Clinically validated mutation scores for HIV-1 resistance to fosamprenavir/ritonavir.

BACKGROUND: We developed clinically relevant genotypic scores for resistance to fosamprenavir/ritonavir in HIV-1 protease inhibitor (PI)-experienced patients. METHODS: PI-experienced patients with virological failure receiving fosamprenavir/ritonavir as the sole PI for at least 3 months and with detectable fosamprenavir plasma levels were included. The impact of baseline protease mutations on virological response (VR, i.e. decrease in plasma HIV-1 RNA between baseline and month 3) was analysed using the Mann-Whitney test. Mutations with prevalence >10% and P value <0.10 were retained. The Jonckheere-Terpstra test was used to select the combination of mutations most strongly associated with VR. The association between score and VR was assessed by multivariate backward regression. RESULTS: In the 73 patients included, the median baseline HIV-1 RNA was 4.6 log(10) copies/mL (range: 2.7-6.9) and the mean decrease at month 3 was -1.07 +/- 1.40 log(10) copies/mL. Ninety per cent of the patients were infected by HIV-1 subtype B variants. Two fosamprenavir/ritonavir mutation scores were constructed: score A (L10F/I/V + L33F + M36I + I54L/M/V/A/T/S + I62V + V82A/F/C/G + I84V + L90M) was based only on mutations associated with a worse VR, whereas score B (L10FIV + L33F + M36I + I54L/M/V/A/T/S + A71V - V77I - N88S + L90M) also took into account favourable mutations. Both scores were independent predictors of VR, however, co-administration of tenofovir was associated with a worse VR and the presence of the N88S protease mutation and co-administration of enfuvirtide with a better VR. CONCLUSIONS: These clinically validated mutation scores should be of interest for the clinical management of PI-experienced patients. The fosamprenavir/ritonavir score A was introduced in the 2006 ANRS algorithm along with isolated mutations I50V and V32I + I47V.

Antiviral responses of human Leydig cells to mumps virus infection or poly I:C stimulation.

BACKGROUND: The immuno-privileged status of the testis is essential to the maintenance of its functions, and innate immunity is likely to play a key role in limiting harmful viral infections, as demonstrated in the rat. In men mumps virus infects Leydig cells and has deleterious effects on testosterone production and spermatogenesis. The aim of this study was to test whether mumps virus infection of isolated human Leydig cells was associated with an inhibition of their innate antiviral defences. METHODS: Leydig cell production of mRNA and protein for interferons (IFNs) and of three antiviral proteins-2'5' oligoadenylate synthetase (2'5'OAS), double-stranded RNA-activated protein kinase (PKR) and MxA-was investigated, in the absence or presence of mumps virus or viral stimuli including poly I:C, a mimetic of RNA viruses replication product. RESULTS: Stimulated or not, human Leydig cells appeared unable to produce routinely detectable IFNs alpha, beta and gamma. Although the level of PKR remained unchanged after stimulation, the expression of 2'5'OAS and MxA was enhanced following either mumps virus or poly I:C exposure (P < 0.05 versus control). CONCLUSIONS: Overall, our results demonstrate that mumps virus replication in human Leydig cells is not associated with a specific inhibition of IFNs or 2'5'OAS, MxA and PKR production and that these cells display relatively weak endogenous antiviral abilities, as opposed to their rat counterparts.

Prevalence of resistance mutations in antiretroviral-naive chronically HIV-infected patients in 1998: a French nationwide study.

OBJECTIVE: To estimate the prevalence of resistance-conferring mutations to antiretroviral drugs in previously untreated patients with chronic HIV-1 infection as a basis for French recommendations on viral genotyping before antiretroviral treatment initiation. DESIGN: Resistance mutations were sought in samples from 404 patients seen in 23 specialized centres throughout metropolitan France in 1998. METHODS: The protease and reverse transcriptase (RT) genes of plasma virions were sequenced. Primary and secondary protease and RT gene mutations were identified from the International AIDS Society resistance testing - USA panel. RESULTS: The prevalence of patients with primary and secondary mutations were 3.7% (95% CI 1.7-5.7) and 50.3% (95% CI 45.0-55.6), respectively. The prevalence of patients with mutations associated with resistance to nucleoside RT inhibitors (NRTI) and non-nucleoside RT inhibitors was 3.3% (95% CI 1.5-5.1) and 0.8% (95% CI 0.0-1.7), respectively. The prevalence of patients with NRTI primary mutations differed according to whether seropositivity had been diagnosed more or less than one year previously (0.2 versus 2.2% P = 0.023). Primary mutations associated with protease inhibitor resistance occurred at a prevalence of 1.9% (95% CI 0.5-3.4) with no difference according to the duration of known seropositivity. CONCLUSION: In France, in 1998, the prevalence of patients with primary mutations associated with resistance to antiretroviral drugs was low. Genotyping before the initiation of therapy was not recommended in chronically HIV-1-infected naive patients. A national sentinel survey of resistance in this clinical setting is performed regularly to update the recommendations for resistance testing.

