Yves Rivière

Molecular characterization of the genomic S RNA segment from lymphocytic choriomeningitis virus

We have used cDNA clones derived from the genomic S RNA segment of lymphocytic choriomeningitis virus (LCMV), Armstrong strain, as hybridization probes to monitor virus gene expression during acute infections. Our results with strand-specific probes confirm the ambisense character of the LCMV S RNA segment and document the presence of both genomic sense and genomic complementary sense RNA species over the time course of infection. We have used nucleotide sequence information to predict primary amino acid sequences for the major viral structural proteins, nucleoprotein (NP) and glycoprotein (GP-C). Antibodies raised against synthetic peptides derived from these predicted protein sequences have indicated that the gene order for the S segment is 3 NP----5 GP-C and provided direct demonstration that the GP-1 portion of the GP-C precursor is encoded nearest the 5 end of the S segment. Comparison of the predicted amino acid sequences for NP and GP-C between the Armstrong CA-1371 strain and the WE strain shows over 90% amino acid identity. This suggests that significant differences described for the pathogenic potential of the Arm and WE strains in C3H mice reside in one or a very few critical amino acid changes.

Kinetics of Lymphocyte Proliferation during Primary Immune Response in Macaques Infected with Pathogenic Simian Immunodeficiency Virus SIVmac251: Preliminary Report of the Effect of Early Antiviral Therapy

ABSTRACT The aim of this study was to evaluate the kinetics of lymphocyte proliferation during primary infection of macaques with pathogenic simian immunodeficiency virus (SIV) and to study the impact of short-term postexposure highly active antiretroviral therapy (HAART) prophylaxis. Twelve macaques were infected by intravenous route with SIVmac251 and given treatment for 28 days starting 4 h postexposure. Group 1 received a placebo, and groups 2 and 3 received combinations of zidovudine (AZT), lamivudine (3TC), and indinavir. Macaques in group 2 received AZT (4.5 mg/kg of body weight), 3TC (2.5 mg/kg), and indinavir (20 mg/kg) twice per day by the oral route whereas macaques in group 3 were given AZT (4.5 mg/kg) and 3TC (2.5 mg/kg) subcutaneously twice per day, to improve the pharmacokinetic action of these drugs, and a higher dose of indinavir (60 mg/kg). The kinetics of lymphocyte proliferation were analyzed by monitoring 5-bromo-2′-deoxyuridine (BrdU) uptake ex vivo and by fluorescence-activated cell sorting analysis. HAART did not protect against SIV infection but did strongly impact on virus loads: viremia was delayed and lowered during antiviral therapy in group 2, with better control after treatment was stopped, and in group 3, viremia was maintained at lower levels during treatment, with virus even undetectable in the blood of some macaques, but there was no evidence of improved control of the virus after treatment. We provide direct evidence that dividing NK cells are detected earlier than dividing T cells in the blood (mostly in CD45RA− T cells), mirroring plasma viremia. Dividing CD8+ T cells were detected earlier than dividing CD4+ T cells, and the highest percentages of proliferating T cells coincided with the first evidence of partial control of peak viremia and with an increase in the percentage of circulating gamma interferon-positive CD8+ T cells. The level of cell proliferation in the blood during SIV primary infection was clearly associated with viral replication levels because the inhibition of viral replication by postexposure HAART strongly reduced lymphocyte proliferation. The results and conclusions in this study are based on experiments in a small numbers of animals and are thus preliminary.

Perturbation of differentiated functions during viral infectionin vivo II. Viral reassortants map growth hormone defect to the S RNA of the lymphocytic choriomeningitis virus genome

Lymphocytic choriomeningitis virus (LCMV) Armstrong caused disordered growth and glucose metabolism secondary to growth hormone deficiency in infected C3H/St mice. In contrast, LCMV strain WE replicated in fewer growth hormone-producing cells, failed to disrupt growth hormone synthesis, and did not cause growth hormone-induced disease in infected, matched controls. The generation of genetic reassortant viruses between the virulent strain of LCMV:Armstrong and the avirulent strain, LCMV:WE, is reported. Using such reassortants and both parental strains of virus in C3H/St mice, the perturbation of growth hormone synthesis was clearly mapped to the S RNA segment of LCMV:ARM.

