NK cells and monocytes modulate primary HTLV-1 infection.

We investigated the impact of monocytes, NK cells, and CD8+ T-cells in primary HTLV-1 infection by depleting cell subsets and exposing macaques to either HTLV-1 wild type (HTLV-1WT) or to the HTLV-1p12KO mutant unable to infect replete animals due to a single point mutation in orf-I that inhibits its expression. The orf-I encoded p8/p12 proteins counteract cytotoxic NK and CD8+ T-cells and favor viral DNA persistence in monocytes. Double NK and CD8+ T-cells or CD8 depletion alone accelerated seroconversion in all animals exposed to HTLV-1WT. In contrast, HTLV-1p12KO infectivity was fully restored only when NK cells were also depleted, demonstrating a critical role of NK cells in primary infection. Monocyte/macrophage depletion resulted in accelerated seroconversion in all animals exposed to HTLV-1WT, but antibody titers to the virus were low and not sustained. Seroconversion did not occur in most animals exposed to HTLV-1p12KO. In vitro experiments in human primary monocytes or THP-1 cells comparing HTLV-1WT and HTLV-1p12KO demonstrated that orf-I expression is associated with inhibition of inflammasome activation in primary cells, with increased CD47 "don't-eat-me" signal surface expression in virus infected cells and decreased monocyte engulfment of infected cells. Collectively, our data demonstrate a critical role for innate NK cells in primary infection and suggest a dual role of monocytes in primary infection. On one hand, orf-I expression increases the chances of viral transmission by sparing infected cells from efferocytosis, and on the other may protect the engulfed infected cells by modulating inflammasome activation. These data also suggest that, once infection is established, the stoichiometry of orf-I expression may contribute to the chronic inflammation observed in HTLV-1 infection by modulating monocyte efferocytosis.

Functional properties and sequence variation of HTLV-1 p13.

Human T cell leukemia virus type-1 (HTLV-1) was the first retrovirus found to cause cancer in humans, but the mechanisms that drive the development of leukemia and other diseases associated with HTLV-1 infection remain to be fully understood. This review describes the functional properties of p13, an 87-amino acid protein coded by HTLV-1 open reading frame II (orf-II). p13 is mainly localized in the inner membrane of the mitochondria, where it induces potassium (K+) influx and reactive oxygen species (ROS) production, which can trigger either proliferation or apoptosis, depending on the ROS setpoint of the cell. Recent evidence indicates that p13 may influence the cell's innate immune response to viral infection and the infected cell phenotype. Association of the HTLV-1 transcriptional activator, Tax, with p13 increases p13's stability, leads to its partial co-localization with Tax in nuclear speckles, and reduces the ability of Tax to interact with the transcription cofactor CBP/p300. Comparison of p13 sequences isolated from HTLV-1-infected individuals revealed a small number of amino acid variations in the domains controlling the subcellular localization of the protein. Disruptive mutations of p13 were found in samples obtained from asymptomatic patients with low proviral load. p13 sequences of HTLV-1 subtype C isolates from indigenous Australian patients showed a high degree of identity among each other, with all samples containing a pattern of 5 amino acids that distinguished them from other subtypes. Further characterization of p13's functional properties and sequence variants may lead to a deeper understanding of the impact of p13 as a contributor to the clinical manifestations of HTLV-1 infection.

Role of HTLV-1 orf-I encoded proteins in viral transmission and persistence.

The human T cell leukemia virus type 1 (HTVL-1), first reported in 1980 by Robert Gallo's group, is the etiologic agent of both cancer and inflammatory diseases. Despite approximately 40 years of investigation, the prognosis for afflicted patients remains poor with no effective treatments. The virus persists in the infected host by evading the host immune response and inducing proliferation of infected CD4+ T-cells. Here, we will review the role that viral orf-I protein products play in altering intracellular signaling, protein expression and cell-cell communication in order to escape immune recognition and promote T-cell proliferation. We will also review studies of orf-I mutations found in infected patients and their potential impact on viral load, transmission and persistence. Finally, we will compare the orf-I gene in HTLV-1 subtypes as well as related STLV-1.

p30 protein: a critical regulator of HTLV-1 viral latency and host immunity.

