The CD8+ T Cell Noncytotoxic Antiviral Responses.

The CD8+ T cell noncytotoxic antiviral response (CNAR) was discovered during studies of asymptomatic HIV-infected subjects more than 30 years ago. In contrast to CD8+ T cell cytotoxic lymphocyte (CTL) activity, CNAR suppresses HIV replication without target cell killing. This activity has characteristics of innate immunity: it acts on all retroviruses and thus is neither epitope specific nor HLA restricted. The HIV-associated CNAR does not affect other virus families. It is mediated, at least in part, by a CD8+ T cell antiviral factor (CAF) that blocks HIV transcription. A variety of assays used to measure CNAR/CAF and the effects on other retrovirus infections are described. Notably, CD8+ T cell noncytotoxic antiviral responses have now been observed with other virus families but are mediated by different cytokines. Characterizing the protein structure of CAF has been challenging despite many biologic, immunologic, and molecular studies. It represents a low-abundance protein that may be identified by future next-generation sequencing approaches. Since CNAR/CAF is a natural noncytotoxic activity, it could provide promising strategies for HIV/AIDS therapy, cure, and prevention.

Genetically edited CD34+ cells derived from human iPS cells in vivo but not in vitro engraft and differentiate into HIV-resistant cells.

Genetic editing of induced pluripotent stem (iPS) cells represents a promising avenue for an HIV cure. However, certain challenges remain before bringing this approach to the clinic. Among them, in vivo engraftment of cells genetically edited in vitro needs to be achieved. In this study, CD34+ cells derived in vitro from iPS cells genetically modified to carry the CCR5Δ32 mutant alleles did not engraft in humanized immunodeficient mice. However, the CD34+ cells isolated from teratomas generated in vivo from these genetically edited iPS cells engrafted in all experiments. These CD34+ cells also gave rise to peripheral blood mononuclear cells in the mice that, when inoculated with HIV in cell culture, were resistant to HIV R5-tropic isolates. This study indicates that teratomas can provide an environment that can help evaluate the engraftment potential of CD34+ cells derived from the genetically modified iPS cells in vitro. The results further confirm the possibility of using genetically engineered iPS cells to derive engraftable hematopoietic stem cells resistant to HIV as an approach toward an HIV cure.

Generation of HIV-1-infected patients' gene-edited induced pluripotent stem cells using feeder-free culture conditions.

OBJECTIVES: The discovery of induced pluripotent stem cells (iPSC) has brought promise to regenerative medicine as it breaks the ethical barrier of using embryonic stem cells. Such cell culture-derived patient-specific autologous stem cells are needed for transplantation. Here we report deriving HIV-1-infected patients' iPSC lines under transgene-free methods and under feeder-free and xeno-free culture conditions to meet the requirement for clinical application. METHODS AND RESULTS: We have reprogrammed patients' peripheral blood mononuclear cells with EBNA1/OriP episomal vectors, or a defective and persistent Sendai virus vector (SeVdp) to ensure a nonintegrating iPSC generation. Both single picked and pooled iPSC lines demonstrated high pluripotency and were able to differentiate into various lineage cells in vivo. The established cell lines could be modified by genetic editing using the TALENs or CRISPR/Cas 9 technology to have a bi-allelic CCR5Δ32 mutations seamlessly. All generated iPSC lines and modified cell lines had no evidence of HIV integration and maintained normal karyotype after expansion. CONCLUSIONS: This study provides a reproducible simple procedure for generating therapeutic grade iPSCs from HIV-infected patients and for engineering these cells to possess a naturally occurring genotype for resistance to HIV-1 infection when differentiated into immune cells.

Genetically-edited induced pluripotent stem cells derived from HIV-1-infected patients on therapy can give rise to immune cells resistant to HIV-1 infection.

