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Biomedical subjects

R Le Grand

Publications and source records attributed to R Le Grand.

At least 37 records · Page 2Linked to original sources
PubMed1998

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.

Amino Acid Sequence↗
PubMed1998

In vivo induction of specific cytotoxic T lymphocytes in mice and rhesus macaques immunized with DNA vector encoding an HIV epitope fused with hepatitis B surface antigen.

DNA immunization offers a novel means to induce humoral and cellular immunity in inbred or in outbred animals. Here we have tested the efficiency of genetic immunization with hepatitis B virus (HBV) envelope-based vectors. In naive primates, injection of a plasmid DNA encoding HBV envelope proteins induced an HBV-specific cytotoxic response and appearance of potentially protective anti-HBs antibodies. Moreover, intramuscular and intradermal injections of a DNA expression vector encoding an epitope of the human immunodeficiency virus envelope fused to the surface protein of the hepatitis B virus (HBsAg) induced strong humoral and cytotoxic responses to antigenic determinants of both viruses in mice and nonhuman primates alike. In addition, in protein-primed Rhesus monkeys B-cell memory was successfully boosted by DNA injection of hybrid vectors and animals subsequently developed a multispecific cellular response. This suggests that DNA-based immunization could be used to boost efficiently and broaden the immune response in individuals immunized with conventional vaccines, regardless of their genetic variability. These results also indicate that it might be possible to rationally design HBsAg-based expression vectors to induce multispecific immune responses for vaccination against hepatitis B and other pathogens.

Animals↗
PubMed1998

[Astrocytes and lentivirus infection in an experimental models of macaque infected with SIVmac251].

The present study demonstrates the susceptibility of astrocytes to infection with SIVmac251. Indeed, primary cultures of astrocytes derived from simian adult brains, can be infected in vitro with the SIVmac251. Results show that SIVmac251 establishes a persistent infection in primary astroglial cultures and that viral replication can be reactivated by TNF-alpha, GM-CSF, IFN-gamma. Viral proteins as Nef, Rev, Vpx and occasionally gp120/160 are evidenced by immunocytochemistry. In vivo SIVmac251 and/or HIV-2 infected astrocytes have been isolated from brains of macaques following ex vivo primary cultures. The whole of these results demonstrated that, in this model, SIV establishes a persistent state of infection of astrocytes, that viral replication can be reactivated by cytokines and moreover suggest strongly an in vivo infection of astrocytes in the brain of these infected macaques.

Animals↗
PubMed1997

Recombinant Mycobacterium bovis BCG producing the N-terminal half of SIVmac251 Env antigen induces neutralizing antibodies and cytotoxic T lymphocyte responses in mice and guinea pigs.

Recombinant Mycobacterium bovis bacillus Calmette-Guérin (rBCG) represents a good candidate for the development of vaccines against AIDS. Several HIV or SIV genes including nef, gag, and env have already been expressed by rBCG strains and shown to induce strong humoral and cellular immune responses in experimental animals. Because a broad immune response directed to multiple HIV/SIV antigens is highly desirable in order to develop effective vaccines, we have also investigated the immune response induced by an rBCG strain expressing a large N-terminal portion of the SIVmac251 Env gp110-encoding gene. The rBCG(SIVmac251Env) strain obtained was able to induce strong CTL responses in mice as well as humoral immune responses in mice and guinea pigs immunized by parenteral routes. The anti-gp110 IgGs produced were able to neutralize in vitro growth of virulent SIVmac251 field isolates. Moreover, guinea pigs immunized by the oral route produced significant levels of anti-gp110 IgAs in the feces, demonstrating that rBCG is able to induce local humoral immunity in the intestinal mucosa. These data provide further evidence of the utility of BCG as a candidate vaccine vector against AIDS.

Animals↗
PubMed1997

Obtention and characterization of primary astrocyte and microglial cultures from adult monkey brains.

Simple methods for obtention of primary cultures of isolated astrocytes and microglia from adult simian brain have been developed. Characterization of these two glial cell populations were performed by morphological observations and by immunocytochemistry. The astroglial cultures were obtained by an indirect method. After L-leucine methyl-ester treatment and trypsinizations, more than 99% of cells expressed glial fibrillary acidic protein (GFAP), whereas no macrophages or microglia could be detected. Likely, the 1% remaining cells were immature astrocytes or cells that lost their GFAP expression. Cultured simian astrocytes expressed vimentin, laminin, and fibronectin. We also found a constitutively low expression of major histocompatibility complex (MHC) class II by cultured astrocytes which was significantly enhanced by lipopolysaccharide (LPS), interferon gamma (IFN-gamma), or tumor necrosis factor alpha (TNF-alpha) treatments. Microglial cultures were obtained by a direct method of isolation using Percoll gradient separations and compared to simian monocyte-derived macrophages or alveolar macrophages. Microglial cells differed from macrophages by their proliferation upon granulocyte-macrophage colony stimulating factor (GM-CSF) treatment and by their typical morphology when observed by scanning electron microscopy. As macrophages, they expressed in vitro CD68, CD64, CD14, CD11b, MHC class II, and fibronectin. However, contrary to macrophages, simian cultured microglia expressed laminin. This observation suggests that microglia represent a new potential source of this extracellular matrix protein in the brain.