The extraordinarily high prevalence of HTLV-1 subtype C (HTLV-1C) in some isolated indigenous communities in Oceania and the severity of the health conditions associated with the virus impress the great need for basic and translational research to prevent and treat HTLV-1 infection. The genome of the virus's most common subtype, HTLV-1A, encodes structural, enzymatic, and regulatory proteins that contribute to viral persistence and pathogenesis. Among these is the p30 protein encoded by the doubly spliced Tax-orf II mRNA, a nuclear/nucleolar protein with both transcriptional and post-transcriptional activity. The p30 protein inhibits the productive replication cycle via nuclear retention of the mRNA that encodes for both the viral transcriptional trans-activator Tax, and the Rex proteins that regulate the transport of incompletely spliced viral mRNA to the cytoplasm. In myeloid cells, p30 inhibits the PU-1 transcription factor that regulates interferon expression and is a critical mediator of innate and adaptive immunity. Furthermore, p30 alters gene expression, cell cycle progression, and DNA damage responses in T-cells, raising the hypothesis that p30 may directly contribute to T cell transformation. By fine-tuning viral expression while also inhibiting host innate responses, p30 is likely essential for viral infection and persistence. This concept is supported by the finding that macaques, a natural host for the closely genetically related simian T-cell leukemia virus 1 (STLV-1), exposed to an HTLV-1 knockout for p30 expression by a single point mutation do not became infected unless reversion and selection of the wild type HTLV-1 genotype occurs. All together, these data suggest that inhibition of p30 may help to curb and eventually eradicate viral infection by exposing infected cells to an effective host immune response.

Complete Rescue of HTLV-1p12KO Infectivity by Depletion of Monocytes Together with NK and CD8+ T Cells.

The transient depletion of monocytes alone prior to exposure of macaques to HTLV-1 enhances both HTLV-1WT (wild type) and HTLV-1p12KO (Orf-1 knockout) infectivity, but seroconversion to either virus is not sustained over time, suggesting a progressive decrease in virus expression. These results raise the hypotheses that either HTLV-1 persistence depends on a monocyte reservoir or monocyte depletion provides a transient immune evasion benefit. To test these hypotheses, we simultaneously depleted NK cells, CD8+ T cells, and monocytes (triple depletion) prior to exposure to HTLV-1WT or HTLV-1p12KO. Remarkably, triple depletion resulted in exacerbation of infection by both viruses and complete rescue of HTLV-1p12KO infectivity. Following triple depletion, we observed rapid and sustained seroconversion, high titers of antibodies against HTLV-1 p24Gag, and frequent detection of viral DNA in the blood and tissues of all animals when compared with depletion of only CD8+ and NK cells, or monocytes alone. The infection of macaques with HTLV-1WT or HTLV-1p12KO was associated with higher plasma levels of IL-10 after 21 weeks, while IL-6, IFN-γ, IL-18, and IL-1ß were only elevated in animals infected with HTLV-1WT. The repeat depletion of monocytes, NK, and CD8+ cells seven months following the first exposure to HTLV-1 did not further exacerbate viral replication. These results underscore the contribution of monocytes in orchestrating anti-viral immunity. Indeed, the absence of orf-1 expression was fully compensated by the simultaneous depletion of CD8+ T cells, NK cells, and monocytes, underlining the primary role of orf-1 in hijacking host immunity.

Cholera toxin B scaffolded, focused SIV V2 epitope elicits antibodies that influence the risk of SIVmac251 acquisition in macaques.

