Bruno Hurtrel

Neuropathology of early HIV-1 infection

Early HIV‐1 invasion of the central nervous system has been demonstrated by many cerebrospinal fluid studies; however, most HIV‐1 carriers remain neurologically unimpaired during the so called “asymptomatic” period lasting from seroconversion to symptomatic AIDS. Therefore, neuropathological studies in the early pre‐AIDS stages are very few, and the natural history of central nervous system changes in HIV‐1 infection remains poorly understood. Examination of brains of asymptomatic HIV‐1 positive individuals who died accidentally and of rare cases with acute fatal encephalopathy revealing HIV infection, and comparison with experimental simian immunodeficiency virus and feline immunodeficiency virus infections suggest that, invasion of the CNS by HIV‐1 occurs at the time of primary infection and induces an immunological process in the central nervous system. This includes an inflammatory T‐cell reaction with vasculitis and leptomeningitis, and immune activation of brain parenchyma with increased number of microglial cells, upregulation of major histocompatibility complex class II antigens and local production of cytokines. Myelin pallor and gliosis of the white matter are usually found and are likely to be the consequence of opening of the blood brain barrier due to vasculitis; direct damage to oligodendrocytes by cytokines may also interfere. These white matter changes may explain, at least partly, the early cerebral atrophy observed, by magnetic resonance imaging, in asymptomatic HIV‐1 carriers. In contrast, cortical damage seems to be a late event in the course of HIV‐1 infection. There is no significant neuronal loss at the early stages of the disease, no accompanying increase in glial fibrillary acid protein staining in the cortex, and only exceptional neuronal apoptosis. Although HIV‐1 proviral DNA may be demonstrated in a number of brains, viral replication remains very low during the asymptomatic stage of HIV‐1 infection. This makes it likely that, although opening of the blood brain barrier may facilitate viral entry into the brain, specific immune responses including both neutralising antibodies and cytotoxic T‐lymphocytes, continuously inhibits viral replication at that stage.

Lack of an Immune Response against the Tetracycline-Dependent Transactivator Correlates with Long-Term Doxycycline-Regulated Transgene Expression in Nonhuman Primates after Intramuscular Injection of Recombinant Adeno-Associated Virus

ABSTRACT We previously documented persistent regulation of erythropoietin (Epo) secretion in mice after a single intramuscular (i.m.) injection of a recombinant adeno-associated virus (rAAV) vector harboring both the tetracycline-dependent transactivator (rtTA) and the Epo cDNA (D. Bohl, A. Salvetti, P. Moullier, and J. M. Heard, Blood 92:1512-1517, 1998). Using the same vector harboring the cynomolgus macaque Epo cDNA instead, the present study evaluated the ability of the tetracycline-regulatable (tetR) system to establish long-term transgene regulation in nonhuman primates. The vector was administered i.m., after which 5-day induction pulses were performed monthly for up to 13 months by using doxycycline (DOX), a tetracycline analog. We show that initial inductions were successful in all individuals and that there was a tight regulation and a rapid deinduction pattern upon DOX withdrawal. For one macaque, regulation of Epo secretion was maintained during the entire experimental period; for the five remaining macaques, secreted Epo became indistinguishable from endogenous Epo upon repeated DOX inductions. We investigated the mechanism involved and showed that, except in the animal in which secretion persisted, delayed humoral and cellular immune responses were directed against the rtTA transactivator protein associated with the reduction of vector DNA in transduced muscles. This study provides some evidence that, when the immune system is not mobilized against the rtTA transactivator, the tetR-regulatable system is able to support long-term transgene regulation in the context of an rAAV in nonhuman primates. In addition, our results suggest potential improvements for vector design.

A Single Injection of Recombinant Measles Virus Vaccines Expressing Human Immunodeficiency Virus (HIV) Type 1 Clade B Envelope Glycoproteins Induces Neutralizing Antibodies and Cellular Immune Responses to HIV

