance of VISTA expression by myeloid cells to antibody-induced arthritis and its potential for relevance in human disease was evaluated. METHODS: VISTA was immunolocalized in normal and arthritic human synovial tissue sections and synovial tissue lysates were subjected to western blot analysis. The collagen antibody-induced arthritis model (CAIA) was performed with DBA/1 J mice treated with antibodies against VISTA and with VISTA-deficient mice (V-KO). Total mRNA from arthritic joints, spleens, and cultured macrophages was analyzed with NanoString arrays. Cytokines secreted by splenic inflammatory macrophages were determined. In-vitro chemotaxis and signal transduction assays were performed with cultured macrophages. RESULTS: VISTA protein was localized to synovial membrane cells, neutrophils, and scattered cells in lymphocyte-rich foci and was detected by western blot analysis in normal synovium and synovium from rheumatoid arthritis patients. Deficiency of VISTA or treatment of mice with anti-VISTA monoclonal antibodies attenuated CAIA. Joint damage and MMP-3 expression were significantly reduced in V-KO mice. Surface expression of C5a receptor was reduced on monocytes, neutrophils, and cultured macrophages from V-KO. Upon Fc receptor engagement in vitro, gene expression by V-KO macrophages was altered profoundly compared to WT, including a significant induction of IL-1 receptor antagonist (IL1rn). CONCLUSIONS: VISTA expression supports immune-complex inflammation in CAIA and VISTA is expressed in human synovium. VISTA supports optimal responses to C5a and modulates macrophage responses to immune complexes.

Toll-like receptor agonists are potent inhibitors of human immunodeficiency virus-type 1 replication in peripheral blood mononuclear cells.

Innate immune responses to microbial pathogens are initiated following the binding of ligand to specific pattern recognition receptors. Each pattern recognition receptor, which includes members of the Toll-like receptor (TLR) family, is specific for a particular type of pathogen associated molecular pattern ensuring that the organism can respond rapidly to a wide range of pathogens including bacteria, viruses, and fungi. We studied the extent to which agonists to endosomal TLR could induce anti-HIV-1 activity in peripheral blood mononuclear cells (PBMCs). When agonists to TLR3, TLR7, TLR8 and TLR9 were added prior to infection with HIV-1, they significantly reduced infection of peripheral blood mononuclear cells. Interestingly, agonists to TLR8 and TLR9 were highly effective at blocking HIV replication even when added as late as 48 h or 72 h, respectively, after HIV-1 infection, indicating that the anti-viral effect was durable and long lasting. Analysis of the induction of anti-viral genes after agonist activation of TLR indicated that all of the agonists induced expression of the type I interferons and interferon stimulated genes, although to variable levels that depended on the agonist used. Interestingly, only the agonist to TLR9, ODN2395 DNA, induced expression of type II interferon and the anti-HIV proteins Apobec3G and SAMHD1. By blocking TLR activity using an inhibitor to the MyD88 adaptor protein, we demonstrated that, at least for TLR8 and TLR9, the anti-HIV activity was not entirely mediated by TLR activation, but likely by the activation of additional anti-viral sensors in HIV target cells. These findings suggest that agonists to the endosomal TLR function to induce expression of anti-HIV molecules by both TLR-mediated and non-TLR-mediated mechanisms. Moreover, the non-TLR-mediated mechanisms induced by these agonists could potentially be exploited to block HIV-1 replication in recently HIV-exposed individuals.

Gardiquimod: a Toll-like receptor-7 agonist that inhibits HIV type 1 infection of human macrophages and activated T cells.

Immune response modifiers are being studied as therapeutic agents for viral infections and cancer. These molecules include agonists for the Toll-like receptors (TLR), a family of innate immune receptors. TLR7 and 8, located in cellular endosomes, bind single-stranded RNA characteristic of viral genomes, and trigger intracellular signaling pathways that induce inflammatory cytokines and antiviral innate immune factors. We studied the anti-HIV-1 effects of gardiquimod, a specific TLR7 agonist when used at concentrations below 10 µM, in macrophages and activated peripheral blood mononuclear cells (PBMCs). Gardiquimod, added prior to or within 2 days after infection with X4, R5, or dual-tropic (R5/X4) strains of HIV-1, significantly reduced infection in these cells. Cocultures of activated PBMCs added to gardiquimod-treated and HIV-1-exposed macrophages demonstrated minimal HIV-1 replication for up to 10 days, suggesting that gardiquimod inhibited activated PBMCs viral amplification from HIV-1-exposed macrophages. Gardiquimod treatment of both activated PBMCs and macrophages induced interferon-alpha (IFN-α) transcription within hours of addition, and sustained IFN-α protein secretion for several days. Treatment of cells with a peptide inhibitor to the MyD88 adaptor protein blocked the induction of IFN-α by gardiquimod, and partially reversed the anti-HIV effects in activated PBMCs. Blocking the IFN-α receptor with a neutralizing antibody also reduced the anti-HIV effect of gardiquimod. Gardiquimod inhibited HIV-1 reverse transcriptase, an early step in the life cycle of HIV-1. These findings suggest that gardiquimod, functioning as both an immune system modifier and a reverse transcriptase inhibitor, could be developed as a novel therapeutic agent to block systemic and mucosal transmission of HIV-1.