OBJECTIVE: To assess the in-vitro CCR5---tropic and CXCR4---tropic HIV---1 infectivity of immune cells, particularly macrophages, derived from CCR5 gene---edited induced pluripotent stem cells (iPSCs) obtained from the peripheral blood mononuclear cells (PBMC) of HIV---infected patients on antiretroviral therapy (ART). DESIGN: PBMC were obtained from six patients who had been HIV---infected for over 20 years and were on ART for 1---12 years prior to this study. METHODS: The PBMC were derived into iPSCs and genetically edited with TALENs or CRISPR---cas9 endonucleases combined with PiggyBac technology to introduce the naturally occurring 32---bp deletion to the CCR5 gene. These iPSCs were differentiated into macrophages, and subsequently challenged with CCR5---tropic or CCR5/CXCR4 dual--- tropic HIV---1 strains. iPSC derivation, gene editing and immune cell differentiation were done in feeder---free, xeno---free in-vitro conditions. RESULTS: Multiple unedited (wild---type) and CCR5 gene---edited (mutant) iPSCs were derived from patients' PBMC. When differentiated into immune cells and HIV---1 challenged, mutant iPSC lines were resistant to CCR5---tropic and to some extent to CCR5/CXCR4 dual---tropic HIV---1 infection when compared to wild---type iPSC lines. CONCLUSION: Our study demonstrates that iPSC---derived, gene---edited immune cells are resistant to distinct HIV---1 strains. These findings have important implications for both in-vitro stem cell development and therapeutic approaches to cure HIV infection.

A randomized, controlled trial of mindfulness-based stress reduction in HIV infection.

OBJECTIVE: Evidence links depression and stress to more rapid progression of HIV-1 disease. We conducted a randomized controlled trial to test whether an intervention aimed at improving stress management and emotion regulation, mindfulness-based stress reduction (MBSR), would improve immunological (i.e. CD4+ T-cell counts) and psychological outcomes in persons with HIV-1 infection. METHODS: We randomly assigned participants with HIV-1 infection and CD4 T-cell counts >350 cells/µl who were not on antiretroviral therapy in a 1:1 ratio to either an MBSR group (n = 89) or an HIV disease self-management skills group (n = 88). The study was conducted at the University of California at San Francisco. We assessed immunologic (CD4, c-reactive protein, IL-6, and d-dimer) and psychological measures (Beck Depression Inventory for depression, modified Differential Emotions Scale for positive and negative affect, Perceived stress-scale, and mindfulness) at 3, 6 and 12 months after initiation of the intervention; we used multiple imputation to address missing values. RESULTS: We observed statistically significant improvements from baseline to 3-months within the MBSR group in depression, positive and negative affect, perceived stress, and mindfulness; between group differences in change were significantly greater in the MBSR group only for positive affect (per item difference on DES-positive 0.25, 95% CI 0.049, 0.44, p = .015). By 12 months the between group difference in positive affect was not statistically significant, although both groups had trends toward improvements compared to baseline in several psychological outcomes that were maintained at 12-months; these improvements were only statistically significant for depression and negative affect in the MBSR group and perceived stress for the control group. The groups did not differ significantly on rates of antiretroviral therapy initiation (MBSR = 39%, control = 29%, p = .22). After 12 months, the mean decrease in CD4+ T-cell count was 49.6 cells/µl in participants in the MBSR arm, compared to 54.2 cells/µl in the control group, a difference of 4.6 cells favoring the MBSR group (95% CI, -44.6, 53.7, p = .85). The between group differences in other immunologic-related outcomes (c-reactive protein, IL-6, HIV-1 viral load, and d-dimer) were not statistically significant at any time point. CONCLUSIONS: MBSR improved positive affect more than an active control arm in the 3 months following the start of the intervention. However, this difference was not maintained over the 12-month follow-up and there were no significant differences in immunologic outcomes between intervention groups. These results emphasize the need for further carefully designed research if we are to translate evidence linking psychological states to immunological outcomes into evidence-based clinical practices.

The CD8+ cell non-cytotoxic antiviral response affects RNA polymerase II-mediated human immunodeficiency virus transcription in infected CD4+ cells.