Introduction: An efficacious HIV vaccine will need to elicit a complex package of innate, humoral, and cellular immune responses. This complex package of responses to vaccine candidates has been studied and yielded important results, yet it has been a recurring challenge to determine the magnitude and protective effect of specific in vivo immune responses in isolation. We therefore designed a single, viral-spike-apical, epitope-focused V2 loop immunogen to reveal individual vaccine-elicited immune factors that contribute to protection against HIV/SIV. Method: We generated a novel vaccine by incorporating the V2 loop B-cell epitope in the cholera toxin B (CTB) scaffold and compared two new immunization regimens to a historically protective 'standard' vaccine regimen (SVR) consisting of 2xDNA prime boosted with 2xALVAC-SIV and 1xΔV1gp120. We immunized a cohort of macaques with 5xCTB-V2c vaccine+alum intramuscularly simultaneously with topical intrarectal vaccination of CTB-V2c vaccine without alum (5xCTB-V2/alum). In a second group, we tested a modified version of the SVR consisting of 2xDNA prime and boosted with 1xALVAC-SIV and 2xALVAC-SIV+CTB-V2/alum, (DA/CTB-V2c/alum). Results: In the absence of any other anti-viral antibodies, V2c epitope was highly immunogenic when incorporated in the CTB scaffold and generated highly functional anti-V2c antibodies in the vaccinated animals. 5xCTB-V2c/alum vaccination mediated non-neutralizing ADCC activity and efferocytosis, but produced low avidity, trogocytosis, and no neutralization of tier 1 virus. Furthermore, DA/CTB-V2c/alum vaccination also generated lower total ADCC activity, avidity, and neutralization compared to the SVR. These data suggest that the ΔV1gp120 boost in the SVR yielded more favorable immune responses than its CTB-V2c counterpart. Vaccination with the SVR generates CCR5- α4ß7+CD4+ Th1, Th2, and Th17 cells, which are less likely to be infected by SIV/HIV and likely contributed to the protection afforded in this regimen. The 5xCTB-V2c/alum regimen likewise elicited higher circulating CCR5- α4ß7+ CD4+ T cells and mucosal α4ß7+ CD4+ T cells compared to the DA/CTB-V2c/alum regimen, whereas the first cell type was associated with reduced risk of viral acquisition. Conclusion: Taken together, these data suggest that individual viral spike B-cell epitopes can be highly immunogenic and functional as isolated immunogens, although they might not be sufficient on their own to provide full protection against HIV/SIV infection.

Vaccine plus microbicide effective in preventing vaginal SIV transmission in macaques.

The human immunodeficiency virus epidemic continues in sub-Saharan Africa, and particularly affects adolescent girls and women who have limited access to antiretroviral therapy. Here we report that the risk of vaginal simian immunodeficiency virus (SIV)mac251 acquisition is reduced by more than 90% using a combination of a vaccine comprising V1-deleted (V2 enhanced) SIV envelope immunogens with topical treatment of the zinc-finger inhibitor SAMT-247. Following 14 weekly intravaginal exposures to the highly pathogenic SIVmac251, 80% of a cohort of 20 macaques vaccinated and treated with SAMT-247 remained uninfected. In an arm of 18 vaccinated-only animals without microbicide, 40% of macaques remained uninfected. The combined SAMT-247/vaccine regimen was significantly more effective than vaccination alone. By analysing immune correlates of protection, we show that, by increasing zinc availability, SAMT-247 increases natural killer cytotoxicity and monocyte efferocytosis, and decreases T-cell activation to augment vaccine-induced protection.

In Vivo Treatment with Insulin-like Growth Factor 1 Reduces CCR5 Expression on Vaccine-Induced Activated CD4+ T-Cells.

At the heart of the DNA/ALVAC/gp120/alum vaccine's efficacy in the absence of neutralizing antibodies is a delicate balance of pro- and anti-inflammatory immune responses that effectively decreases the risk of SIVmac251 acquisition in macaques. Vaccine efficacy is linked to antibodies recognizing the V2 helical conformation, DC-10 tolerogenic dendritic cells eliciting the clearance of apoptotic cells via efferocytosis, and CCR5 downregulation on vaccine-induced gut homing CD4+ cells. RAS activation is also linked to vaccine efficacy, which prompted the testing of IGF-1, a potent inducer of RAS activation with vaccination. We found that IGF-1 changed the hierarchy of V1/V2 epitope recognition and decreased both ADCC specific for helical V2 and efferocytosis. Remarkably, IGF-1 also reduced the expression of CCR5 on vaccine-induced CD4+ gut-homing T-cells, compensating for its negative effect on ADCC and efferocytosis and resulting in equivalent vaccine efficacy (71% with IGF-1 and 69% without).

