Geretti, A.M.

Detection of drug resistance mutations at low plasma HIV-1 RNA load in a European multicentre cohort study (Article)

2011-08-01T00:00:00Z

Background and objectives: Guidelines indicate a plasma HIV-1 RNA load of 500-1000 copies/mL as the minimal threshold for antiretroviral drug resistance testing. Resistance testing at lower viral load levels may be useful to guide timely treatment switches, although data on the clinical utility of this remain limited. We report here the influence of viral load levels on the probability of detecting drug resistance mutations (DRMs) and other mutations by routine genotypic testing in a large multicentre European cohort, with a focus on tests performed at a viral load <1000 copies/mL. Methods: A total of 16511 HIV-1 reverse transcriptase and protease sequences from 11492 treatment-experienced patients were identified, and linked to clinical data on viral load, CD4 T cell counts and antiretroviral treatment history. Test results from 3162 treatment-naive patients served as controls. Multivariable analysis was employed to identify predictors of reverse transcriptase and protease DRMs. Results: Overall, 2500/16 511 (15.14%) test results were obtained at a viral load <1000 copies/mL. Individuals with viral load levels of 1000-10000 copies/mL showed the highest probability of drug resistance to any drug class. Independently from other measurable confounders, treatment-experienced patients showed a trend for DRMs and other mutations to decrease at viral load levels <500 copies/mL. Conclusions: Genotypic testing at low viral load may identify emerging antiretroviral drug resistance at an early stage, and thus might be successfully employed in guiding prompt management strategies that may reduce the accumulation of resistance and cross-resistance, viral adaptive changes, and larger viral load increases.

European guidelines on the clinical management of HIV-1 tropism testing (Article)

2011-05-01T00:00:00Z

Viral tropism is the ability of viruses to enter and infect specific host cells and is based on the ability of viruses to bind to receptors on those cells. Testing for HIV tropism is recommended before prescribing a chemokine receptor blocker. In most European countries, HIV tropism is identified with tropism phenotype testing. New data support genotype analysis of the HIV third hypervariable loop (V3) for the identification of tropism. The European Consensus Group on clinical management of tropism testing was established to make recommendations to clinicians and clinical virologists. The panel recommends HIV-tropism testing for the following groups: drug-naive patients in whom toxic effects are anticipated or for whom few treatment options are available; patients who have poor tolerability to or toxic effects from current treatment or who have CNS pathology; and patients for whom therapy has failed and a change in treatment is considered. In general, an enhanced sensitivity Trofile assay and V3 population genotyping are the recommended methods. Genotypic methods are anticipated to be used more frequently in the clinical setting because of their greater accessibility, lower cost, and faster turnaround time than other methods. For the interpretation of V3 loop genotyping, clinically validated systems should be used when possible. Laboratories doing HIV tropism tests should have adequate quality assurance measures. Similarly, close collaboration between HIV clinicians and virologists is needed to ensure adequate diagnostic and treatment decisions.

Performance of a nurse-led paediatric point of care service for respiratory syncytial virus testing in secondary care (Article)

2011-01-01T00:00:00Z

Objectives: To evaluate respiratory syncytial virus (RSV)-point-of-care-testing (POCT) performance among paediatric patients with respiratory symptoms, using the BinaxNOW® RSV assay performed by trained nurses on the paediatric ward, and compare results with those obtained by real-time polymerase chain reaction (PCR). Methods: Four paediatric nurses were trained and certified in using RSV-POCT. Between October 2008 and March 2009, all hospitalised children below 5 years of age presenting with a suspected RSV infection had nasopharyngeal swabs (NPS) tested by RSV-POCT by the nurses and a real-time PCR targeting common respiratory viruses by laboratory staff. Results: Among 159 NPS, 21 (13.2%) were RSV-POCT positive and 138 (86.8%) negative. All 21 RSV-POCT positive samples were positive by PCR, yielding a specificity of 100% (95% CI 95.7%, 100.0%). Of 138 RSV-POCT negative samples, 30 (21.7%) were RSV positive by PCR (sensitivity 41.2%; 95% CI: 27.9%, 55.8%). The positive and negative predictive values for RSV-POCT were 100% (95% CI 80.8%, 100.0%) and 78.3% (95% CI 70.3%, 84.6%) respectively. Other respiratory viruses were detected in 52/138 (39.9%) NPS. Conclusions: A POCT for RSV run by trained nurses can be used reliably as a first screening step in symptomatic children. Negative samples should be analysed for RSV and other respiratory pathogens by real-time PCR.

European guideline for the management of genital herpes, 2010 (Article)

2011-01-01T00:00:00Z

This is the guideline for genital herpes simplex virus (HSV) management for the IUSTI/WHO Europe, 2010. They describe the epidemiology, diagnosis, clinical features, treatment and prevention of genital HSV infection. They include details on the management of HSV in pregnancy, those who are immunocompromised and the clinical investigation and management of suspected HSV-resistant disease.

