Joseph W. Adelsberger

HIV-1 induces phenotypic and functional perturbations of B cells in chronically infected individuals

A number of perturbations of B cells has been described in the setting of HIV infection; however, most remain poorly understood. To directly address the effect of HIV replication on B cell function, we investigated the capacity of B cells isolated from HIV-infected patients to respond to a variety of stimuli before and after reduction of viremia by effective antiretroviral therapy. B cells taken from patients with high levels of plasma viremia were defective in their proliferative responses to various stimuli. Viremia was also associated with the appearance of a subpopulation of B cells that expressed reduced levels of CD21. After fractionation into CD21high- and CD21low-expressing B cells, the CD21low fraction showed dramatically reduced proliferation in response to B cell stimuli and enhanced secretion of immunoglobulins when compared with the CD21high fraction. Electron microscopic analysis of each fraction revealed cells with plasmacytoid features in the CD21low B cell population but not in the CD21high fraction. These results indicate that HIV viremia induces the appearance of a subset of B cells whose function is impaired and which may be responsible for the hypergammaglobulinemia associated with HIV disease.

Cellular Immune Responses to Human Papillomavirus (HPV)-16 L1 in Healthy Volunteers Immunized with Recombinant HPV-16 L1 Virus-Like Particles

The causal association between papillomavirus (HPV) infection and cervical cancer has been demonstrated; the development of a prophylactic vaccine to protect against HPV infection may therefore reduce the incidence of this cancer worldwide. Noninfectious HPV-like particles (VLPs), composed of the L1 major capsid protein, are current candidate vaccines for prevention of HPV infection and cervical neoplasia. Although neutralizing antibodies have a pivotal role in the prevention of initial infection, cellular immune responses to HPV antigens may have an important role in viral clearance. A phase II trial was conducted to further evaluate the immunogenicity of a recombinant HPV-16 L1 VLP vaccine administered intramuscularly, without adjuvant, at 0, 1, and 6 months. Cell-mediated immune responses (lymphoproliferation and cytokine production) to HPV-16 L1 VLPs were evaluated in peripheral blood mononuclear cells (PBMCs) from 43 individuals receiving the L1 VLP vaccine and from 10 individuals receiving placebo. Vaccination resulted, at months 2 and 7 (i.e., 1 month after the second immunization and 1 month after third immunization, respectively), in increases in T cell-proliferative response to HPV-16 L1 VLPs (P<.001). In addition, significant increases in cytokine (interferon-gamma, interleukin [IL]-5 and IL-10) responses to L1 VLPs were observed after vaccination (P<.001). The strongest cytokine responses at month 7 were observed in individuals with high antibody titers at month 2, suggesting that neutralizing antibodies generated by initial vaccination may augment T cell responses to subsequent booster vaccinations. No significant increases in lymphoproliferative or cytokine responses to L1 VLPs were observed in individuals receiving placebo. In summary, the HPV-16 L1 vaccine induces not only robust B cell responses but also L1-specific T cell responses detectable by proliferation of both CD4+ and CD8+ T cells and in vitro production of both Th1- and Th2-type cytokines. Future efficacy studies are needed to evaluate whether and/or how VLP vaccines confer protection against genital HPV infection and associated disease.

Induction of prolonged survival of CD4+ T lymphocytes by intermittent IL-2 therapy in HIV-infected patients

HIV infection leads to decreases in the number of CD4 T lymphocytes and an increased risk for opportunistic infections and neoplasms. The administration of intermittent cycles of IL-2 to HIV-infected patients can lead to profound increases (often greater than 100%) in CD4 cell number and percentage. Using in vivo labeling with 2H-glucose and BrdU, we have been able to demonstrate that, although therapy with IL-2 leads to high levels of proliferation of CD4 as well as CD8 lymphocytes, it is a remarkable preferential increase in survival of CD4 cells (with half-lives that can exceed 3 years) that is critical to the sustained expansion of these cells. This increased survival was time-dependent: the median half-life, as determined by semiempirical modeling, of labeled CD4 cells in 6 patients increased from 1.7 weeks following an early IL-2 cycle to 28.7 weeks following a later cycle, while CD8 cells showed no change in the median half-life. Examination of lymphocyte subsets demonstrated that phenotypically naive (CD27+CD45RO-) as well as central memory (CD27+CD45RO+) CD4 cells were preferentially expanded, suggesting that IL-2 can help maintain cells important for host defense against new antigens as well as for long-term memory to opportunistic pathogens.