HIV vaccine candidate efficacy in female macaques mediated by cAMP-dependent efferocytosis and V2-specific ADCC.

The development of an effective vaccine to protect against HIV acquisition will be greatly bolstered by in-depth understanding of the innate and adaptive responses to vaccination. We report here that the efficacy of DNA/ALVAC/gp120/alum vaccines, based on V2-specific antibodies mediating apoptosis of infected cells (V2-ADCC), is complemented by efferocytosis, a cyclic AMP (cAMP)-dependent antiphlogistic engulfment of apoptotic cells by CD14+ monocytes. Central to vaccine efficacy is the engagement of the CCL2/CCR2 axis and tolerogenic dendritic cells producing IL-10 (DC-10). Epigenetic reprogramming in CD14+ cells of the cyclic AMP/CREB pathway and increased systemic levels of miRNA-139-5p, a negative regulator of expression of the cAMP-specific phosphodiesterase PDE4D, correlated with vaccine efficacy. These data posit that efferocytosis, through the prompt and effective removal of apoptotic infected cells, contributes to vaccine efficacy by decreasing inflammation and maintaining tissue homeostasis.

Transient Viral Activation in Human T Cell Leukemia Virus Type 1-Infected Macaques Treated With Pomalidomide.

Human T cell leukemia virus type 1 (HTLV-1) persists in the host despite a vigorous immune response that includes cytotoxic T cells (CTL) and natural killer (NK) cells, suggesting the virus has developed effective mechanisms to counteract host immune surveillance. We recently showed that in vitro treatment of HTLV-1-infected cells with the drug pomalidomide (Pom) increases surface expression of MHC-I, ICAM-1, and B7-2, and significantly increases the susceptibility of HTLV-1-infected cells to NK and CTL killing, which is dependent on viral orf-I expression. We reasoned that by restoring cell surface expression of these molecules, Pom treatment has the potential to reduce virus burden by rendering infected cells susceptible to NK and CTL killing. We used the rhesus macaque model to determine if Pom treatment of infected individuals activates the host immune system and allows recognition and clearance of HTLV-1-infected cells. We administered Pom (0.2 mg/kg) orally to four HTLV-1-infected macaques over a 24 day period and collected blood, urine, and bone marrow samples throughout the study. Pom treatment caused immune activation in all four animals and a marked increase in proliferating CD4+, CD8+, and NK cells as measured by Ki-67+ cells. Activation markers HLA-DR, CD11b, and CD69 also increased during treatment. While we detected an increased frequency of cells with a memory CD8+ phenotype, we also found an increased frequency of cells with a Treg-like phenotype. Concomitant with immune activation, the frequency of detection of viral DNA and the HTLV-1-specific humoral response increased as well. In 3 of 4 animals, Pom treatment resulted in increased antibodies to HTLV-1 antigens as measured by western blot and p24Gag ELISA. Consistent with Pom inducing immune and HTLV-1 activation, we measured elevated leukotrienes LTB4 and LTE4 in the urine of all animals. Despite an increase in plasma LTB4, no significant changes in plasma cytokine/chemokine levels were detected. In all cases, however, cellular populations, LTB4, and LTE4 decreased to baseline or lower levels 2 weeks after cessation of treatment. These results indicated that Pom treatment induces a transient HTLV-1-specific immune activation in infected individuals, but also suggest Pom may not be effective as a single-agent therapeutic.

Anti-V2 antibodies virus vulnerability revealed by envelope V1 deletion in HIV vaccine candidates.