Virus replication and evolution drive the kinetics and specificity of SIV-specific cytotoxic T lymphocyte. (Article)

2001-12-01T00:00:00Z

SIV (simian immunodeficiency virus) infection of cynomolgus macaques provides an excellent model for investigating the basis of protective immunity against HIV (human immunodeficiency virus). We explored the protective role of cytotoxic T lymphocytes (CTL) against the pathogenic molecular clone SIVmac-J5. Vaccine-induced CTL precursors (CTLp) against Env, Gag or Nef did not protect macaques against intravenous challenge. However, detection of Rev-specific CTLp in infected macaques was associated with effective virus containment. Furthermore, CTL against an immunodominant Gag/p26 epitope (amino acids 242-250) resulted in the emergence of a mutant virus that uniformly replaced wild-type virus in the spleen and partially escaped recognition. During primary infection, CTLp detection in blood coincided with decreasing viremia. After 12 months, two outcomes emerged. In one group of macaques, persistent viremia was associated with high viral load in lymphoid organs and declining CD4+ T-cell counts. CTLp were maintained in asymptomatic macaques, but declined in the symptomatic phase of infection. In a second group, loss of detectable viremia was associated with low-level virus reservoirs in lymphoid organs, asymptomatic status and maintained CD4+ T-cell counts. CTLp peaked in the first 4 months of infection and subsequently declined in this group. These studies provide insights into the complex interplay between virus replication and host immunity.

Decline of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocytes in the peripheral blood of long-term nonprogressing macaques infected with SIVmac32H-J5. (Article)

1999-12-01T00:00:00Z

The evolution of simian immunodeficiency virus (SIV)–specific cytotoxic T lymphocyte precursors (CTLps) and their relationship with virus replication were studied in SIV‐infected macaques. After primary viremia, 3 of 8 macaques lost culturable virus and polymerase chain reaction–detectable provirus in peripheral blood. Although proviral DNA persisted in the spleen and lymph nodes, virus loads were below or barely above detection levels. Throughout the study, the 3 macaques remained asymptomatic, with stable CD4+ cell counts. These findings were associated with the detection of CTLps directed against both structural and regulatory SIV proteins. The response peaked during the first 7 months of infection but waned subsequently. CTLps increased after rechallenge of 1 macaque, suggesting that limited antigenic stimulation contributed to their disappearance from circulation. Transient viremia with increasing CTLp frequencies and antibody titers also suggested at least partial susceptibility to reinfection. These findings bear implications for vaccination strategies aimed at inducing protective CTLs against lentiviruses.

Towards an HIV-1 vaccine: Lessons from studies in macaque models. (Article)

1998-05-01T00:00:00Z

The development of a safe and effective vaccine for the prevention of AIDS has thus far proven to be extremely difficult, at least in part due to complexities associated with HIV-1 and its pathogenesis. The recent description of individuals transiently infected with HIV-1, as well as persons who survived HIV-1 infection for more than 15 years, indicates the ability of the immune response of certain individuals to control HIV-1 infection. Moreover, vaccination-challenge experiments in macaques infected with simian immunodeficiency virus have shown that protection against infection or development of disease may be achieved in the absence of sterilizing immunity, suggesting that the goals for AIDS vaccine development may have to be redefined. In addition, evaluation of new lentivirus vaccine strategies may largely benefit from the use of the newly developed chimeric simian-human immunodeficiency viruses, allowing the testing of HIV-1 antigen based vaccines in macaques.

Cytotoxic T lymphocytes in AIDS pathogenesis: lessons to be learned from the macaque model of simian immunodeficiency virus infection (Article)

1998-01-01T00:00:00Z

Role of cytotoxic T lymphocyte responses in the control of primate immunodeficiency viruses (Doctoral Thesis)

1997-10-29T00:00:00Z

CD8+ cytotoxic T lymphocytes of a cynomolgus macaque infected with simian immunodeficiency virus (SIV)mac32H-J5 recognize a nine amino acid epitope in SIV Gag p26. (Article)

1997-01-01T00:00:00Z

A detailed analysis of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocyte (CTL) responses and the identification of the proteins and epitopes they target may improve the design of immunotherapeutic interventions and provide insights into AIDS pathogenesis. Here, we identified a new CTL epitope in the SIV Gag protein, recognized by CD8+ and MHC class I-restricted CTL clones from a long-term asymptomatic cynomolgus macaque (Macaca fascicularis) infected with SIVmac32H-J5. Using overlapping synthetic peptides, the optimal minimal epitope was characterized as a nine amino acid peptide representing amino acids 242-250 of p26 (SVDEQIQWM). CTL recognition was shown to be abolished by amino acid substitutions observed within homologous human immunodeficiency virus (HIV)-1 and HIV-2 sequences.

