Early divergence in lymphoid tissue apoptosis between pathogenic and nonpathogenic simian immunodeficiency virus infections of nonhuman primates.

The events that contribute to the progression to AIDS during the acute phase of a primate lentiviral infection are still poorly understood. In this study, we used pathogenic and nonpathogenic simian models of simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) and African green monkeys (AGMs), respectively, to investigate the relationship between apoptosis in lymph nodes and the extent of viral replication, immune activation, and disease outcome. Here, we show that, in SIVmac251-infected RMs, a marked increased in lymphocyte apoptosis is evident during primary infection at the level of lymph nodes. Interestingly, the levels of apoptosis correlated with the extent of viral replication and the rate of disease progression to AIDS, with higher apoptosis in RMs of Indian genetic background than in those of Chinese origin. In stark contrast, no changes in the levels of lymphocyte apoptosis were observed during primary infection in the nonpathogenic model of SIVagm-sab infection of AGMs, despite similarly high rates of viral replication. A further and early divergence between SIV-infected RMs and AGMs was observed in terms of the dynamics of T- and B-cell proliferation in lymph nodes, with RMs showing significantly higher levels of cycling cells (Ki67(+)) in the T-cell zones in association with relatively low levels of Ki67(+) in the B-cell zones, whereas AGMs displayed a low frequency of Ki67(+) in the T-cell area but a high proportion of Ki67(+) cells in the B-cell area. As such, this study suggests that species-specific host factors determine an early immune response to SIV that predominantly involves either cellular or humoral immunity in RMs and AGMs, respectively. Taken together, these data are consistent with the hypotheses that (i) high levels of T-cell activation and lymphocyte apoptosis are key pathogenic factors during pathogenic SIV infection of RMs and (ii) low T-cell activation and apoptosis are determinants of the AIDS resistance of SIVagm-infected AGMs, despite high levels of SIVagm replication.

Early and persistent bone marrow hematopoiesis defect in simian/human immunodeficiency virus-infected macaques despite efficient reduction of viremia by highly active antiretroviral therapy during primary infection.

The hematological abnormalities observed in human immunodeficiency virus (HIV)-infected patients appear to be mainly due to bone marrow dysfunction. A macaque models of AIDS could greatly facilitate an in vivo approach to the pathogenesis of such dysfunction. Here, we evaluated in this model the impact of infection with a pathogenic simian/human immunodeficiency virus (SHIV) on bone marrow hematopoiesis. Three groups of macaques were inoculated with 50 50% median infective doses of pathogenic SHIV 89.P, which expresses env of dual-tropic HIV type 1 (HIV-1) 89.6 primary isolate. During the primary phase of infection, animals were treated with either a placebo or highly active antiretroviral therapy (HAART) combining zidovudine, lamivudine, and indinavir, initiated 4 or 72 h postinfection (p.i.) and administered twice a day until day 28 p.i. In both placebo-treated and HAART-treated animals, bone marrow colony-forming cells (CFC) progressively decreased quite early, during the first month p.i. One year p.i., both placebo- and HAART-treated animals displayed decreases in CFC to about 56% of preinfection values. At the same time, a dramatic decrease (greater than 77%) of bone marrow CD34(+) long-term culture-initiating cells was noted in all animals were found. No statistically significant differences between placebo- and HAART-treated monkeys were found. These data argue for an early and profound alteration of myelopoiesis at the level of the most primitive CD34(+) progenitor cells during SHIV infection, independently of the level of viremia, circulating CD4(+) cell counts, or antiviral treatment.

Longitudinal analysis of CD8(+) T-cell phenotype and IL-7, IL-15 and IL-16 mRNA expression in different tissues during primary simian immunodeficiency virus infection.

Infection of macaques with pathogenic isolates of simian immunodeficiency virus (SIV) represents a useful model of HIV infection that offers the unique opportunity to investigate the very early modifications that affect CD8(+) T-lymphocyte subsets and related cytokines during lentiviral infection. Herein, three cynomolgus macaques were inoculated intravenously with a pathogenic isolate of SIVmac 251. In fresh isolated mononuclear cells from blood, lymph node and bronchoalveolar lavage, we analyzed changes in the phenotype of CD8(+) T cells and we used reverse transcription-PCR to monitor the expression of IL-7, IL-15 and IL-16 mRNA. We demonstrated that an expansion of CD8(+)CD28(-) T cells occurs from the third week of infection on in the peripheral blood and in the lung, whereas CD8(+)CD28(+) T cells expand in the lymph nodes. Concomitantly, we evidenced mRNA modulations in IL-16, IL-15 and IL-7 expression in the three compartments studied. The containment of systemic viral replication was associated with an overexpression of IL-16 mRNA in the lung and in the peripheral blood. Given the immunomodulatory properties of IL-15 and IL-7 and the potential antiviral ability of IL-16, these perturbations could have important implications in early viral dissemination and HIV immunopathogenesis.