The efficacy of ALVAC-based HIV and SIV vaccines in humans and macaques correlates with antibodies to envelope variable region 2 (V2). We show here that vaccine-induced antibodies to SIV variable region 1 (V1) inhibit anti-V2 antibody-mediated cytotoxicity and reverse their ability to block V2 peptide interaction with the α4ß7 integrin. SIV vaccines engineered to delete V1 and favor an α helix, rather than a ß sheet V2 conformation, induced V2-specific ADCC correlating with decreased risk of SIV acquisition. Removal of V1 from the HIV-1 clade A/E A244 envelope resulted in decreased binding to antibodies recognizing V2 in the ß sheet conformation. Thus, deletion of V1 in HIV envelope immunogens may improve antibody responses to V2 virus vulnerability sites and increase the efficacy of HIV vaccine candidates.

A potential robust antiviral defense state in the common vampire bat: Expression, induction and molecular characterization of the three interferon-stimulated genes -OAS1, ADAR1 and PKR.

Bats are known to harbor many zoonotic viruses, some of which are pathogenic to other mammals while they seem to be harmless in bats. As the interferon (IFN) response represents the first line of defense against viral infections in mammals, it is hypothesized that activation of the IFN system is one of the mechanisms enabling bats to co-exist with viruses. We have previously reported induction of type I IFN in a cell line from the common vampire bat, Desmodus rotundus, upon polyinosinic:polycytidylic acid (poly(I:C)) stimulation. To deepen our knowledge on D. rotundus' IFN-I antiviral response, we molecularly characterized three interferon-stimulated genes (ISGs), OAS1, PKR and ADAR1, closely implicated in the IFN-I antiviral response, and tested their functionality in our cellular model. We first found that D. rotundus encoded two OAS1 paralogs, OAS1a and OAS1b, and that the functional domains of the four ISGs characterized were highly conserved with those of other mammals. Despite their significant transcription level in the absence of stimulation, the transcription of the four ISGs characterized was enhanced by poly(I:C). In addition, the transcription of OAS1a and OAS1b appears to be differentially regulated. These findings demonstrate an active ISG antiviral response in D. rotundus in which OAS1b may play an important role.

Development of molecular and cellular tools to decipher the type I IFN pathway of the common vampire bat.

Though the common vampire bat, Desmodus rotundus, is known as the main rabies virus reservoir in Latin America, no tools are available to investigate its antiviral innate immune system. To characterize the IFN-I pathway, we established an immortalized cell line from a D. rotundus fetal lung named FLuDero. Then we molecularly characterized some of the Toll-like receptors (TLR3, 7, 8 and 9), the three RIG-I-like receptor members, as well as IFNα1 and IFNß. Challenging the FLuDero cell line with poly (I:C) resulted in an up-regulation of both IFNα1 and IFNß and the induction of expression of the different pattern recognition receptors characterized. These findings provide evidence of the intact dsRNA recognition machinery and the IFN-I signaling pathway in our cellular model. Herein, we generated a sum of insightful specific molecular and cellular tools that will serve as a useful model to study virus-host interactions of the common vampire bat.

Increased osteopontin expression in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patient cells is associated with IL-17 expression.

BACKGROUND: HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurological inflammatory disease associated with a predominant infiltration of CD4+ T lymphocytes, which are the main subset of HTLV-1-infected cells. It has been demonstrated that in cell line the viral Tax protein transcriptionnally regulate expression of osteopontin, an inflammatory cytokine associated with Th17-related pathologies. OBJECTIVES: The aim of the study was to explore osteopontin expression in HTLV-1 asymptomatic carriers and in HAM/TSP patients and consequences on IL17 expression. STUDY DESIGN: We quantified Tax, osteopontin, RORγ, IL17 and IL22 mRNA expressions in cells from 10 HAM/TSP patients, 6 asymptomatic HTLV-1 carriers (ASY) and 4 HTLV-1-negative healthy donors during ex vivo culture. RESULTS: We observed that the expression of osteopontin was higher in HAM/TSP patients and correlated with Tax expression levels. Positive regulation of RORγ, IL17 and IL22 were also observed during cell culture. CONCLUSIONS: Our results propose a new mechanism which could contribute to HAM/TSP pathogenesis.