Interferon-alpha and -gamma expression in the rat testis.

Interferon-alpha (IFN alpha), -beta, and -gamma are well known for their antiviral, antiproliferative, and immunoregulatory activities. Although several studies suggest an involvement of IFNs in the spermatogenic process, nothing is known about the possible production of these molecules within the testis. Moreover, the antiviral capabilities of testicular cells have not yet been explored despite their importance in the context of sexually transmissible diseases. Using reverse transcription-polymerase chain reaction, a cytopathic inhibition micromethod assay, and an enzyme-linked immunosorbent assay, the present study demonstrates for the first time that IFN alpha and -gamma are produced by testicular cells. IFN alpha protein and corresponding messenger RNA are expressed by peritubular, Sertoli, and germ cells. In vitro, IFN alpha production by Sertoli cells, peritubular cells, and early spermatids was inducible by the Sendai virus, whereas pachytene spermatocyte IFN alpha production was not triggered by this virus. Of all the testicular cell types tested, Sertoli cells by far produced the highest concentrations of IFN alpha/beta, followed by peritubular cells. Both IFN gamma messenger RNA and IFN gamma protein were found in early spermatids, but, in contrast, were not produced by peritubular cells, Sertoli cells, or pachytene spermatocytes. In conclusion, our study establishes the cellular distribution of IFNs within the seminiferous tubules and provides the basis for research into the possible involvement of IFNs in regulation of the spermatogenic process. To the best of our knowledge, our results afford the first insight on how the testicular antiviral defense system is organized.

Safety and efficacy of ritonavir and saquinavir in combination with zidovudine and lamivudine.

BACKGROUND: Ritonavir is a potent inhibitor of cytochrome P4503A4 that strongly increases saquinavir bioavailability. In this study we assessed the safety and antiretroviral efficacy of the combination of these two compounds in patients pretreated and receiving continued treatment with zidovudine and lamivudine who were protease inhibitor naive and who had a CD4 cell counts below 200/mm3. METHODS: In this 48-week pilot study, all patients received 600 mg ritonavir and 400 mg saquinavir twice daily. Administration of zidovudine and lamivudine was continued without a change in previous doses. Viral load, CD4 cell count, and the emergence of resistance to the two protease inhibitors were evaluated repeatedly up to week 48. RESULTS: Sixteen patients were included in the study. Previous nucleoside analog treatment duration was 48+/-22 months (mean +/- SD). Two patients quit taking both protease inhibitors within 2 weeks. The ritonavir dose had to be reduced in 10 other patients because of side effects. Between inclusion and week 48, plasma viremia varied from 4.87+/-0.43 to 3.00+/-1.29 log10 copies/mL and CD4 cell counts ranged from 98+/-61 to 250+/-139/mm3. Ten patients (63%) had viral loads below 200 copies/mL and 7 (44%) had viral loads below 50 copies/mL. A single key mutation that conferred ritonavir resistance I84V and V82A/V developed in two patients. A mutation at codon 54 developed in another patient. These mutations were associated with repeated cessations of antiretroviral treatment. No lipodystrophy was observed. CONCLUSION: Ritonavir and saquinavir in combination are quite well tolerated and induce a high and sustained antiretroviral efficacy. A four-drug combination that includes these two protease inhibitors should be considered as a first line of treatment in patients with low CD4 cell counts.

Zidovudine resensitization and dual HIV-1 resistance to zidovudine and lamivudine in the delta lamivudine roll-over study.