ABSTRACT The anchored and secreted forms of the human immunodeficiency virus type 1 (HIV-1) 89.6 envelope glycoprotein, either complete or after deletion of the V3 loop, were expressed in a cloned attenuated measles virus (MV) vector. The recombinant viruses grew as efficiently as the parental virus and expressed high levels of the HIV protein. Expression was stable during serial passages. The immunogenicity of these recombinant vectors was tested in mice susceptible to MV and in macaques. High titers of antibodies to both MV and HIV-Env were obtained after a single injection in susceptible mice. These antibodies neutralized homologous SHIV89.6p virus, as well as several heterologous HIV-1 primary isolates. A gp160 mutant in which the V3 loop was deleted induced antibodies that neutralized heterologous viruses more efficiently than antibodies induced by the native envelope protein. A high level of CD8+ and CD4+ cells specific for HIV gp120 was also detected in MV-susceptible mice. Furthermore, recombinant MV was able to raise immune responses against HIV in mice and macaques with a preexisting anti-MV immunity. Therefore, recombinant MV vaccines inducing anti-HIV neutralizing antibodies and specific T lymphocytes responses deserve to be tested as a candidate AIDS vaccine.

A molecularly cloned Schwarz strain of measles virus vaccine induces strong immune responses in macaques and transgenic mice

ABSTRACT Live attenuated RNA viruses make highly efficient vaccines. Among them, measles virus (MV) vaccine has been given to a very large number of children and has been shown to be highly efficacious and safe. Therefore, this vaccine might be a very promising vector to immunize children against both measles and other infectious agents, such as human immunodeficiency virus. A vector was previously derived from the Edmonston B strain of MV, a vaccine strain abandoned 25 years ago. Sequence analysis revealed that the genome of this vector diverges from Edmonston B by 10 amino acid substitutions not related to any Edmonston subgroup. Here we describe an infectious cDNA for the Schwarz/Moraten strain, a widely used MV vaccine. This cDNA was constructed from a batch of commercial vaccine. The extremities of the cDNA were engineered in order to maximize virus yield during rescue. A previously described helper cell-based rescue system was adapted by cocultivating transfected cells on primary chicken embryo fibroblasts, the cells used to produce the Schwarz/Moraten vaccine. After two passages the sequence of the rescued virus was identical to that of the cDNA and of the published Schwarz/Moraten sequence. Two additional transcription units were introduced in the cDNA for cloning foreign genetic material. The immunogenicity of rescued virus was studied in macaques and in mice transgenic for the CD46 MV receptor. Antibody titers and T-cell responses (ELISpot) in animals inoculated with low doses of rescued virus were identical to those obtained with commercial Schwarz MV vaccine. In contrast, the immunogenicity of the previously described Edmonston B strain-derived MV clone was much lower. This new molecular clone will allow for the production of MV vaccine without having to rely on seed stocks. The additional transcription units allow expressing heterologous antigens, thereby providing polyvalent vaccines based on an approved, safe, and efficient MV vaccine strain that is used worldwide.

IL-7 Induces Immunological Improvement in SIV-Infected Rhesus Macaques under Antiviral Therapy

Despite efficient antiretroviral therapy (ART), CD4+ T cell counts often remain low in HIV-1-infected patients. This has led to IL-7, a crucial cytokine involved in both thymopoiesis and peripheral T cell homeostasis, being suggested as an additional therapeutic strategy. We investigated whether recombinant simian IL-7-treatment enhanced the T cell renewal initiated by ART in rhesus macaques chronically infected with SIVmac251. Six macaques in the early chronic phase of SIV infection received antiretroviral treatment. Four macaques also received a 3-wk course of IL-7 injections. Viral load was unaffected by IL-7 treatment. IL-7 treatment increased the number of circulating CD4+ and CD8+ memory T cells expressing activation (HLA-DR+, CD25+) and proliferation (Ki-67+) markers. It also increased naive (CD45RAbrightCD62L+) T cell counts by peripheral proliferation and enhanced de novo thymic production. The studied parameters returned to pretreatment values by day 29 after the initiation of treatment, concomitantly to the appearance of anti-IL-7 neutralizing Abs, supporting the need for a nonimmunogenic molecule for human treatment. Thus, IL-7, which increases T cell memory and de novo renewal of naive T cells may have additional benefits in HIV-infected patients receiving ART.

