Human Immunodeficiency Virus Replication In CD4 Research Articles

CD8 T Cell Virus Inhibition Assay Protocol.

The human immunodeficiency virus (HIV)-1 viral inhibition assay (VIA) measures CD8+ T cell-mediated inhibition of HIV replication in CD4+ T cells and is increasingly used for clinical testing of HIV vaccines and immunotherapies. Different VIAs that differ in length of CD8:CD4 T cell culture periods (6-13 days), purity of CD4 cultures [isolated CD4+ T cells or CD8+ depleted peripheral blood mononuclear cells (PBMCs)], HIV strains (laboratory strains, isolates, reporter viruses) and read-outs of virus inhibition (p24 ELISA, intracellular measurement of p24, luciferase reporter expression, and viral gag RNA) have been reported. Here, we describe multiple modifications to a 7-day VIA protocol, the most impactful being the introduction of independent replicate cultures for both HIV infected-CD4 (HIV-CD4) and HIV-CD4:CD8 T cell cultures. Virus inhibition was quantified using a ratio of weighted averages of p24+ cells in replicate cultures and the corresponding 95% confidence intervals. We identify methodological and analysis changes that could be incorporated into other protocols to improve assay reproducibility. We found that in people living with HIV (PLWH) on antiretroviral therapy (ART), CD8 T cell virus inhibition was largely stable over time, supporting the use of this assay and/or analysis methods to examine therapeutic interventions. Graphic abstract.

Expression of Chimeric Receptor CD4ζby Natural Killer Cells Derived from Human Pluripotent Stem Cells Improves In Vitro Activity but Does Not Enhance Suppression of HIV Infection In Vivo

Cell-based immunotherapy has been gaining interest as an improved means to treat human immunodeficiency virus (HIV)/AIDS. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) could become a potential resource. Our previous studies have shown hESC and iPSC-derived natural killer (NK) cells can inhibit HIV-infected targets in vitro. Here, we advance those studies by expressing a HIV chimeric receptor combining the extracellular portion of CD4 to the CD3ζ intracellular signaling chain. We hypothesized that expression of this CD4ζ receptor would more efficiently direct hESC- and iPSC-derived NK cells to target HIV-infected cells. In vitro studies showed the CD4ζ expressing hESC- and iPSC-NK cells inhibited HIV replication in CD4+ T-cells more efficiently than their unmodified counterparts. We then evaluated CD4ζ expressing hESC (CD4ζ-hESC)- and iPSC-NK cells in vivo anti-HIV activity using a humanized mouse model. We demonstrated significant suppression of HIV replication in mice treated with both CD4ζ-modified and -unmodified hESC-/iPSC-NK cells compared with control mice. However, we did not observe significantly increased efficacy of CD4ζ expression in suppression of HIV infection. These studies indicate that hESC/iPSC-based immunotherapy can be used as a unique resource to target HIV/AIDS.

CpG Oligodeoxynucleotides Block Human Immunodeficiency Virus Type 1 Replication in Human Lymphoid Tissue Infected Ex Vivo

Oligodeoxynucleotides (ODNs) with immunomodulatory motifs control a number of microbial infections in animal models, presumably by acting through toll-like receptor 9 (TLR9) to induce a number of cytokines (e.g., alpha interferon and tumor necrosis factor alpha). The immunomodulatory motif consists of unmethylated sequences of cytosine and guanosine (CpG motif). ODNs without CpG motifs do not trigger TLR9. We hypothesized that triggering of TLR9 generates a cellular environment unfavorable for human immunodeficiency virus (HIV) replication. We tested this hypothesis in human lymphocyte cultures and found that phosphorothioate-modified ODN CpG2006 (type B ODNs) inhibited HIV replication nearly completely and prevented the loss of CD4(+) T cells. ODNs CpG2216 and CpG10 (type A ODNs) were less effective. CpG2006 blocked HIV replication in purified CD4(+) T cells and T-cell lines; CpG10 was ineffective in this setting, indicating that type A ODNs may inhibit HIV replication in CD4(+) T-cell lines indirectly through a separate cell subset. However, control ODNs without CpG motifs also showed anti-HIV effects, indicating that these effects are nonspecific and not due to TLR9 triggering. The mechanism of action is not clear. CpG2006 and its control ODN blocked syncytium formation in a cell fusion-based assay, but CpG10, CpG2216, and their control ODNs did not. The latter types interfered with the HIV replication cycle during disassembly or reverse transcription. In contrast, CpG2006 and CpG2216 specifically induced cytokines critical to initiation of the innate immune response. In summary, the nonspecific anti-HIV activity of CpG ODNs, their ability to stimulate HIV replication in latently infected cells, potentially resulting in their elimination, and their documented ability to link the innate and adaptive immune responses make them attractive candidates for further study as anti-HIV drugs.

CD4(+) and CD8(+) T cell death during human immunodeficiency virus infection in vitro.