Relationships Between HIV Disease History and Blood HIV-1 DNA Load in Perinatally Infected Adolescents and Young Adults: The ANRS-EP38-IMMIP Study

BACKGROUNDnOur aim was to study the impact of lifelong human immunodeficiency virus (HIV) disease history on the current immune and virological status of perinatally infected patients reaching adulthood. We evaluated blood cell-associated HIV DNA load as an indicator of cell-associated HIV reservoirs and an independent predictor of disease progression.nnnMETHODSnThe ANRS-EP38-IMMIP Study included 93 patients aged 15-24 years who were infected with HIV during the perinatal period. HIV DNA load was quantified by real-time polymerase chain reaction.nnnRESULTSnEighty-five percent of patients were receiving highly active antiretroviral therapy (HAART), and HIV RNA was undetectable in the plasma of 75% of these patients. The median HIV DNA load was 2.84 (interquartile range, 2.51-3.16) log(10) copies per 10(6) peripheral blood mononuclear cells. In patients with viral suppression, HIV DNA load was independently associated with cumulative HIV RNA viremia over the last 5 years. HIV DNA load was negatively correlated with CD4 cell count in patients with active replication but not in those with undetectable HIV RNA.nnnCONCLUSIONSnIn perinatally infected youths who are successfully treated, sustained viral suppression is associated with a low HIV DNA load. The absence of association between current HIV DNA load and CD4 cell counts suggests that the unique physiological characteristics of pediatric infection persist after adolescence.nnnCLINICAL TRIALS REGISTRATIONnNCT01055873.

Perturbation of differentiated functions in Vivo during persistent viral infection III. Decreased growth hormone mRNA

Lymphocytic choriomeningitis virus (LCMV) persistent infection that results from the inoculation of C3H/St newborn mice causes growth hormone (GH) deficiency and associated disease characterized by both reduced weight and serum glucose levels. Molecular analysis of pituitary nucleic acids shows GH deficient mice have, on average, fivefold reduced levels of GH mRNA although the histopathology of such GH producing cells is normal. Northern blots indicate that the length of GH mRNA is comparable in the GH deficient, virus infected mice and the GH normal, uninfected age-matched controls. Hence, truncated GH mRNA cannot account for hormonal defect. Mice infected congenitally through mating of persistently infected parents have normal growth and blood glucose levels. GH mRNA levels in pituitaries of these mice are equivalent to those of uninfected age-matched controls but significantly greater than those seen in neonatally infected GH deficient mice. Although infectious virus titers in the sera are equivalent in congenitally and neonatally infected age- and sex-matched mice, virus titers are significantly lower in pituitaries and brains of the congenitally infected mice when compared to neonatally inoculated mice. Additionally, the number of GH-producing pituitary cells expressing viral proteins is less in congenitally infected mice relative to those in neonatally inoculated mice. Hence there is a direct association between viral replication in GH-producing cells, lowered GH mRNA, and GH deficiency.

A long-term follow-up of an HIV type 1-infected patient reveals a coincidence of nef-directed cytotoxic T lymphocyte effectors and high incidence of epitope-deleted variants

Cytotoxic T lymphocytes (CTL) play a critical role in controlling human immunodeficiency virus-1 (HIV-1) and simian immunodeficiency virus (SIV) infections. However, in spite of developing a strong CTL response most HIV-1-infected patients eventually progress to AIDS. Amino acid changes in CTL epitope have been previously described and may permit HIV to escape from CTL immune responses. The importance of CTL selection pressure in controlling the course of viral evolution in HIV-infected patient remains debatable. For over a 10-year period, we longitudinally followed a patient for bulk unstimulated effector (eCTL) and stimulated memory CTL responses (mCTL) against the viral proteins Gag, Pol, and Nef. The patient showed a strong CTL response against Nef in unstimulated peripheral blood mononuclear cells with a peak during Month 40 of the follow-up. The mCTL response was also higher against Nef than Gag and Pol. PCR amplification and nucleotide sequencing of the plasma viral variants showed a viral variant with the epitope deletion that was detected early during the follow-up and essentially replaced the wild-type virus during the peak eCTL response. These studies support the importance of Nef epitope deletion as a mechanism for HIV-1 escape from CTL immune pressure.

In untreated HIV-1-infected children, PBMC-associated HIV DNA levels and cell-free HIV RNA levels are correlated to distinct T-lymphocyte populations.