Nanoparticles containing siRNA to silence CD4 and CCR5 reduce expression of these receptors and inhibit HIV-1 infection in human female reproductive tract tissue explants.

Human Immunodeficiency Virus-type 1 (HIV-1) binds to CD4 and CCR5 receptors on target cells in the human female reproductive tract. We sought to determine whether reducing levels of messenger RNA (mRNA) transcripts that encode these receptors in female reproductive tract cells could protect mucosal tissue explants from HIV-1 infection. Explants prepared from the endometrium, endocervix, and ectocervix of hysterectomy tissues from HIV-1 sero-negative women were exposed to nanoparticles containing CD4- and CCR5-specific short-interfering RNA (siRNA) sequences. Explants were then exposed two days later to HIV-1, and HIV-1 reverse transcripts were measured five days post-infection. Explants treated with nanoparticles containing CD4- and CCR5-specific siRNA showed reduced levels of CD4 and CCR5 transcripts, and significantly lower levels of HIV-1 reverse transcripts compared to those treated with an irrelevant siRNA. In female reproductive tract explants and in peripheral blood cell cultures, siRNA transfection induced the secretion of IFN-alpha (IFN-α), a potent antiviral cytokine. In female mice, murine-specific Cd4-siRNA nanoparticles instilled within the uterus significantly reduced murine Cd4 transcripts by day 3. Our findings demonstrate that siRNA nanoparticles reduce expression of HIV-1 infectivity receptors in human female reproductive tract tissues and also inhibit HIV-1 infection. Murine studies demonstrate that nanoparticles can penetrate the reproductive tract tissues in vivo and silence gene expression. The induction of IFN-α after siRNA transfection can potentially contribute to the antiviral effect. These findings support the therapeutic development of nanoparticles to deliver siRNA molecules to silence host cell receptors in the female reproductive tract as a novel microbicide to inhibit mucosal HIV-1 transmission.

Innate factors in human breast milk inhibit cell-free HIV-1 but not cell-associated HIV-1 infection of CD4+ cells.

BACKGROUND: Transmission of HIV from mother to child through breast-feeding remains a global health challenge, particularly in developing countries. Breast milk from an HIV-infected women may contain both cell-free HIV-1 and cell-associated virus; however, the impact of human breast milk on HIV infection and replication in CD4 cells remain poorly understood. OBJECTIVES: In the present study, we evaluated the effects of breast milk in vitro on infection of CD4 cells with cell-free HIV-1, including effects on HIV-1 receptor expression, reverse transcription, integration, and viral transcription. Additionally, we evaluated the ability of breast milk to inhibit cell-associated transmission of HIV-1 from infected CD4 T lymphocytes. RESULTS: Our results demonstrate that breast milk potently inhibits infection with cell-free HIV-1 in vitro independently of viral tropism and significantly decreases HIV-1 reverse transcription and integration in CD4 cells. However, the inhibitory effect of breast milk on HIV-1 infection of CD4 cells was lost during extended culture, and direct coculture of HIV-infected CD4 T lymphocytes with susceptible target cells revealed that breast milk was ineffective at blocking cell-associated HIV-1 infection. CONCLUSIONS: Our findings suggest that breast milk may provide a protective function against cell-free HIV-1 but may be less effective at blocking infection by cell-associated virus.

HIV type 1 infection in women: increased transcription of HIV type 1 in ectocervical tissue explants.

BACKGROUND: Mucosal surfaces of the female reproductive tract are the main routes of heterosexual transmission of human immunodeficiency virus type 1 (HIV-1), but the contribution of each of the reproductive sites to mucosal transmission is unknown. METHODS: We compared levels of HIV-1 transcription between ectocervical and endometrial tissue explants infected ex vivo with HIV-1. RESULTS: We detected higher levels of HIV-1 transcription in the ectocervix. Although CD45 expression was also increased at this site, higher levels of HIV-1 transcription could not be accounted for exclusively by differences in CD45 expression. This suggests that factors other than CD45 levels regulate HIV-1 transcription within the ectocervix. We detected higher levels of interleukin (IL)-6 at this site. Furthermore, addition of recombinant IL-6 to tissue explants enhanced HIV-1 transcription to a much greater degree in the ectocervix than in the endometrium. CONCLUSIONS: This is, to our knowledge, the first study to compare ectocervix and endometrium in a tissue explant model of HIV-1 infection and to demonstrate greater HIV-1 transcription in the ectocervix. Our results suggest that the ectocervix is more conducive to HIV-1 replication than is the endometrium and that IL-6 enhances HIV-1 transcription at this site. Thus, the ectocervix is an important site to be considered in heterosexual transmission of HIV-1.