A CD8+ cell non-cytotoxic antiviral response (CNAR), mediated by a CD8+ cell antiviral factor (CAF), is associated with a long-term healthy state in human immunodeficiency virus (HIV) infection. CNAR/CAF reduces viral transcription without a known effect on specific viral sequences in the HIV genome. In studies to define the mechanism involved in the block in viral transcription, we now report that transcription from the HIV-LTR reporter is reduced in infected CD4+ cells upon treatment with CAF. In agreement with this observation, the amount of RNA polymerase II (RNAPII) on the HIV promoter and other viral regions was strongly diminished in HIV-infected CD4+ cells co-cultivated with CNAR-expressing CD8+ cells. These results demonstrate further that CNAR/CAF has a specific role in regulating HIV transcription and a step during the preinitiation complex assembly appears to be sensitive to CNAR/CAF.

Dispelling myths and focusing on notable concepts in HIV pathogenesis.

Since the discovery of HIV over three decades ago, major efforts have been made to control and perhaps eliminate HIV infection worldwide. During these studies, certain myths or misconceptions about this infectious disease have been emphasized and other potentially beneficial concepts have received less attention. A true long-term solution to HIV infection merits an appreciation of alternative ideas and findings that could be beneficial in the ultimate control of HIV/AIDS. Here, I discuss six issues and call for more attention to the science of HIV and well-designed clinical trials.

Conserved epitopes on HIV-1, FIV and SIV p24 proteins are recognized by HIV-1 infected subjects.

Cross-reactive peptides on HIV-1 and FIV p24 protein sequences were studied using peripheral blood mononuclear cells (PBMC) from untreated HIV-1-infected long-term survivors (LTS; >10 y of infection without antiretroviral therapy, ART), short-term HIV-1 infected subjects not on ART, and ART-treated HIV-1 infected subjects. IFNγ-ELISpot and CFSE-proliferation analyses were performed with PBMC using overlapping HIV-1 and FIV p24 peptides. Over half of the HIV-1 infected subjects tested (22/31 or 71%) responded to one or more FIV p24 peptide pools by either IFNγ or T-cell proliferation analysis. PBMC and T cells from infected subjects in all 3 HIV(+) groups predominantly recognized one FIV p24 peptide pool (Fp14) by IFNγ production and one additional FIV p24 peptide pool (Fp9) by T-cell proliferation analysis. Furthermore, evaluation of overlapping SIV p24 peptide sequences identified conserved epitope(s) on the Fp14/Hp15-counterpart of SIV, Sp14, but none on Fp9-counterpart of SIV, Sp9. The responses to these FIV peptide pools were highly reproducible and persisted throughout 2-4 y of monitoring. Intracellular staining analysis for cytotoxins and phenotyping for CD107a determined that peptide epitopes from Fp9 and Fp14 pools induced cytotoxic T lymphocyte-associated molecules including perforin, granzyme B, granzyme A, and/or expression of CD107a. Selected FIV and corresponding SIV epitopes recognized by HIV-1 infected patients indicate that these protein sequences are evolutionarily conserved on both SIV and HIV-1 (e.g., Hp15:Fp14:Sp14). These studies demonstrate that comparative immunogenicity analysis of HIV-1, FIV, and SIV can identify evolutionarily-conserved T cell-associated lentiviral epitopes, which could be used as a vaccine for prophylaxis or immunotherapy.

Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection.

Individuals homozygous for the C-C chemokine receptor type 5 gene with 32-bp deletions (CCR5Δ32) are resistant to HIV-1 infection. In this study, we generated induced pluripotent stem cells (iPSCs) homozygous for the naturally occurring CCR5Δ32 mutation through genome editing of wild-type iPSCs using a combination of transcription activator-like effector nucleases (TALENs) or RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 together with the piggyBac technology. Remarkably, TALENs or CRISPR-Cas9-mediated double-strand DNA breaks resulted in up to 100% targeting of the colonies on one allele of which biallelic targeting occurred at an average of 14% with TALENs and 33% with CRISPR. Excision of the piggyBac using transposase seamlessly reproduced exactly the naturally occurring CCR5Δ32 mutation without detectable exogenous sequences. We differentiated these modified iPSCs into monocytes/macrophages and demonstrated their resistance to HIV-1 challenge. We propose that this strategy may provide an approach toward a functional cure of HIV-1 infection.