[Evaluation of the effect of early and massive tritherapy on the expression of cellular factors potentially implicated in antiretroviral therapy resistance]. / Evaluation de l'effet d'une trithérapie précoce et massive sur l'expression des facteurs cellulaires potentiellement impliqués dans l'échappement à la thérapeutique antirétrovirale.

Treatment of the human immunodeficiency virus (HIV) is restricted by therapeutic escape. The biological mechanisms of this chemoresistance rely notably on the modulation of cell kinase and P-glycoprotein (P-gp) expression. In this study, we investigated, in cynomolgus macaques, the roles of SHIV89.6P infection and of HAART in the mRNA expression of these cell factors. SHIV infection, or associated pathophysiological disorders, increase both thymidine kinase and thymidylate kinase mRNA expression and decrease those of P-gp. On the other hand, the expression of other cell kinases is not modulated. In parallel, HAART accentuates the decrease of P-gp expression and attenuates the increase of kinase expression. On the whole, such metabolic disorders, evidenced herein an animal model of HIV infection, could be involved in HIV-infected patients.

Immune responses following simian/human immunodeficiency virus (SHIV) challenge of rhesus macaques after human immunodeficiency virus (HIV)-1 third variable domain (V3) loop-based genetic immunization.

Following DNA immunization of rhesus macaques with a plasmid encoding the human immunodeficiency virus (HIV)-1 third variable domain (V3) loop, presented by pseudo-viral envelope particles of hepatitis B virus, specific immune responses were induced. The primates were then inoculated with a chimeric simian/human immunodeficiency virus (SHIV). All the animals were infected, but the V3-specific immunization provided a relative attenuation of the acute phase of infection in the absence of neutralizing antibody. In all animals, SHIV-specific cytotoxic T-lymphocyte precursors (CTLp) were detected early in peripheral blood and lymph nodes. The viremia peak correlated significantly with the decrease in CD4+ T cells and with a transient increase in the percentage of natural killer cells. The infection induced an oligoclonalization of the CD8+ T-cell variable beta chain repertoire in the blood. Surprisingly, HIV envelope-specific CTLp generated by genetic immunization may be governed by distinct circulation rules compared to SHIV-specific CTLp induced by infection.

Secretion of beta-chemokines by bronchoalveolar lavage cells during primary infection of macaques inoculated with attenuated nef-deleted or pathogenic simian immunodeficiency virus strain mac251.

Primary infection of macaques with simian immunodeficiency virus (SIV) as a model of human immunodeficiency virus (HIV) infection represents a unique opportunity to investigate early lentivirus-host interactions. In order to gain insight into immunopathogenic events taking place in the lung during lentiviral infection, we analysed lymphocyte expansion in the lung and chemokine secretion by mononuclear cells obtained by bronchoalveolar lavage (BALMCs) during primary infection by a pathogenic and a non-pathogenic SIV. Two groups of cynomolgus macaques were inoculated intravenously with a fully pathogenic isolate of SIVmac251 or with an attenuated, nef-deleted, molecular clone of SIVmac251. Spontaneous MIP-1alpha, MIP-1beta and RANTES production was assessed by ELISA in supernatants of short-term cultured BALMCs. Kinetics of haematological, virological and immunological parameters were investigated simultaneously. All 11 inoculated animals became infected. Monkeys inoculated with the nef-deleted SIV clone exhibited a significantly reduced plasma virus load and a less pronounced accumulation of lymphocytes in the lung compared to monkeys infected with the pathogenic SIVmac251 isolate. Compared to pre-infection levels, we observed an increase in the levels of RANTES, MIP1-alpha and MIP1-beta production in the two groups of monkeys, by the time of peak viraemia. Strikingly, a greater enhancement of RANTES and MIP-1alpha production was detected in monkeys infected with the attenuated virus. Given the potential influence of beta-chemokines on the immune response and virus replication, such results suggest that RANTES, MIP1-alpha and MIP1-beta could contribute to the singular features of the immune response elicited during infection of macaques with an attenuated SIV.

