Patricia Brochard

Chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that induces in humans a disease characterized by fever, rash, and pain in muscles and joints. The recent emergence or reemergence of CHIKV in the Indian Ocean Islands and India has stressed the need to better understand the pathogenesis of this disease. Previous CHIKV disease models have used young or immunodeficient mice, but these do not recapitulate human disease patterns and are unsuitable for testing immune-based therapies. Herein, we describe what we believe to be a new model for CHIKV infection in adult, immunocompetent cynomolgus macaques. CHIKV infection in these animals recapitulated the viral, clinical, and pathological features observed in human disease. In the macaques, long-term CHIKV infection was observed in joints, muscles, lymphoid organs, and liver, which could explain the long-lasting CHIKV disease symptoms observed in humans. In addition, the study identified macrophages as the main cellular reservoirs during the late stages of CHIKV infection in vivo. This model of CHIKV physiopathology should allow the development of new therapeutic and/or prophylactic strategies.

Primary infection with simian immunodeficiency virus: plasmacytoid dendritic cell homing to lymph nodes, type I interferon, and immune suppression.

Plasmacytoid dendritic cells (pDCs) are antigen-presenting cells that develop into type-I interferon (IFN-I)-producing cells in response to pathogens. Their role in human immunodeficiency virus (HIV) pathogenesis needs to be understood. We analyzed their dynamics in relation to innate and adaptive immunity very early during the acute phase of simian immunodeficiency virus (SIV) infection in 18 macaques. pDC counts decreased in blood and increased in peripheral lymph nodes, consistent with early recruitment in secondary lymphoid tissues. These changes correlated with the kinetic and intensity of viremia and were associated with a peak of plasma IFN-I. IFN-I and viremia were positively correlated with functional activity of the immune suppression associated enzyme indoleamine-2,3-dioxygenase (IDO) and FoxP3(+)CD8(+) T cells, which both negatively correlated with SIV-specific T-cell proliferation and CD4(+) T-cell activation. These data suggest that pDCs and IFN-I play a key role in shaping innate and adaptive immunity toward suppressive pathways during the acute phase of SIV/HIV primary infection.

Infection of Macaques after Vaginal Exposure to Cell-Associated Simian Immunodeficiency Virus

BACKGROUND The contribution of infected semen cells to sexual transmission of human immunodeficiency virus (HIV) is still debated. We addressed this issue in the model of experimental infection of macaques with simian immunodeficiency virus (SIV). METHODS Frozen stocks of cells obtained from the spleen of macaques at the peak of SIVmac251 viremia were prepared. After being thawed and washed, cells were deposited at different concentrations in the vaginas of adult macaques treated with medroxyprogesterone acetate (Depo-Provera). To unravel mechanisms of infection, stock cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) were inoculated intravaginally. Follow-up testing of samples from the mucosa and different lymphoid tissues obtained 21 and 45 h later was performed by flow cytometry, immunohistochemical analysis, and in situ hybridization. RESULTS Systemic and persistent infection was achieved after vaginal exposure of macaques to SIV-infected cells. The dose needed to infect 50% of females was 6.69 x 10(5)+/-2.08 x 10(5) viral DNA copies. At days 1 and 2 after exposure to cell-associated SIV labeled with CFSE, SIV-positive cells were detected in proximal and distal lymphoid tissues. CONCLUSIONS Infection with SIV after exposure of vaginal and cervical mucosa to cell-associated virus represents a new mechanism of sexual transmission of HIV and SIV that may have significant impacts in the development of preventive approaches like microbicides.

FOXP3 CD25 CD8 T-CELL INDUCTION DURING PRIMARY SIMIAN IMMUNODEFICIENCY VIRUS INFECTION IN CYNOMOLGUS MACAQUES CORRELATES WITH LOW CD4 T-CELL ACTIVATION AND HIGH VIRAL LOAD

ABSTRACT The early immune response fails to prevent the establishment of chronic human immunodeficiency virus (HIV) infection but may influence viremia during primary infection, thereby possibly affecting long-term disease progression. CD25+ FoxP3+ regulatory T cells may contribute to HIV/simian immunodeficiency virus (SIV) pathogenesis by suppressing efficient antiviral responses during primary infection, favoring high levels of viral replication and the establishment of chronic infection. In contrast, they may decrease immune activation during chronic infection. CD4+ regulatory T cells have been studied in the most detail, but CD8+ CD25+ FoxP3+ T cells also have regulatory properties. We monitored the dynamics of CD25+ FoxP3+ T cells during primary and chronic SIVmac251 infection in cynomolgus macaques. The number of peripheral CD4+ CD25+ FoxP3+ T cells paralleled that of memory CD4+ T cells, with a rapid decline during primary infection followed by a rebound to levels just below baseline and gradual depletion during the course of infection. No change in the proportion of CD25+ FoxP3+ T cells was observed in peripheral lymph nodes. A small number of CD4+ CD25+ FoxP3+ T cells at set point was associated with a high plasma viral load. In contrast, peripheral CD8+ CD25+ FoxP3+ T cells were induced a few days after peak plasma viral load during primary infection. The number of these cells was positively correlated with viral load and negatively correlated with CD4+ T-cell activation, SIV antigen-specific proliferative responses during primary infection, and plasma viral load at set point, with large numbers of CD8+ CD25+ FoxP3+ T cells being indicative of a poor prognosis.