Animals↗
PubMed1997

Production and characterization of monoclonal antibodies to simian immunodeficiency virus envelope glycoproteins.

Twelve monoclonal antibodies (MAbs), TB1 to TB12, were produced against a soluble vaccinia recombinant envelope glycoprotein (gp140) from simian immunodeficiency virus SIVmac251. These MAbs recognized SIV gp140 with a relatively high affinity (K0.5 from 6.7 x 10(-8) to 4 x 10(-9) M). All the MAbs except TB9, TB11, and TB12 cross-reacted with HIV-2 envelope glycoproteins, but none of the 12 MAbs recognized those from HIV-1. Using a panel of 87 overlapping synthetic peptides containing 20 amino acid residues, with an overlap of 10 amino acids and spanning the entire primary sequence of gp140, 3 linear epitopes were identified. The first mapped with a neutralizing MAb, TB12, which recognized a linear sequence around amino acids 28-31 within the N-terminal end of the external envelope glycoprotein. The two other new nonneutralizing MAbs recognized linear epitopes around amino acid sequence 380-381 by MAbs TB1, TB2, and TB3, and at the transmembrane glycoprotein amino acids 581-600 by MAb TB6. Seven of the 12 MAbs, TB4, TB5, TB7-9, TB10, and TB11, failed to bind the linear synthetic peptides in ELISA. Moreover, among these seven MAbs only MAbs TB4, TB5, TB9, and TB10 failed to recognize SIV envelope glycoproteins in Western blot (WB) or ELISA after reduction of disulfide bridges by dithiothreitol (DTT), suggesting that they are directed against conformational or discontinuous epitopes. It is of interest to note that MAb TB10 can block the binding of gp140 to the CD4 receptor when the MAb is previously incubated with gp140. Consistent with this result, MAb TB10 cannot bind to gp140 that has been previously complexed with the CD4 receptor. All these results suggest that MAb TB10 recognizes a conformational or discontinuous epitope overlapping or close to the CD4-binding site. These properties are probably implicated in the neutralizing activity observed with this MAb.

Amino Acid Sequence↗
PubMed1997

Direct ex vivo simian immunodeficiency virus (SIV)-specific cytotoxic activity detected from small intestine intraepithelial lymphocytes of SIV-infected macaques at an advanced stage of infection.

Human immunodeficiency virus (HIV) induces a profound disorganization of the lymphoid tissues with marked abnormalities of the immune system at the terminal stage of infection. Since the digestive mucosal immune system is by far the largest lymphoid organ of the body, we attempted to evaluate its functional activity in advanced stages of simian immunodeficiency virus (SIV) infection in the SIV-macaque model of HIV infection. Two chronically intravenously SIV-infected macaques, including one at the AIDS stage, were studied. Intestinal intraepithelial lymphocytes (IEL) were isolated, analyzed, and compared to lymphocytes obtained from blood, spleen, and different lymph nodes: IEL were predominantly CD8+ T lymphocytes expressing the alphaE beta7 integrin and lacking the CD28 coactivatory molecule. A direct ex vivo SIV-specific cytotoxic activity was prominently found in the IEL of both macaques and was weaker or absent in the other sites. To our knowledge, this is the first report of SIV-specific cytotoxic activity from small intestine IEL in SIV-infected macaques. Considering the high similitude of the SIV-macaque model with the HIV infection in humans, these results may be highly important for the pathogenesis of HIV infection and more generally important for the characterization and function of digestive CD8+ IEL population.

Animals↗
PubMed1996

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.

Acute Disease↗
PubMed1996

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.

AIDS Vaccines↗
PubMed1996

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.

Acute Disease↗
PubMed1996

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.

Acute Disease↗
PubMed1996

Granulocyte macrophage colony stimulating factor stimulates in vitro proliferation of astrocytes derived from simian mature brains.

In the brain, granulocyte-macrophage colony stimulating factor (GM-CSF) may be released by infiltrated cells of the immune system including T and B lymphocytes and mononuclear phagocytes, but also by nervous system resident cells such as microglia and astrocytes. Astrocyte-secreted GM-CSF may play an important role in enhancing the local inflammatory response to central nervous system (CNS) injury and in recruting microglia and activated macrophages. In this study, we demonstrated that GM-CSF, as TNF alpha and IL 6, stimulates in vitro proliferation of simian astrocytes in primary cultures. Results were confirmed by blocking experiments performed with a specific neutralizing mAb directed against GM-CSF. Furthermore, we demonstrated that GM-CSF mediates its effect on these cells through the alpha subunit of the GM-CSF receptor which is constitutively expressed at the membrane of the cultured simian astrocytes as assessed by immunofluorescence. GM-CSF effects on astrocytes could be involved in astrocytosis, a hallmark of various neurological injuries and in inflammatory processes in an autocrine manner.