RANTES, IFN-gamma, CCR1, and CCR5 mRNA expression in peripheral blood, lymph node, and bronchoalveolar lavage mononuclear cells during primary simian immunodeficiency virus infection of macaques.

Primary infection of macaques with pathogenic isolates of simian immunodeficiency virus (SIV) (as a model of HIV infection in humans) represents a unique opportunity to study early lentivirus/host interactions. We sought to determine whether there is a temporal relationship linking SIV replication and dissemination and the expression of the chemokine RANTES (regulated upon activation normal T cell expressed and secreted) and the SIV/HIV coreceptor CCR5 in different tissues during acute SIV infection of macaques. Four cynomolgus macaques were inoculated intravenously with a pathogenic primary isolate of SIVmac251. RT-PCR was used to monitor the expression of RANTES and CCR5 mRNA in fresh isolated mononuclear cells from blood, lymph node, and bronchoalveolar lavages. These expressions were compared to those of IFN-gamma as an indicator of the development of the immune response and to another receptor for RANTES, CCR1, which is not described as a coreceptor for SIV/HIV-1 entry. An enhancement of CCR1/CCR5 mRNA expression was noticed during primary SIVmac251 infection of macaques, mainly in tissue. In the three different compartments investigated, IFN-gamma and RANTES overexpression was noticed by the time of systemic viral replication containment. Our results put CCR5 and RANTES mRNA expression back in the context of inflammatory and immune responses to SIV primary infection.

Selective quasispecies transmission after systemic or mucosal exposure of macaques to simian immunodeficiency virus.

Sexual transmission is the major cause of the AIDS epidemic. For the development of new antiviral and vaccine strategies, we therefore need to understand the mechanisms by which lentiviruses cross the mucosal barrier and the subsequent pathogenic consequences. For this purpose, experimental approaches are greatly facilitated by the development of relevant animal models. In this study, macaques were inoculated intravenously, intrarectally, or intravaginally with a pathogenic cell-free isolate of SIVmac251. Patterns of virological and immunological events significantly differed between vaginally (transient viremia, late seroconversion) and intravenously or intrarectally inoculated monkeys (persistent viremia and early seroconversion). Two weeks after infection, analysis of the env gene nucleotide sequences of proviruses recovered from PBMCs demonstrated that most of the differences were observed in the V1 loop. Three viral variants were specifically associated with vaginal transmission, whereas no such selection was evidenced after intravenous or intrarectal transmissions. These results are in favor of specific mechanisms associated with vaginal transmission, implicating viral envelope structure.

Variation in virological parameters and antibody responses in macaques after atraumatic vaginal exposure to a pathogenic primary isolate of SIVmac251.

We developed an animal model for the male-to-female transmission of human immunodeficiency virus, consisting of an atraumatic vaginal application of simian immunodeficiency virus onto the intact vaginal mucosa of cynomolgus macaques. Different doses of a pathogenic isolate of SIVmac251, with or without seminal plasma, were infused into the vaginas of female macaques. Infection of macaques could be achieved after a single exposure to the virus. Two patterns of infection were underscored with no relation to the virus dose inoculated: in 50% of the monkeys, SIV was persistently recovered and a strong antibody response to SIV was evidenced in blood and vaginal secretions. In the other infected animals, SIV infection was only transiently evidenced and a weak systemic antibody response was detected. It appeared that the presence of seminal plasma may be implicated in this variability only when low doses of virus are inoculated. Sequence analysis of the env gene of SIV revealed that most of the persistently viraemic animals were infected with a viral variant different from that of transiently viraemic macaques.

Consequences of ddI-induced reduction of acute SIVmac251 virus load on cytokine profiles in cynomolgus macaques.

This study evaluates the consequences of antiretroviral treatment of the acute simian immunodeficiency virus (SIV) primary infection on virus load and cytokine responses. Four cynomolgus macaques were inoculated intravenously with a pathogenic primary isolate (SIVmac251). Animals were pretreated with 10.8 mg/kg/day of dideoxyinosine (ddI) from 4 days before inoculation, and treatment was continued for 28 days. Proinflammatory (IL6, IL1 beta and TNF alpha) and antiinflammatory (IL10) cytokine and lymphokine (IL2, IL4 and IFN gamma) polymerase chain reaction (PCR) ratios were monitored in unmanipulated peripheral blood mononuclear cells (PBMCs) during acute infection by using a semiquantitative reverse transcription (RT)-PCR method. PBMC-associated virus loads were dramatically reduced compared to those of placebo-treated macaques. Nevertheless, a transient rise in IL6, IL1 beta, TNF alpha and IL10 mRNA expression was observed in PBMCs. IL2, IL4 and IFN gamma mRNAs were either undetectable or weakly detectable throughout the study, with no major changes. Despite a dramatic reduction in the acute viral loads in ddI-treated monkeys, early cytokine mRNA profiles were comparable to those of untreated SIVmac251-infected monkeys. Contrary to what was previously evidenced during primary infection with an attenuated SIV clone, no increase in IL2 and IL4 mRNA was detected in PBMCs of the ddI-treated monkeys, although these monkeys exhibited virus loads similar to those evidenced in macaques infected by attenuated SIV. These data indicate that differential lymphokine expression patterns found in pathogenic and Nef-truncated SIV-infected monkeys may not be strictly dependent on virus load levels.