OBJECTIVE: To study zidovudine resensitization and dual resistance to zidovudine/lamivudine in HIV-1 isolates from nucleoside reverse transcriptase (RT) inhibitor-experienced patients during selective pressure exerted by zidovudine/lamivudine combination therapy. DESIGN AND METHODS: HIV-1 isolates from 29 patients receiving zidovudine/lamivudine combination therapy in the Delta roll-over study were analysed at entry and during a 1 year follow-up period for phenotypic susceptibility to zidovudine and lamivudine in the ANRS PBMC assay. The RT gene from codon 20 to 230 and at codon 333 was analysed by nucleotide sequencing of the corresponding isolates. RESULTS: HIV-1 isolates from 23 of the 29 patients were phenotypically resistant to zidovudine at baseline; 61% of these patients showed significant zidovudine resensitization during follow-up. The zidovudine IC50 value correlated positively with log10 plasma HIV-1 RNA (P = 0.02) and negatively with the CD4 cell count (P = 0.004). Zidovudine resensitization (related to acquisition of the M184V mutation) was transient, with evolution towards dual resistance to zidovudine and lamivudine in 20 of the 29 patients. The phenotype of certain dually resistant isolates coincided with the emergence of multiple mutations in the 5' part of the RT gene. CONCLUSIONS: M184V-mediated zidovudine resensitization of HIV-1 is transient in most patients who are given zidovudine/lamivudine combination therapy when zidovudine resistance has already emerged. The subsequent evolution towards dual phenotypic resistance to zidovudine/lamivudine corresponds to complex genotypic profiles.

The role of human spleen interferon on natural killer activity of peripheral blood lymphocytes enriched in large granular lymphocytes.

The elimination of monocytes as well as B- and T-lymphocytes by forming rosettes with high affinity for sheep red blood cells yielded an enriched population of both natural killer (NK) activity (cytotoxicity: 65.4 +/- 9.9% with an E/T ratio of 12:1, P less than 0.005) and large granular lymphocytes (LGL: 76 +/- 13%) compared to the untreated lymphocyte population where NK activity is 35.7 +/- 17.3% (E/T 12:1) and the percentage of LGL of 26 +/- 6%. We studied the action of type I interferon (IFN) obtained from human spleens, on NK activity of 9 peripheral blood lymphocyte populations and 9 enriched in LGL. NK activity of the total lymphocyte population is significantly increased (P less than or equal to 0.05) in 6 out of 9 cases after treatment by interferon. Cell populations enriched in LGL showed increased NK activity in only one case after treatment by interferon, but no increased activity was found in the other cases. These results are compatible with the notion of cellular cooperation in increased NK activity by interferon.

Longitudinal study of HIV-specific cytotoxic lymphocytes in HIV type 1-infected patients: relative balance between host immune response and the spread of HIV type 1 infection.

To evaluate the contribution of a specific cytotoxic response in the control of HIV infection in relation to clinical status, we performed serial analysis of anti-Env and anti-Gag cytotoxic activity in 13 infected individuals over a 6- to 10-year period, using cryopreserved peripheral blood mononuclear cells (PBMCs). Autologous EBV-transformed B cell lines infected in vitro with recombinant vaccinia viruses expressing HIV-1 env and gag genes were used as targets. Without any stimulation of the effector cells, we were able to show an anti-HIV cytotoxic activity in the PBMCs of 12 of 13 HIV-1-infected patients, consistent with chronic immune activation in HIV infection. Different patterns of HIV-specific cytotoxic activity were observed, and the extent of this cytotoxic response varied between the clinically defined groups of individuals. No direct relationship was observed with the number of CD4 and CD8 lymphocytes during the observation period. However, patients who remained asymptomatic had a more vigorous cytotoxic response than patients with clinical deterioration during the observation period, and a significant difference was observed for HIV Gag-specific CTL activity. From these data, we suggest that the HIV-specific cytotoxic response has a protective role in the course of HIV infection.

HIV RNA and HIV DNA in peripheral blood mononuclear cells are consistent markers for estimating viral load in patients undergoing long-term potent treatment.

The aim of this study was to evaluate residual viral replication by assessing the HIV load of circulating infected cells in patients given an effective antiprotease-containing treatment for 1 year. PBMC HIV RNA and HIV DNA was quantified by techniques standardized and evaluated by interlaboratory quality control testing. Viral markers identified in a multicenter study were validated in a cross-sectional study of 121 patients beginning treatment. A longitudinal study of 3 viral markers was carried out in 18 patients, each of whom had fewer than 200 copies of HIV RNA per milliliter of plasma after 12 months of treatment. The cross-sectional study showed that viral replication in PBMCs was correlated with the number of circulating infected cells (Spearman rank correlation; p = 0.0001, r = 0.35) and the concentration of virus particles in the plasma (Spearman; p = 0.0001, r = 0.54). The longitudinal study showed that the decrease in HIV RNA levels was smaller in PBMCs than in the plasma. The largest decrease in HIV DNA levels after 12 months of treatment was recorded in patients with low levels of intracellular replication (Spearman; p = 0.005, r = 0.69). PBMC HIV RNA and HIV DNA levels were very informative markers, complementary to plasma HIV RNA levels. They should be used in future trials evaluating the long-term efficacy of new associations of highly active antiretroviral treatments.