Exposure to Cigarette Smoke Disrupts CCL20-Mediated Antimicrobial Activity in Respiratory Epithelial Cells.

Cigarette smoke (CS) exposure is known to increase infection rates, but the mechanisms are not well understood. These studies tested the hypothesis that CS exposure would impair antimicrobial activity of apical conditioned media from human airway (BEAS-2B) cultures by reducing induction and release of the antimicrobial peptide CCL20. BEAS-2B cultures were exposed to CS extract and assayed for temporal and physical characteristics of release as well as for antimicrobial activity. E. coli were exposed to Beas-2B-conditioned media (BCM) and subsequent bacterial colonies were enumerated. In time course studies TLR-agonist-induced CCL20 transcription and release were rapid, of short duration and release was consistently targeted to the apical/luminal compartment. Cells treated with CS extract had diminished release of CCL20 under both constitutive and toll-like receptor (TLR) agonist stimulating conditions. Exposure of the cells to CS significantly reduced the antimicrobial activity in BCM and neutralizing antibodies to CCL20 brought antibacterial activity back to baseline levels demonstrating that antimicrobial activity in this culture system was primarily attributable to CCL20. These studies add to the understanding of CCL20 as a mucosal antimicrobial and improve insight into a likely mechanism linking infection to CS exposure.

Estradiol and progesterone regulate HIV type 1 replication in peripheral blood cells.

Endogenous levels of estradiol and progesterone fluctuate in the peripheral blood of premenopausal women during the reproductive cycle. We studied the effects of these sex hormones on HIV-1 replication in peripheral blood mononuclear cells (PBMCs). We compared HIV-1 replication in PBMCs infected in the presence of mid-secretory (high concentrations) and mid-proliferative (low concentrations) or in the absence of sex hormones. With PBMCs from men, we used concentrations of estradiol and progesterone that are normally present in their plasma. Our findings demonstrate that mid-proliferative phase conditions increased, and mid-secretory phase conditions decreased, HIV-1 replication. To determine if sex hormones affect specific stages of the viral life cycle we performed real-time PCR assays and found decreased levels of HIV-1 integration in the mid-secretory phase and increased levels viral transcription in the mid-proliferative phase. No significant effects on HIV-1 reverse transcription or on CCR5 expression were found. In addition, we assessed hormonal regulation of the HIV-1 LTR in the absence of the viral regulatory protein Tat. We observed that mid-proliferative hormone levels enhanced, whereas mid-secretory hormone concentrations reduced, the activity of the LTR. These findings demonstrate that in HIV-1-infected cells, estradiol and progesterone regulate HIV-1 replication most likely by directly altering HIV-1 transcriptional activation. An additional indirect mechanism of sex hormone regulation of cytokine and chemokine secretion cannot be excluded.

The murine SP-C promoter directs type II cell-specific expression in transgenic mice.

Genomic DNA from the mouse pulmonary surfactant protein C (SP-C) gene was analyzed in transgenic mice to identify DNA essential for alveolar type II cell-specific expression. SP-C promoter constructs extending either 13 or 4.8 kb upstream of the transcription start site directed lung-specific expression of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. In situ hybridization analysis demonstrated alveolar cell-specific expression in the lungs of adult transgenic mice, and the pattern of 4.8 SP-C-CAT expression during development paralleled that of the endogenous SP-C gene. With the use of deletion constructs, lung-specific, low-level CAT activity was detected in tissue assays of SP-C-CAT transgenic mice retaining 318 bp of the promoter. In transient and stable cell transfection experiments, the 4.8-kb SP-C promoter was 90-fold more active as a stably integrated gene. These findings indicate that 1) the 4.8-kb SP-C promoter is sufficient to direct cell-specific and developmental expression, 2) an enhancer essential for lung-specific expression maps to the proximal 318-bp promoter, and 3) the activity of the 4.8-kb SP-C promoter construct is highly dependent on its chromatin environment.

Promoters of the murine embryonic beta-like globin genes Ey and betah1 do not compete for interaction with the beta-globin locus control region.

Mammalian beta-globin loci contain multiple beta-like genes that are expressed at different times during development. The murine beta-globin locus contains two genes expressed during the embryo stage, Ey and betah1, and two genes expressed at both the fetal and postnatal stages, beta-major and beta-minor. Studies of transgenic human beta-like globin loci in mice have suggested that expression of one gene at the locus will suppress expression of other genes at the locus. To test this hypothesis we produced mouse lines with deletions of either the Ey or betah1 promoter in the endogenous murine beta-globin locus. Promoter deletion eliminated expression of the mutant gene but did not affect expression of the remaining embryonic gene or the fetal-adult beta-globin genes on the mutant allele. These results demonstrate a lack of competitive effects between individual mouse embryonic beta-globin gene promoters and other genes in the locus. The implication of these findings for models of beta-globin gene expression are discussed.