HIV infection-associated immune activation occurs by two distinct pathways that differentially affect CD4 and CD8 T cells

HIV infection is characterized by a brisk immune activation that plays an important role in the CD4 depletion and immune dysfunction of patients with AIDS. The mechanism underlying this activation is poorly understood. In the current study, we tested the hypothesis that this activation is the net product of two distinct pathways: the inflammatory response to HIV infection and the homeostatic response to CD4 T cell depletion. Using ex vivo BrdU incorporation of PBMCs from 284 patients with different stages of HIV infection, we found that CD4 proliferation was better predicted by the combination of CD4 depletion and HIV viral load (R2 = 0.375, P < 0.001) than by either parameter alone (CD4 T cell counts, R2 = 0.202, P < 0.001; HIV viremia, R2 = 0.302, P < 0.001). Interestingly, CD8 T cell proliferation could be predicted by HIV RNA levels alone (R2 = 0.334, P < 0.001) and this predictive value increased only slightly (R2 = 0.346, P < 0.001) when CD4 T cell depletion was taken into account. Consistent with the hypothesis that CD4 T cell proliferation is driven by IL-7 as a homeostatic response to CD4 T cell depletion, levels of phosphorylated STAT-5 were found to be elevated in naive subsets of CD4 and CD8 T cells from patients with HIV infection and in the central memory subset of CD4 T cells. Taken together these data demonstrate that at least two different pathways lead to immune activation of T cells in patients with HIV infection and these pathways differentially influence CD4 and CD8 T cell subsets.

IL-27, a novel anti-HIV cytokine, activates multiple interferon-inducible genes in macrophages

Objective: IL-27 is a novel anti-HIV cytokine that inhibits HIV-1 replication in both CD4 T cells and monocyte-derived macrophages (MDM) as IFN-α does. To elucidate the mechanism of the antiviral activity, we compared the activity and the gene expression profile of IL-27-treated cells with that of IFN-α-treated cells. Methods: CD4 T cells and monocytes were isolated from peripheral blood mononuclear cells of healthy donors. CD4 T cells were stimulated with phytohemagglutinin, and MDM were induced from monocytes using macrophage-colony stimulating factor. HIV-1 replication was monitored by p24 antigen capture assay. The gene expression profiles were analysed using DNA microarray analysis. The increase in the expression of IFN-inducible genes (IFIG) was confirmed by the Quantigene plex assay. Results: Both cytokines preferentially inhibited HIV-1 replication in MDM compared with CD4 T cells. Quantitative real time polymerase chain reaction, enzyme-linked immunosorbent assay and neutralization assay using anti-IFN indicated that IFN-α, IFN-β and IFN-γ had no significant impact on IL-27-mediated HIV inhibition. DNA microarray analysis illustrated that IFN-α induced 33 and 18 IFIG in MDM and CD4 T cells, respectively. IL-27 induced 28 IFIG in MDM and five IFIG in CD4 T cells. The quantitative assay confirmed that IL-27 activated genes of RNA-dependent kinase, oligoadenylate synthetase, myxovirus protein, and apolipoprotein B messenger RNA-editing enzyme-catalytic polypeptide-like 3G. Conclusion: IL-27 differentially regulates the gene expression between CD4 T cells and MDM. IL-27 significantly induces antiviral genes in MDM as does IFN-α, suggesting that IL-27 inhibits HIV replication in MDM via mechanism(s) similar to that of IFN-α.

Increased peripheral expansion of naive CD4+ T cells in vivo after IL-2 treatment of patients with HIV infection.