New types of primers (stair primers) for PCR amplification of the variable V3 region of the human immunodeficiency virus.

The aim of the study was to develop a reliable PCR method for the detection of viral genomes with frequent mutations like HIV and hepatitis C virus. A system of 'stair' primers is suggested which allows amplification of a genomic sequence despite the presence of mutations in the region of the primers. In this system, classical primers are replaced with primers composed of a mixture of equimolar oligonucleotides in which the 5' end remains constant (single sized fragment) and the 3' end is displaced base by base. By PCR, 'stair' primers (HIV set) were compared to single-sequence primers of 20 and 25 nucleotides chosen in the same hypervariable region of the HIV gp120 (on both sides of V3 region), as well as to classical primers chosen in the conserved pol (polV2) and gag (SK38-39) regions of the genome. Of 17 HIV isolates obtained by co-culture of lymphocytes from HIV-seropositive patients, 17/17 (100%) were amplified using stair primers, 14/17 (82%) with 25-nucleotide primers, and 12/17 (70%) with 20-nucleotide primers. Amplification occurred in 17/17 instances with polV2 primers and in 16/17 instances with SK38-39. In addition, 55 other isolates were tested comparatively using stair, polV2 and SK38-39 primers. All isolates were amplified using stair and SK38-39 primers and 54/55 isolates with polV2 primers. When applied to 22 extracts of patients' lymphocytes DNA, stair primers amplified all 22 extracts to the same degree as polV2 and SK38-39, whereas the 20 and 25 nucleotide primers chosen in the variable region were not as reliable. This new primer system allows reliable detection of variable genomic regions of the HIV genome and amplification of such regions directly in patient leukocytes. In addition, the contribution of this system to microbiology and human genetics in general may be important.

[Beta interferon in multiple sclerosis: pharmacological differences]. / Les interférons bêta dans la sclérose en plaques: les différences pharmacologiques.

Interferons beta (INF) would be a good recombinant therapeutic choice for the management of relapsing remitting multiple sclerosis (RRMS). Three forms are approved in various countries including one IFN beta-1b (Betaféron) and two IFN beta-1a (Avonex and Ribif). These agents are apparently similar although they are not identical. Differences may be of clinical relevance. Important differences are described here in terms of pharmacokinetics, the spectrum of side effects and molecular chemistry. The clinical consequences on the benefit/risk ratio are discussed. This review recalls the difficulty in developing such compounds and in reaching marketing approval. An improvement in the acceptability and efficiency of IFN beta could by obtained when analyzing basic research data.

[Human spleen: a new and potent source of human interferon for experimental and therapeutic use]. / La rate humaine: une nouvelle et puissante source d'interféron humain pour usage thérapeutique et expérimental.

Cells of a single human spleen, cultivated in vitro and induced by Sendai virus, produce constantly more than 10(8) units of interferon per spleen. This interferon can be used for therapeutic assays and new basic investigations.

[Herpes simplex virus in the saliva of immunosuppressed patients (author's transl)]. / Précocité de l'isolement du virus herpétique dans la salive de malades immunodéprimés, antérieurement infectés

Attempts to isolate Herpes Simplex Virus (HSV) from the saliva of patients submitted to immunosuppressive drugs after renal transplantation were undertaken in the early part of the week following the graft. Results showed that the virus constantly appeared in the salvia of patients with serological positive tests to HSV. The presence of HSV was generally detected a few days before the appearance of the clinical disease.

[Effect of interferon on cell populations enriched in NK activity]. / Effet de l'interféron sur des populations cellulaires enrichies en activité NK.

We have studied the NK activity after either interferon action or not, in total lymphocyte populations, as well as in cellular populations enriched with NK activity. (LGL = 76 +/- 13%). The incubation with interferon lasts 16 hours at 37 degrees C. The result obtained is an increase of the NK activity of the total lymphocyte population, while the cellular population, formerly enriched with NK activity, is not affected. These results are in favour of a necessary cellular cooperation on the interferon action on NK cells.