Background:Clinical studies support biologically independent roles of cell-free HIV particles and HIV-infected cells in disease progression. The associations between the level of infected cells and immune markers have been poorly studied, particularly in perinatally infected children. Objective:We tested the hypothesis that independent roles of cell-free virus and infected cells in HIV pathogenesis should be revealed by different associations between each of them and specific immune markers. Methods:Levels of HIV RNA and DNA, HIV-specific CD8 T lymphocytes, activated and naive/memory T lymphocytes were determined in 44 untreated HIV-1-infected children. Pearson partial correlation coefficients were used to assess associations between the variables. Results:Here we provide new information, by showing a direct correlation between the percentages of CD4+HLA-DR+ lymphocytes and HIV DNA levels. Furthermore, higher HIV-specific CD8 T-lymphocyte frequencies were associated with lower HIV DNA levels. In contrast, CD8+38+ lymphocytes and memory CD4 lymphocytes were correlated only to the HIV RNA level. All correlations were independent of age and CD4 depletion. Conclusions:Several immune markers were correlated to either the HIV RNA or the HIV DNA level, but never to both of them, supporting the concept that cell-free virus and infected cells play different roles in HIV-1 immunopathogenesis.

The use of lymphocytic choriomeningitis virus reassortants to map viral genes causing virulence

Lymphocytic choriomeningitis virus (LCMV) is the prototypic arenavirus. The genome consists of two single strand RNA species, L and S, with approximate molecular weight of 2.85 x 106 and 1.35 x 106, respectively [1]. Several individual strains of LCMV can be distinguished by biochemical and immunological studies. In addition, variation in the pathogenicity among LCMV strains has been reported [2, 3]. For example, C3H newborn mice inoculated with LCMV Armstrong (Arm) develop growth retardation and disordered glucose metabolism secondary to growth hormone deficiency, but infection with LCMV WE strain has no effect on growth development or blood sugar levels [4]. Adult guinea pigs resist infection when inoculated with the Arm strain but succumb when challenged with the WE strain [2, 3]. Since the genome of LCMV is segmented it is possible to generate reassortants between two different strains. We have recently obtained and characterized intertypic reassortants between the Arm and WE and the Arm and Traub strains of LCMV. Using such reassortants, monoclonal antibodies specific for the nucleoprotein (NP) and the glycoproteins (GP-1 and GP-2) of Arm and WE, and specific cDNA probes of LCMV-Arm, we have demonstrated that the small RNA segment of LCMV codes for the three major structural polypeptides [5]. Utilizing reassortant and both parental strains of virus we have shown that the perturbation of growth hormone synthesis in C3H/St mice maps to the S RNA of LCMVArm [6, Table 1] and that the L RNA of LCMV WE is important for virus replication in vivo and is associated with fatal acute disease following infection of adult guinea pigs [7, Table 1]. Both of these disease states occur independently of anti-LCMV immune responses [4, 6, 7]. Other experiments, not discussed here, show that the induction and activity of virus-specific H-2-restricted cytotoxie T lymphocytes is associated only with LCMV gene products encoded by the S RNA segment [8]. Recently we noted that LCMV reassortants with the WE/Arm or the Traub/Arm genotype caused a lethal disease following neonatal inoculation of BALB/c mice while, in contrast, parental strains or reciprocal reassortants did not. The pathogenesis of the disease which is characterized by inhibition of growth and death, is the presumed generation of interferon, causing liver necrosis. Disease and liver necrosis were prevented by the administration of a potent sheep-globulin against mouse ~ +/3 interferon (Table

Loss of reactivity of vaccine-induced CD4 T cells in immunized monkeys after SIV/HIV challenge.

Background:Immunization protocols involving priming with DNA and boosting with recombinant live virus vectors such as recombinant modified Vaccinia Ankara (rMVA) are considered as vaccine candidates against HIV. Such protocols improve the outcome of simian/human immunodeficiency virus (SHIV) pathogenic challenge in Rhesus monkeys. Objectives:To investigate the fate of vaccine-induced T cells after a mucosal SHIV challenge. Methods:We immunized Rhesus monkeys (Macaca mulatta) by DNA priming followed by rMVA boost. After intrarectal challenge with SHIV 89.6P, immunized animals demonstrated early control of viral replication and stable CD4 T-cell counts. We monitored T-cell responses by measuring IFN-γ secretion and proliferation. Results:Immunization induced strong and sustained SHIV-specific CD4 and CD8 T-cell responses. CD8 T-cell responses were recalled during acute infection, whereas none of the vaccine-induced SHIV-specific CD4 T-cell responses were recalled. Moreover, most of the CD4 T-cell responses became undetectable in peripheral blood or lymph nodes even after in-vitro peptide stimulation. In contrast, we persistently detected CD4 T-cell responses specific for control recall antigens in infected animals. Conclusion:SHIV 89.6P challenge results in a lack of reactivity of vaccine-induced SHIV-specific CD4 T cells. These results may have important implications in the AIDS vaccine field, especially for the evaluation of new vaccine candidates, both in preventive and therapeutic trials.