Intermittent interleukin-2 (IL-2) therapy has been shown to increase the number of CD4+ T cells, preferentially cells with a naive phenotype, in patients with HIV infection. For this report we investigated the mechanism underlying this expansion by studying the relative roles of peripheral expansion and thymic output. In a cohort of six patients receiving IL-2 over a period of 1 year, the mean number of naive CD4+ T cells increased from 139 to 387 cells per μl while levels of T cell receptor rearrangement excision circles (TRECs) declined from 47,946 to 26,510 copies per 106 naive T cells, thus making it unlikely that the CD4+ T cell count increases were secondary to increase in thymic output. To examine directly the impact of IL-2 on peripheral expansion, peripheral blood mature, naive CD4+ T cells were labeled ex vivo with 5-bromodeoxyuridine as well as stained directly for Ki67. These studies revealed a 7-fold increase in the percentage of 5-bromodeoxyuridine-positive cells and a 20–40-fold increase in Ki67 staining in the naive CD4+ T cell pool in the setting of IL-2 administration. This degree of increase in mature CD4+ T cell turnover induced by IL-2 does not compromise the future replicative potential of these cells, because longitudinal measurements of telomere length went from 6,981 to 7,153 bp after 1 year of IL-2 therapy. These data strongly suggest that much of the increase in CD4+ cells associated with IL-2 treatment is caused by peripheral expansion of existing naive CD4+ T cells rather than increased thymic output and that these increases occur without compromising the potential of these cells for further cell division.

Naïve T-Cell Dynamics in Human Immunodeficiency Virus Type 1 Infection: Effects of Highly Active Antiretroviral Therapy Provide Insights into the Mechanisms of Naïve T-Cell Depletion

ABSTRACT Both naïve CD4+ and naïve CD8+ T cells are depleted in individuals with human immunodeficiency virus type 1 (HIV-1) infection by unknown mechanisms. Analysis of their dynamics prior to and after highly active antiretroviral therapy (HAART) could reveal possible mechanisms of depletion. Twenty patients were evaluated with immunophenotyping, intracellular Ki67 staining, T-cell receptor excision circle (TREC) quantitation in sorted CD4 and CD8 cells, and thymic computed tomography scans prior to and ∼6 and ∼18 months after initiation of HAART. Naïve T-cell proliferation decreased significantly during the first 6 months of therapy (P < 0.01) followed by a slower decline. Thymic indices did not change significantly over time. At baseline, naïve CD4+ T-cell numbers were lower than naive CD8+ T-cell numbers; after HAART, a greater increase in naïve CD4+ T cells than naïve CD8+ T cells was observed. A greater relative change (n-fold) in the number of TREC+ T cells/μl than in naïve T-cell counts was observed at 6 months for both CD4+ (median relative change [n-fold] of 2.2 and 1.7, respectively; P < 0.01) and CD8+ T cell pools (1.4 and 1.2; P < 0.01). A more pronounced decrease in the proliferation than the disappearance rate of naïve T cells after HAART was observed in a second group of six HIV-1-infected patients studied by in vivo pulse labeling with bromodeoxyuridine. These observations are consistent with a mathematical model where the HIV-1-induced increase in proliferation of naïve T cells is mostly explained by a faster recruitment into memory cells.

Discovery of Small-Molecule Human Immunodeficiency Virus Type 1 Entry Inhibitors That Target the gp120-Binding Domain of CD4

ABSTRACT The interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and the CD4 receptor is highly specific and involves relatively small contact surfaces on both proteins according to crystal structure analysis. This molecularly conserved interaction presents an excellent opportunity for antiviral targeting. Here we report a group of pentavalent antimony-containing small molecule compounds, NSC 13778 (molecular weight, 319) and its analogs, which exert a potent anti-HIV activity. These compounds block the entry of X4-, R5-, and X4/R5-tropic HIV-1 strains into CD4+ cells but show little or no activity in CD4-negative cells or against vesicular stomatitis virus-G pseudotyped virions. The compounds compete with gp120 for binding to CD4: either immobilized on a solid phase (soluble CD4) or on the T-cell surface (native CD4 receptor) as determined by a competitive gp120 capture enzyme-linked immunosorbent assay or flow cytometry. NSC 13778 binds to an N-terminal two-domain CD4 protein, D1/D2 CD4, immobilized on a surface plasmon resonance sensor chip, and dose dependently reduces the emission intensity of intrinsic tryptophan fluorescence of D1/D2 CD4, which contains two of the three tryptophan residues in the gp120-binding domain. Furthermore, T cells incubated with the compounds alone show decreased reactivity to anti-CD4 monoclonal antibodies known to recognize the gp120-binding site. In contrast to gp120-binders that inhibit gp120-CD4 interaction by binding to gp120, these compounds appear to disrupt gp120-CD4 contact by targeting the specific gp120-binding domain of CD4. NSC 13778 may represent a prototype of a new class of HIV-1 entry inhibitors that can break into the gp120-CD4 interface and mask the gp120-binding site on the CD4 molecules, effectively repelling incoming virions.