Effect of SIVmac infection on plasmacytoid and CD1c+ myeloid dendritic cells in cynomolgus macaques

Dendritic cells (DCs) are known to be essential for the induction and regulation of immune responses. Non‐human primates are essential in biomedical research and contribute to our understanding of the involvement of DCs in human infectious diseases. However, no direct single‐platform method for quantifying DC precursors has yet been optimized in macaques to give accurate absolute blood counts of these rare‐event cell populations in the blood. We adapted a rapid whole‐blood assay for the absolute quantification of DCs in cynomolgous macaques by four‐colour flow cytometry, using a single‐platform assay compatible with human blood. Cynomolgus macaque plasmacytoid DCs (pDCs) and CD1c+ myeloid DCs (CD1c+ mDCs) were quantified in the blood of 34 healthy macaques and the results obtained were compared with those for blood samples from 11 healthy humans. In addition, circulating absolute numbers of pDCs were quantified in cynomolgus macaques chronically infected with SIVmac. During infection, pDC counts decreased whereas circulating CD1c+ mDC counts increased. Information regarding absolute pDC and mDC counts in non‐human primates may improve our understanding of the role of these cells in SIV/HIV infection and in other infectious diseases.

Dynamics of T-Cell Responses and Memory T Cells during Primary Simian Immunodeficiency Virus Infection in Cynomolgus Macaques

ABSTRACT Cellular immune responses make an important contribution to both the control of human immunodeficiency virus (HIV) replication and disease progression. We used a pathogenic model of SIVmac251 infection of cynomolgus macaques to longitudinally evaluate cellular immune responses in association with various rates of disease progression. We found an inverse relationship between plasma viral load and the simian immunodeficiency virus (SIV)-specific T cells responses in peripheral blood and lymph nodes. SIV-specific T-cell responses in peripheral blood were transient during primary infection, with the highest responses detected around 3 months after infection. There was also a transient increase of central memory CD8+ T cells in peripheral blood during primary infection, and effector memory T-cell counts in peripheral lymph nodes were increased. This study emphasizes the importance of the early virus-specific immune responses in the outcome of HIV/SIV disease and provides details about the changes of virus-specific immune responses over time.

Dynamics of viral replication in blood and lymphoid tissues during SIVmac251 infection of macaques

BackgroundExtensive studies of primary infection are crucial to our understanding of the course of HIV disease. In SIV-infected macaques, a model closely mimicking HIV pathogenesis, we used a combination of three markers -- viral RNA, 2LTR circles and viral DNA -- to evaluate viral replication and dissemination simultaneously in blood, secondary lymphoid tissues, and the gut during primary and chronic infections. Subsequent viral compartmentalization in the main target cells of the virus in peripheral blood during the chronic phase of infection was evaluated by cell sorting and viral quantification with the three markers studied.ResultsThe evolutions of viral RNA, 2LTR circles and DNA levels were correlated in a given tissue during primary and early chronic infection. The decrease in plasma viral load principally reflects a large decrease in viral replication in gut-associated lymphoid tissue (GALT), with viral RNA and DNA levels remaining stable in the spleen and peripheral lymph nodes. Later, during chronic infection, a progressive depletion of central memory CD4+ T cells from the peripheral blood was observed, accompanied by high levels of viral replication in the cells of this subtype. The virus was also found to replicate at this point in the infection in naive CD4+ T cells. Viral RNA was frequently detected in monocytes, but no SIV replication appeared to occur in these cells, as no viral DNA or 2LTR circles were detected.ConclusionWe demonstrated the persistence of viral replication and dissemination, mostly in secondary lymphoid tissues, during primary and early chronic infection. During chronic infection, the central memory CD4+ T cells were the major site of viral replication in peripheral blood, but viral replication also occurred in naive CD4+ T cells. The role of monocytes seemed to be limited to carrying the virus as a cargo because there was an observed lack of replication in these cells. These data may have important implications for the targeting of HIV treatment to these diverse compartments.

CCR5 or CXCR4 use influences the relationship between CD4 cell depletion, NKp44L expression and NK cytotoxicity in SHIV-infected macaques.