Lack of interleukin 10 expression in monocyte-derived macrophages in response to in vitro infection by HIV type 1 isolates.

Interleukin 10 is a pleiotropic cytokine that is overexpressed in HIV-infected patients. Here, we investigated IL-10 expression in primary cultures of monocyte-derived macrophages (MDMs) in response to in vitro infection by HIV-1/Ba-L or two macrophage-tropic HIV-1 primary isolates. Whatever the multiplicity of infection used, and in spite of high replication levels and an increase in HIV-infected cell frequency, neither significant IL-10 secretion nor IL-10 mRNA overexpression was induced in HIV-1-infected MDMs. Moreover, identical results were obtained with HIV-1-infected 1-day monocytes. These results show that MDM infection by HIV is not sufficient by itself for inducing IL-10 synthesis.

Chemoattractant factors (IP-10, MIP-1alpha, IL-16) mRNA expression in mononuclear cells from different tissues during acute SIVmac251 infection of macaques.

We have used semiquantitative RT-PCR to monitor the expression of mRNA encoding chemoattractant factors IP-10, MIP-1alpha, and IL-16 in freshly isolated peripheral blood mononuclear cells (PBMCs), lymph node mononuclear cells (LNMCs), and mononuclear cells obtained after bronchoalveolar lavages (BALMCs) of two cynomolgus macaques inoculated intravenously with a pathogenic isolate of simian immunodeficiency virus, SIVmac251. Concomitant with the peak of systemic viral replication (two weeks after experimental inoculation) and proinflammatory cytokine IL-6 mRNA expression, high levels of MIP-1alpha and IP-10 mRNA were produced in LNMCs and BALMCs. In BALMCs, in which we have reported a marked progressive overexpression of IFN-gamma mRNA coinciding with an increase in the CD8+ lymphocyte percentages, we noticed a progressive overexpression of IL-16 mRNA. Our results suggest the role of chemokines IP-10, MIP-1alpha, and IL-16 in the development of inflammatory and immune responses during the early stages of lentiviral infection.

Cytokine mRNA expression in mononuclear cells from different tissues during acute SIVmac251 infection of macaques.

We used semiquantitative RT-PCR to monitor the expression of mRNA encoding cytokines (IL-1 beta, IL-6, TNF-alpha, and IL-10) and IFN-gamma in fresh isolated peripheral blood mononuclear cells (PBMCs), lymph node mononuclear cells (LNMCs), and mononuclear cells obtained after bronchoalveolar lavages (BALMCs), of four cynomolgus macaques inoculated intravenously with a pathogenic isolate of simian immunodeficiency virus (SIVmac251). To investigate the effects of the viral load on the expression of the cytokines, two monkeys received 30 mg kg-1 day-1 of didanosine (ddI). The two nontreated monkeys became infected and seroconverted, whereas the ddI-treated monkeys were completely protected as demonstrated by all criteria of diagnosis of SIV infection. Concomitant with the peak of viral replication (2 weeks after the experimental inoculation), high levels of IL-6 mRNA were produced in PBMCs, LNMCs, and BALMCs of the two placebotreated infected monkeys. Overexpression of TNF-alpha and IL-10 mRNAs was sometimes observed in LNMCs and BALMCs. A progressive overexpression of IFN-gamma mRNA, starting 2 weeks after experimental inoculation, was observed in BALMCs from infected animals. Concurrently, a marked increase in the CD8+ lymphocyte percentage in the BAL fluids was detected by FACS analysis. Thus, our results emphasize the importance of a comparative study of the expression of cytokines in different tissues. They suggest the interactions of monocyte/macrophage monokine production with viral replication, as well as the role of IFN-gamma in the development of lung cellular immunity to SIV infection.