Actinomycin D Induces High-Level Resistance to Thymidine Analogs in Replication of Human Immunodeficiency Virus Type 1 by Interfering with Host Cell Thymidine Kinase Expression

ABSTRACT Actinomycin D (ActD) is a transcription inhibitor and has been used in the treatment of certain forms of cancer. ActD has been reported to be a potential inhibitor of human immunodeficiency virus type 1 (HIV-1) replication due to its ability to inhibit reverse transcription. In contrast to what was expected, low concentrations of ActD (1 to 10 nM) upregulated HIV-1 replication 8- to 10-fold in MT-2 cells and had no effect on HIV-2 replication or on HIV-1 replication in MT-4, Jurkat, or peripheral blood mononuclear cells. The upregulation of HIV-1 replication was associated with an increase in HIV-1 transcription and a decrease in CD4 and CXCR4 expression. To further evaluate the effects of ActD on emergence of drug resistance in HIV-1 replication, a series of drug resistance assays were performed. Of interest, treatment of MT-2 cells with ActD also led to a high level of resistance to thymidine analogs (>1,000-fold increase in resistance to zidovudine and >250-fold to stavudine) but not to other nucleoside reverse transcriptases (RT), nonnucleoside RT, or protease inhibitors. This resistance appeared to be due to a suppression of host cell thymidine kinase-1 (TK-1) expression. These results indicate that ActD leads to a novel form of thymidine analog resistance by suppressing host cell TK-1 expression. These results suggest that administration of combination drugs to HIV-1-infected patients may induce resistance to antiretroviral compounds via a modification of cellular factors.

Interleukin-2 Inhibits HIV-1 Replication in Some Human T Cell Lymphotrophic Virus-1-infected Cell Lines via the Induction and Incorporation of APOBEC3G into the Virion *

Background: Some cytokines exhibit dichotomous effects on HIV replication in different cell types. Results: IL-2 inhibits HIV-1 replication in MT-2 cells via intracellular induction and virion incorporation of the host restriction factor, APOBEC3G. Conclusion: Stimulation of MT-2 cell with IL-2 produces viruses with impaired replication competency. Significance: This new insight could open the door to additional host-based strategies to suppress HIV-1 replication. IL-2 has been used in culture of primary T cells to maintain cell proliferation. We have previously reported that IL-27 inhibits HIV-1 replication in primary T cells in the presence of IL-2. To gain a better understanding of the mechanisms involved in this inhibitory effect, we attempted to investigate in detail the effects of IL-27 and IL-2 using several cell lines. Unexpectedly, IL-27 did not inhibit HIV-1 in T cell lines, whereas IL-2 inhibited HIV-1 replication in the human T cell lymphotrophic virus (HTLV)-1-transformed T cell lines, MT-2, MT-4, SLB-1, and ATL-2. No effects were seen in HTLV-1-negative cell lines. Utilizing MT-2 cells, we demonstrated that IL-2 treatment inhibited HIV-1 syncytia-inducing ability and dose-dependently decreased supernatant p24 antigen levels by >90%. Using real time PCR and Western blot analysis, we observed that IL-2 treatment induced the host restriction factor, APOBEC3G with accumulation into the lower molecular mass active form as characterized by FPLC. Further analysis revealed that the virus recovered from IL-2-treated MT-2 cells had impaired replication competency. This was found to be due to incorporation of APOBEC3G into the virion despite the presence of Vif. These findings demonstrate a novel role for IL-2 in regulating production of infectious HIV-1 virions in HTLV-1-infected cells through the induction of APOBEC3G.