Objective:HIV-1 infection is characterized by a progressive decline of CD4 cell count, the underlying mechanisms of which are still debated. We recently found that during HIV-1 infection, CD4 T cells overexpress a ligand of the NK activating receptor NKp44 (NKp44L) and are sensitized to NK cytolytic activity. The expression of NKp44L is triggered by a highly conserved motif of gp41 (3S) and is inhibited by anti-3S antibodies. Design:To assess whether viral tropism can affect NKp44L expression, NK cytotoxicity, and anti-3S antibodies production, 10 macaques were infected either with the CCR5 tropic SHIV162P3 or with a CXCR4/CCR5 dual-tropic SHIV89.6P. Results:In SHIV162P3-infected macaques, expression of NKp44L was inversely correlated with anti-3S antibodies, in relation to CD4 depletion and NK cytotoxicity. By contrast, no such correlation was found in macaques infected with SHIV89.6Pwhich, induced a rapid decline of CD4 T cells. Conclusions:These results highlight the key role played by NK cells in CD4 cell count decline with respect to coreceptor usage, and provided the setting to investigate new strategies for preventive and/or therapeutic immunization to stimulate anti-3S antibodies.

Prevention of vaginal simian immunodeficiency virus transmission in macaques by postexposure prophylaxis with zidovudine, lamivudine and indinavir.

Objective:To evaluate the efficacy of postexposure prophylaxis with a combination of zidovudine (ZDV), lamivudine (3TC) and indinavir (IDV), after vaginal exposure to HIV. Design:Experimental intravaginal exposure of female cynomolgus macaques to SIVmac251. Methods:ZDV/3TC/IDV treatment was initiated 4 h after exposure and continued for 28 days. Groups of six animals received a placebo or a combination of oral ZDV (4.5 mg/kg), 3TC (2.5 mg/kg) and IDV (20 mg/kg) twice daily or subcutaneous ZDV (4.5 mg/kg) and 3TC (2.5 mg/kg) twice daily, and a higher dose of IDV (60 mg/kg) administered orally twice daily. Results:In the placebo group, all animals were infected. Antiretroviral association protected one of the six animals if all drugs were administered orally and four of the six animals if ZDV and 3TC were administered subcutaneously and IDV was given orally at triple dose. In infected animals, viremia was significantly delayed and lowered in treated animals than in animals given placebo, and high CD4 cell counts were maintained in the treated animals, at least in the medium term. Antiretroviral dosages made in macaques receiving the same treatments showed that protection efficacy could be linked to antiretroviral plasmatic concentration. Conclusion:This study shows, for the first time in macaques, that the postexposure prophylaxis recommended for humans may be effective after vaginal exposure. Improvements in pharmacokinetic parameters significantly increased treatment efficiency.

Pre-clinical development of a combination microbicide vaginal ring containing dapivirine and darunavir

OBJECTIVES Combination microbicide vaginal rings may be more effective than single microbicide rings at reducing/preventing sexual transmission of HIV. Here, we report the pre-clinical development and macaque pharmacokinetics of matrix-type silicone elastomer vaginal rings containing dapivirine and darunavir. METHODS Macaque rings containing 25 mg dapivirine, 100 mg dapivirine, 300 mg darunavir or 100 mg dapivirine+300 mg darunavir were manufactured and characterized by differential scanning calorimetry. In vitro release was assessed into isopropanol/water and simulated vaginal fluid. Macaque vaginal fluid and blood serum concentrations for both antiretrovirals were measured during 28 day ring use. Tissue levels were measured on day 28. Ex vivo challenge studies were performed on vaginal fluid samples and IC50 values were calculated. RESULTS Darunavir caused a concentration-dependent reduction in the dapivirine melting temperature in both solid drug mixes and in the combination ring. In vitro release from rings was dependent on drug loading, the number of drugs present and the release medium. In macaques, serum concentrations of both microbicides were maintained between 10(1) and 10(2) pg/mL. Vaginal fluid levels ranged between 10(3) and 10(4) ng/g and between 10(4) and 10(5) ng/g for dapivirine and darunavir, respectively. Both dapivirine and darunavir showed very similar concentrations in each tissue type; the range of drug tissue concentrations followed the general rank order: vagina (1.8 × 10(3)-3.8 × 10(3) ng/g)  > cervix (9.4 × 10(1)-3.9 × 10(2) ng/g)  > uterus (0-108 ng/g)  > rectum (0-40 ng/g). Measured IC50 values were >2 ng/mL for both compounds. CONCLUSIONS Based on these results, and in light of recent clinical progress of the 25 mg dapivirine ring, a combination vaginal ring containing dapivirine and darunavir is a viable second-generation HIV microbicide candidate.