Superinfection of HIV-2-preinfected macaques after rectal exposure to a primary isolate of SIVmac251.

To test the protection afforded by a weakly pathogenic HIV-2 isolate against the superinfection or development of SIV-induced disease, we intrarectally challenged six HIV-2-preinfected rhesus monkeys with a pathogenic isolate of SIVmac251. At the time of SIV challenge, none of these HIV-2-infected animals was positive for virus isolation, p27-Gag antigenemia, or HIV-2 provirus detection in PBMCs or peripheral lymph nodes. However, all monkeys exhibited anti-HIV-2 antibody titers ranging from 10(2) to 10(3). Neutralizing antibodies against the challenge SIV strain were also detected in two animals. After rectal exposure to SIVmac251, five of the six HIV-2-preinfected macaques were superinfected. SIVmac251 DNA sequences were detected repeatedly in the PBMCs of the five superinfected animals and the two controls, whereas no HIV-2 provirus was detected for 14 months postchallenge. The one monkey that resisted superinfection was negative for all SIV infection criteria. This monkey exhibited the highest anti-SIV ELISA and cross-neutralizing antibody titers on the day of SIV challenge. Preinfection with a weakly pathogenic HIV-2 ROD isolate protected one of six macaques from infection with the closely related pathogenic SIVmac251 isolate, but no protection from the progression of disease was evidenced in the other five.

Comparative interleukin (IL-2)/interferon IFN-gamma and IL-4/IL-10 responses during acute infection of macaques inoculated with attenuated nef-truncated or pathogenic SICmac251 virus.

Comparison of immune responses to infection by a pathogenic or a nonpathogenic immunodeficiency virus in macaques may provide insights into pathogenetic events leading to simian AIDS. This work is aimed at exploring cytokine expression during infection by simian immunodeficiency virus (SIV). We used semiquantitative reverse transcription-PCR to monitor interleukin (IL)-2/interferon (IFN)-gamma (Th1-like), and IL-4/IL-10 (Th2-like) expression in unmanipulated peripheral blood mononuclear cells (PBMCs), during the acute phase of infection of eight cynomolgus macaques (Macaca fascicularis) with a pathogenic primary isolate of SIVmac251 (full-length nef), and of four other cynomolgus macaques by an attenuated molecular clone of SIVmac251 (nef-truncated). All the monkeys became infected, as clearly shown by the presence of infected PBMCs and by seroconversion. Nevertheless, PBMC-associated virus loads and p27 antigenemia in monkeys infected by the attenuated virus clone remained lower than those observed in animals infected with the pathogenic SIVmac251 isolate. A rise of IL-10 mRNA expression occurred in both groups of monkeys coincident with the peak of viral replication. In monkeys infected with the pathogenic SIVmac251, IL-2, IL-4, and IFN-gamma mRNAs were either weakly detectable or undetectable. On the contrary, animals infected by the attenuated virus exhibited an overexpression of these cytokine mRNAs during the first weeks after inoculation. The lack of expression of these cytokines in monkeys infected with the pathogenic primary isolate may reflect early immunodeficiency.

Interleukin 1 beta, interleukin 6, tumor necrosis factor alpha, and interleukin 10 responses in peripheral blood mononuclear cells of cynomolgus macaques during acute infection with SIVmac251.

HIV infection ultimately leads to AIDS, despite the immune responses elicited soon after infection. In addition to the observed changes in lymphoid cell subsets, alteration of the cytokine network most likely accompanies and/or contributes to the lack of protective immune responses. In an attempt to delineate the early events in the immune response to lentivirus infection, we have sequentially monitored levels of proinflammatory (IL-1 beta, IL-6, and TNF-alpha) and antiinflammatory (IL-10) cytokine mRNAs in PBMCs of cynomolgus macaques during primary SIVmac infection. Eight monkeys were infected i.v. with 4 AID50 of cell-free SIVmac251. All monkeys seroconverted between days 16 and 21 postinfection (p.i.), and had detectable peripheral viremia. The viral load peaked between days 12 and 16 p.i., and fell sharply thereafter. A marked increase in the expression of IL-6 mRNA was observed in all macaques during the first weeks following infection. An increase in the levels of expression of IL-1 beta, TNF-alpha, and IL-10 mRNA was also determined in six, six, and five of the eight monkeys, respectively. While IL-6, TNF-alpha, and IL-10 increased transiently, increased levels of IL-1 beta mRNA expression were sustained over 44 days in most monkeys.