Comparison of early and late feline immunodeficiency virus encephalopathies

DesignThe study of the early and late stages of encephalopathy following infection by the feline immunodeficiency virus (FIV) was carried out with laboratory and naturally infected cats. InterventionsAnimals infected experimentally were injected with three different isolates of the virus, administered either intracerebrally or intravenously, and sacrificed at 7 days, 1 and 6 months (intracerebral injection), and 2, 6 and 12 months (intravenous injection) post-inoculation, respectively. ConclusionsGeneral features of encephalopathy were found to be identical, regardless of the method of inoculation or the viral strain used. Moderate gliosis and glial nodules, sometimes associated with perivascular infiltrates and white matter pallor, were observed at 1 month (intracerebral injection) and 2 months (intravenous injection), and remained unchanged until 12 months post-inoculation. The fact that these initial stages are identical for intravenously and intracerebrally inoculated cats suggests that the virus enters the brain very quickly in intravenously infected animals. Encephalopathy in cats naturally infected with FIV only consisted of gliosis, glial nodules, white matter pallor, meningeal perivascular calcification and meningitis. These lesions were more frequent and more severe in the group coinfected with feline leukaemia virus and feline infectious peritonitis virus. Although multinucleated cells were rare, the strong similarities between HIV and simian immunodeficiency virus encephalopathies at comparable stages support the view that FIV infection may represent an interesting model for a physiopathological approach of HIV infection of the central nervous system.

IL-7 stimulates T cell renewal without increasing viral replication in simian immunodeficiency virus-infected macaques

The main failure of antiretroviral therapy is the lack of restoration of HIV-specific CD4+ T cells. IL-7, which has been shown to be a crucial cytokine for thymopoiesis, has been envisaged as an additive therapeutic strategy. However, in vitro studies suggest that IL-7 might sustain HIV replication in thymocytes and T lymphocytes. Therefore, in the present study, we evaluated the effect of IL-7 on both T cell renewal and viral load in SIVmac-infected young macaques in the absence of antiretroviral therapy. This evaluation was conducted during the asymptomatic phase in view of a potential treatment of HIV patients. We show that IL-7 induces both a central renewal and a peripheral expansion of T lymphocytes associated with cell activation. No alarming modulation of the other hemopoietic cells was observed. No increase in the viral load was shown in blood or lymph nodes. These data strengthen the rationale for the use of IL-7 as an efficient immunotherapy in AIDS.

Early Divergence in Lymphoid Tissue Apoptosis between Pathogenic and Nonpathogenic Simian Immunodeficiency Virus Infections of Nonhuman Primates

ABSTRACT 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.

Nonpathogenesis of Simian Immunodeficiency Virus Infection Is Associated with Reduced Inflammation and Recruitment of Plasmacytoid Dendritic Cells to Lymph Nodes, Not to Lack of an Interferon Type I Response, during the Acute Phase

ABSTRACT Divergent Toll-like receptor 7 (TLR7) and TLR9 signaling has been proposed to distinguish pathogenic from nonpathogenic simian immunodeficiency virus infection in primate models. We demonstrate here that increased expression of type I interferon in pathogenic rhesus macaques compared to nonpathogenic African green monkeys was associated with the recruitment of plasmacytoid dendritic cells in the lymph nodes and the presence of an inflammatory environment early after infection, instead of a difference in the TLR7/9 response.

Viral load in tissues during the early and chronic phase of non‐pathogenic SIVagm infection

Abstract:  African green monkeys (AGMs) persistently infected with SIVagm do not develop AIDS, although their plasma viremia levels can reach those reported for pathogenic HIV‐1 and SIVmac infections. In contrast, the viral burden in lymph nodes in SIVagm‐infected AGMs is generally lower in comparison with HIV/SIVmac pathogenic infections, at least during the chronic phase of SIVagm infection. We searched for the primary targets of viral replication, which might account for the high viremias in SIVagm‐infected AGMs. We evaluated for the first time during primary infection SIVagm dissemination in various lymphoid and non‐lymphoid tissues. Sixteen distinct organs at a time point corresponding to maximal virus production were analyzed for viral RNA and DNA load. At days 8 and 9 p.i., viral RNA could be detected in a wide range of tissues, such as jejunum, spleen, mesenteric lymph nodes, thymus and lung. Quantification of viral DNA and RNA as well as of productively infected cells revealed that viral replication during this early phase takes place mainly in secondary lymphoid organs and in the gut (5 × 104–5 × 108 RNA copies/106 cells). By 4 years p.i., RNA copy numbers were below detection level in thymus and lung. Secondary lymphoid organs displayed 6 × 102–2 × 106 RNA copies/106 cells, while some tissue fragments of ileum and jejunum still showed high viral loads (up to 109 copies/106 cells). Altogether, these results indicate a rapid dissemination of SIVagm into lymphoid tissues, including the small intestine. The latter, despite showing marked regional variations, most likely contributes significantly to the high levels of viremia observed during SIVagm infection.