We have evaluated the death of CD4(+) and CD8(+) T cells during in vitro human immunodeficiency virus (HIV) infection of peripheral blood mononuclear cells (PBMC) and tonsilar tissue. Acute infections with several X4 and R5 HIV isolates induced a decrease in cell viability that was higher in infections with X4 viruses and correlated with an increased rate of CD4(+) T-cell death. In CD4(+) T cells, the primary X4 isolate AOM induced higher levels of death than the laboratory X4 isolates IIIB and NL4-3 or the R5 isolates BaL and MDM. An effect on CD8(+) T-cell viability was exclusively observed in infections by X4 viruses, including the NL4-3 strain, in both PBMC and tonsilar tissue. This effect was dependent on the env gene of the infecting isolate and required productive HIV replication in CD4(+) but not in CD8(+) T cells. Our results suggest that X4 and R5 HIV isolates depleted CD4(+) T cells to a different extent and that CD8(+) T-cell viability may also be affected by mechanisms other than those acting in CD4(+) T cells.

CD8(+)-Cell antiviral factor activity is not restricted to human immunodeficiency virus (HIV)-specific T cells and can block HIV replication after initiation of reverse transcription.

CD8(+) lymphocytes from human immunodeficiency virus (HIV)-infected patients can suppress in vitro HIV replication in CD4(+) T cells by a noncytolytic mechanism involving secreted CD8(+)-cell antiviral factor(s) (CAF). Using an HIV Nef-specific cytotoxic-T-lymphocyte (CTL) line and autologous CD4(+) T cells infected with a nef-deleted HIV-1 virus, we demonstrated that, after a priming antigenic stimulation, this suppression does not require the presence of the specific antigen during the effector phase. Furthermore, using an Epstein-Barr virus (EBV)-specific CTL line from an HIV-seronegative donor, we demonstrated that the ability to inhibit HIV replication in a noncytolytic manner is not restricted to HIV-specific effector cells; indeed, EBV-specific CTL were as efficient as HIV-specific effectors in suppressing R5 or X4 HIV-1 strain replication in vitro. This HIV-suppressive activity mediated by a soluble factor(s) present in the culture supernatant was detectable for up to 14 days following stimulation of EBV-specific CD8(+) cells with the cognate epitope peptide. Following acute infection of CEM cells with an X4 strain of HIV-1, EBV-specific CTL line supernatant containing HIV-suppressive activity did not block virus entry but was shown to interfere with virus replication after the first template switching of reverse transcription. Our results suggest that the noncytolytic control of HIV replication by EBV-specific CD8(+) T lymphocytes corresponded to a CAF-like activity and thus demonstrate that CAF production may not be restricted to CTL induced during HIV disease. Moreover, CAF acts after reverse transcription at least for X4 isolate replication inhibition.

CD8(+) cell noncytotoxic anti-human immunodeficiency virus response inhibits expression of viral RNA but not reverse transcription or provirus integration.

CD8(+) T cells from human immunodeficiency virus (HIV)-infected individuals can suppress HIV replication in CD4(+) cells by a noncytotoxic mechanism that inhibits the expression of viral RNA. The present study examined whether other step(s) in the virus replicative cycle could be affected by the CD8(+) cells. Culturing HIV-infected CD4(+) T cells with antiviral CD8(+) T cells did not significantly reduce the amounts of (i) early HIV DNA reverse transcripts (detected by LTR-U3/R), (ii) total nuclear HIV gag DNA, or (iii) integrated proviral DNA. However, exposure to the CD8(+) T cells did cause a reduction in the amount of multiply spliced tat and full-length gag mRNA expressed by the infected CD4(+) T cells, confirming previous observations. The levels of glyceraldehyde-3-phosphate dehydrogenase and interleukin-2 receptor-alpha mRNA were not affected. The results support the conclusion that the noncytotoxic anti-HIV response of CD8(+) T cells, demonstrable in vitro, does not affect any of the virus replication steps leading to the integration of proviral HIV, but specifically interrupts the expression of viral RNA.

HIV replication in CD4+ T cells of HIV-infected individuals is regulated by a balance between the viral suppressive effects of endogenous beta-chemokines and the viral inductive effects of other endogenous cytokines.

This study demonstrates that the beta-chemokines macrophage inflammatory proteins 1 alpha and 1 beta (MIP-1 alpha and MIP-1 beta) and, RANTES (regulated on activation, normally T-cell expressed and secreted) inhibit human immunodeficiency virus (HIV) replication in anti-CD3 or recall antigen-stimulated peripheral blood mononuclear cells (PBMCs) of asymptomatic HIV-infected subjects. Significant levels of beta-chemokines were produced by both CD4+ and CD8+ PBMC subsets from HIV-infected individuals. Neutralization of endogenous MIP-1 alpha, MIP-1 beta, and RANTES did not rescue HIV replication in cultures to which greater than 10% CD8+ T cells had been added, indicating that the HIV suppressor activity of CD8+ T cells cannot be explained entirely by the beta-chemokines. However, significant enhancement of viral replication was observed upon neutralization of endogenous beta-chemokines in CD8-depleted or CD4+ PBMCs from most donors, particularly in cultures with low inducible levels of HIV production. In contrast, certain endogenous proinflammatory cytokines induced HIV replication in these same cells. These data suggest that the levels of HIV replication in CD4+ PBMC reflect the balance of the opposing effects of endogenous suppressive factors, such as the beta-chemokines, and HIV-inducing cytokines, such as tumor necrosis factor alpha and interleukin 1 beta.