Chemically modified peptides based on the membrane-proximal external region of the HIV-1 envelope induce high-titer, epitope-specific nonneutralizing antibodies in rabbits.

Broadly neutralizing monoclonal antibodies (bNAbs) 2F5 and 4E10 bind to the membrane proximal external region (MPER) of gp41 and also cross-react with phospholipids. In this study, we investigated if chemical modifications on the MPER adjacent to 2F5 and 4E10 epitopes using mimetics of inflammation-associated posttranslational modifications to induce 2F5- and 4E10-like bNAbs can break tolerance. We synthesized a series of chemically modified peptides spanning the MPER. The serine, threonine, and tyrosine residues in the peptides were modified with sulfate, phosphate, or nitrate moieties and presented in liposomes for rabbit immunizations. All immunizations resulted in high antisera titers directed toward both the modified and unmodified immunogens. Tyrosine modification was observed to significantly suppress antiepitope responses. Sera with strong anti-gp140 titers were purified by affinity chromatography toward the MPER peptide and found to possess a higher affinity toward the MPER than did the bNAbs 2F5 and 4E10. Modest neutralization was observed in the H9 neutralization assay, but neutralization was not observed in the TZM-bl cell or peripheral blood mononuclear cell (PBMC) neutralization assay platforms. Although neutralizing antibodies were not induced by this approach, we conclude that chemical modifications can increase the immune responses to poorly immunogenic antigens, suggesting that chemical modification in an appropriate immunization protocol should be explored further as an HIV-1 vaccine strategy.

Evolutionarily conserved epitopes on human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus reverse transcriptases detected by HIV-1-infected subjects.

Anti-human immunodeficiency virus (HIV) cytotoxic T lymphocyte (CTL)-associated epitopes, evolutionarily conserved on both HIV type 1 (HIV-1) and feline immunodeficiency virus (FIV) reverse transcriptases (RT), were identified using gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) and carboxyfluorescein diacetate succinimide ester (CFSE) proliferation assays followed by CTL-associated cytotoxin analysis. The peripheral blood mononuclear cells (PBMC) or T cells from HIV-1-seropositive (HIV(+)) subjects were stimulated with overlapping RT peptide pools. The PBMC from the HIV(+) subjects had more robust IFN-γ responses to the HIV-1 peptide pools than to the FIV peptide pools, except for peptide-pool F3. In contrast, much higher and more frequent CD8(+) T-cell proliferation responses were observed with the FIV peptide pools than with the HIV peptide pools. HIV-1-seronegative subjects had no proliferation or IFN-γ responses to the HIV and FIV peptide pools. A total of 24% (40 of 166) of the IFN-γ responses to HIV pools and 43% (23 of 53) of the CD8(+) T-cell proliferation responses also correlated to responses to their counterpart FIV pools. Thus, more evolutionarily conserved functional epitopes were identified by T-cell proliferation than by IFN-γ responses. In the HIV(+) subjects, peptide-pool F3, but not the HIV H3 counterpart, induced the most IFN-γ and proliferation responses. These reactions to peptide-pool F3 were highly reproducible and persisted over the 1 to 2 years of testing. All five individual peptides and epitopes of peptide-pool F3 induced IFN-γ and/or proliferation responses in addition to inducing CTL-associated cytotoxin responses (perforin, granzyme A, granzyme B). The epitopes inducing polyfunctional T-cell activities were highly conserved among human, simian, feline, and ungulate lentiviruses, which indicated that these epitopes are evolutionarily conserved. These results suggest that FIV peptides could be used in an HIV-1 vaccine.

Nevirapine inhibits the anti-HIV activity of CD8+ cells.