Animals↗
PubMed1996

Cytokine mRNA levels in unmanipulated (ex vivo) and in vitro stimulated monkey PBMCs using a semi-quantitative RT-PCR and high sensitivity fluorescence-based detection strategy.

To investigate the spectrum of cytokines expressed by peripheral blood mononuclear cells (PBMC) from cynomolgus macaques (Macaca fascicularis), we used a semi-quantitative RT-PCR to determine levels of mRNA coding for IL-1 beta, IL-2, IL-4, IL-6, IL-10, IFN-gamma, and TNF-alpha. The PCR products were labelled and quantified using a new fluorescent tag TOTO-1 (thiazole orange dimer) and an automated fluorescence-based electrophoretic instrument. Using this assay, the base line levels of cytokine mRNA expression in unmanipulated PBMCs (ex vivo) from 10 healthy monkeys were compared with the mRNA levels for the same cytokines in PBMC samples from two pre-immunized monkeys following culture with previously defined optimal concentrations of purified protein derivative (PPD), tetanus toxoid (TT) and the mitogen concanavalin A (con-A). While transcripts or IL-2, IL-4 and IFN-gamma were either low or not detected in unmanipulated PBMCs, varying levels of IL-1 beta, IL-5, IL-10, and TNF-alpha were readily detected in the same samples. With the exception of IL-10, the mitogen con-A induced the highest levels of cytokine expression, followed by levels induced by culture with TT. The levels of cytokine expression induced by PPD however, were not significantly elevated, despite the fact that the cells showed marked proliferative responses. This assay is a simple and convenient method for evaluating the levels of cytokine expression in small PBMC samples and will allow for the concurrent evaluation of immune profiles with functional immune analyses.

Animals↗
PubMed1995

Specificity and neutralizing capacity of three monoclonal antibodies produced against the envelope glycoprotein of simian immunodeficiency virus isolate 251.

Three mouse monoclonal antibodies (mAb) were produced against soluble recombinant vaccinia virus gp140 from SIV-mac251. Two mAbs (1B9 and 6C11) were mapped at the aa 411-430 sequence within the V4 domain, and the third mAb (3C8) recognizes a conformation-dependent epitope on the external envelope glycoprotein. This was shown by its loss of reactivity in Western blot and ELISA with dithiothreitol-reduced gp140. mAb 3C8, but not 1B9 and 6C11, cross-reacts well with gp140 and gp125 from HIV-2ROD, indicating that this discontinuous epitope includes conserved regions localized within the external envelope glycoprotein. Analysis of the neutralizing activities of the mAbs showed that only mAb 1B9 is able to inhibit both syncytium formation and SIVmac251 infection of human peripheral blood lymphocytes.

Animals↗
PubMed1995

Highly attenuated SIVmac142 is immunogenic but does not protect against SIVmac251 challenge.

We report here the use of the highly attenuated SIVmac142 clone, unable to establish permanent infection of rhesus macaques, in a vaccine trial. Four rhesus macaques were immunized over a long time period with HUT-78 cells infected with wild-type SIVmac142 or, in order to reinforce the safety use of the vaccine, a deleted mutant with similar in vitro infectivity. The first two injections were done with living cells and the remaining boosts with cells emulsified in muramyl dipeptide adjuvant. Three control macaques were injected with uninfected HUT-78 cells. Over 3 years, we have been unable except once to detect viral infections by three methods. However, antibodies directed against the viral Gag proteins and envelope glycoproteins were detected by immunoblots and/or in vitro neutralization assays. All macaques were challenged intravenously with a low dose (10 animal infectious doses) of a highly pathogenic biological clone of SIVmac251 grown on macaque PBMCs. All seven animals became persistently viremic following challenge. The cell-associated viral loads of the vaccinated monkeys were not reduced relative to those of unvaccinated controls during the first weeks postchallenge even if vaccinated monkeys did not present a transient CD4 decrease. Thus, our data reinforced the notion that the efficacy of live attenuated SIV requires the establishment of persistent infection.

Animals↗
PubMed1995

Functional consequences of macrophage infection by human immunodeficiency virus: bispecific antibody targeting of HIV-1-infected cells to Fc gamma RI expressing effector cells.

Human monocytes/macrophages play a major role in pathogenesis of human immunodeficiency virus (HIV) infection. These cells have been suspected of acting as a reservoir for the virus and are important in viral dissemination and persistence in infected individual. Furthermore, several biologic and clinical features indicate that monocytes/macrophages from HIV-1-seropositive patients have characteristics of an activation status, including the ability to secrete high levels of cytokines. Dysregulation of the cytokine network may influence the level and the consequences of viral replication in infected monocytes/macrophages. Therefore, the development of virus-specific agents that may interfere with viral replication could help to slow down the fatal course of HIV infection. In this article, we try to further quantify the early and late kinetic patterns of the cytokine network during HIV-1 macrophage infection and report the biologic effects of virus-specific bispecific antibody (MDX-240) in HIV-1 macrophage infection.

Antibodies, Bispecific↗