Simian immunodeficiency virus (SIV)-specific CD8+ cytotoxic T lymphocyte responses of naive and vaccinated cynomolgus macaques infected with (SIV)mac32H(J5): quantitative analysis by in vitro antigenic stimulation. (Article)

1997-01-01T00:00:00Z

Detailed analyses of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocyte (CTL) responses in vaccinated and infected macaques may help to clarify the role of CTL immunity in protection against lentiviruses. Here, the optimal conditions for the measurement of SIV Gag-specific CTL were investigated by bulk and limiting dilution assays of peripheral blood mononuclear cells (PBMC) from naive and vaccinated cynomolgus macaques (Macaca fascicularis) infected with SIVmac32H(J5). In vitro restimulation was generally required for CTL detection. Selective activation of CD8+ and MHC-restricted SIV Gag-specific CTL was induced by stimulation with autologous para-formaldehyde-fixed B-lymphoblastoid cell lines infected with a recombinant vaccinia virus expressing SIV Gag. Applied to limiting dilution assays, antigenic stimulation reproducibly demonstrated SIV Gag-specific CTL precursors (CTLp) in PBMC of all animals studied, including those lacking significant responses in standard bulk CTL assays.

Vaccine-induced virus-neutralizing antibodies and cytotoxic T cells do not protect macaques from experimental infection with simian immunodeficiency virus SIVmac32H (J5). (Article)

1995-01-01T00:00:00Z

To gain further insight into the ability of subunit vaccines to protect monkeys from experimental infection with simian immunodeficiency virus (SIV), two groups of cynomolgus macaques were immunized with either recombinant SIVmac32H-derived envelope glycoproteins (Env) incorporated into immune-stimulating complexes (iscoms) (group A) or with these SIV Env iscoms in combination with p27gag iscoms and three Nef lipopeptides (group B). Four monkeys immunized with recombinant feline immunodeficiency virus Env iscoms served as controls (group C). Animals were immunized intramuscularly at weeks 0, 4, 10, and 16. Two weeks after the last immunization, monkeys were challenged intravenously with 50 monkey 50% infectious doses of virus derived from the J5 molecular clone of SIVmac32H propagated in monkey peripheral blood mononuclear cells. High titers of SIV-neutralizing antibodies were induced in the monkeys of groups A and B. In addition, p27gag-specific antibodies were detected in the monkeys of group B. Vaccine-induced cytotoxic-T-lymphocyte precursors against Env, Gag, and Nef were detected on the day of challenge in the monkeys of group B. Env-specific cytotoxic-T-lymphocyte precursors were detected in one monkey from group A. In spite of the observed antibody and T-cell responses, none of the monkeys was protected from experimental infection. In addition, longitudinal determination of cell-associated virus loads at weeks 2, 4, 6, 9, and 12 postchallenge revealed no significant differences between vaccinated and control monkeys. These findings illustrate the need to clarify the roles of the different arms of the immune system in conferring protection against primate lentivirus infections.

Selective in vitro expansion of HLA class I-restricted HIV-1 gag-specific CD8+ T cells: cytotoxic T-lymphocyte epitopes and precursor frequencies. (Article)

1993-01-01T00:00:00Z

OBJECTIVE: To identify HIV-1 Gag cytotoxic T-lymphocyte (CTL) epitopes and HLA restriction of their recognition, and to define precursor frequencies of HIV-1 Gag-specific CTL in the blood of seropositive individuals. METHODS: B-lymphoblastoid cell lines (B-LCL) infected with recombinant vaccinia viruses (rVV) containing a gene coding for HIV-1 Gag (rVV-Gag) were fixed with paraformaldehyde (PFA) and used as antigen-presenting cells (APC) to stimulate peripheral blood mononuclear cells (PBMC) from asymptomatic HIV-seropositive individuals. Specific CTL activity was determined in 51Cr-release assays using B-LCL as targets after infection with rVV-Gag or after pulsing with partially overlapping peptides spanning the Gag sequence. RESULTS: In vitro stimulation resulted in an increased number of CD8+ T cells and CD45R0+ and HLA-DR+ cells. Gag-specific cytotoxicity, mediated predominantly by HLA class I-restricted CD8+ CTL, was observed in all seven individuals studied. Multiple HLA-restricted CTL epitopes were identified with a single culture from one of the individuals. Gag-expressing APC were successfully used as stimulator cells in limiting dilution analysis to determine CTL precursor (CTLp) frequencies. CONCLUSION: PFA-fixed rVV-Gag-infected autologous B-LCL can be used as stimulator cells in bulk PBMC cultures to identify CTL epitopes and to determine CTLp frequencies. This method will facilitate the analysis of HIV-1-specific CTL responses in HIV-infected and vaccinated individuals.