Antiretroviral therapy (ART) significantly reduced the CD8 cell noncytotoxic anti-HIV response in 12 HIV-1-infected subjects (P < 0.0001). In separate experiments, CD8(+) cells from long-term survivors were cocultured with HIV-infected CD4(+) cells using varying concentrations of anti-HIV drugs. The antiviral function of CD8(+) cells from 4 of the 14 LTSs was reduced with exposure to 10 µM of nevirapine (P < 0.05). The antiviral activity of CD8(+) cells from 2 LTSs was inhibited by 5 µM of zidovudine. These studies indicate that nevirapine and probably zidovudine can inhibit the anti-HIV activity of CD8(+) cells and thus could influence the effectiveness of antiretroviral therapy.

Plasmacytoid dendritic cell number and responses to Toll-like receptor 7 and 9 agonists vary in HIV Type 1-infected individuals in relation to clinical state.

In HIV-1 infection, plasmacytoid dendritic cell (PDC) numbers and function are decreased. No detailed comparisons of PDC responses to various stimuli in HIV-1-infected patients are available. Using for the first time purified PDCs, we compared PDC responses [interferon (IFN)-α production/cell] to various stimuli in a large number (n=48) of HIV-1-infected patients and healthy volunteers (n=19). Toll-like receptor (TLR)7- and TLR9-induced expression of PDC surface activation and maturation markers was also compared in the two populations. We have confirmed that PDC number coincides with CD4(+) T cell counts and clinical state. Notably, we have shown that a direct association of PDC function in terms of IFN-α production/cell exists with PDC numbers and CD4(+) cell counts when PDCs are exposed to a TLR9 ligand and HIV-infected cells, but not with a TLR7 ligand. Moreover, in the HIV-infected subjects but not the healthy controls, the magnitude of IFN-α release per PDC in response to the TLR7 ligand is significantly (p<0.01) lower than that to the TLR9 ligand. However, in both study populations, the TLR7 stimulation in comparison to TLR9 stimulation induced higher expression of PDC surface activation and maturation markers and significantly (p<0.05) decreased the expression of BDCA-2, a negative regulator of interferon. Furthermore, the cross-ligation of BDCA-2 significantly (p<0.05) inhibited TLR9- but not TLR7-induced IFN-α production by PDCs from both clinical groups. These findings suggest that differences exist in TLR7- and TLR9-induced IFN-α production by PDCs in HIV-infected individuals that are not directly related to BDCA-2 down-modulation.

Differential transmission of HIV traversing fetal oral/intestinal epithelia and adult oral epithelia.

While human immunodeficiency virus (HIV) transmission through the adult oral route is rare, mother-to-child transmission (MTCT) through the neonatal/infant oral and/or gastrointestinal route is common. To study the mechanisms of cell-free and cell-associated HIV transmission across adult oral and neonatal/infant oral/intestinal epithelia, we established ex vivo organ tissue model systems of adult and fetal origin. Given the similarity of neonatal and fetal oral epithelia with respect to epithelial stratification and density of HIV-susceptible immune cells, we used fetal oral the epithelium as a model for neonatal/infant oral epithelium. We found that cell-free HIV traversed fetal oral and intestinal epithelia and infected HIV-susceptible CD4(+) T lymphocytes, Langerhans/dendritic cells, and macrophages. To study the penetration of cell-associated virus into fetal oral and intestinal epithelia, HIV-infected macrophages and lymphocytes were added to the surfaces of fetal oral and intestinal epithelia. HIV-infected macrophages, but not lymphocytes, transmigrated across fetal oral epithelia. HIV-infected macrophages and, to a lesser extent, lymphocytes transmigrated across fetal intestinal epithelia. In contrast to the fetal oral/intestinal epithelia, cell-free HIV transmigration through adult oral epithelia was inefficient and virions did not infect intraepithelial and subepithelial HIV-susceptible cells. In addition, HIV-infected macrophages and lymphocytes did not transmigrate through intact adult oral epithelia. Transmigration of cell-free and cell-associated HIV across the fetal oral/intestinal mucosal epithelium may serve as an initial mechanism for HIV MTCT.