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Introduction: The availability of new classes of antiretroviral drugs is critical for treatment-experienced patients due to drug resistance to and unwanted side effects from current drugs. Our aim was therefore to evaluate the anti-HIV-1 activity of a new set of antivirals, dipeptides (WG-am or VQ-am) combined with a single-stranded oligonucleotide (ssON). The dipeptides were identified as naturally occurring and enriched in feces and systemic circulation in HIV-1-infected elite controllers and were proposed to act as entry inhibitors by binding to HIV-1 gp120. The ssON is DNA 35-mer, stabilized by phosphorothioate modifications, which acts on the endocytic step by binding to cell host receptors and inhibiting viruses through interference with binding to nucleolin. Methods: Chou–Talalay’s Combination Index method for quantifying synergism was used to evaluate the drug combinations. Patient-derived chimeric viruses encoding the gp120 (envregion) were produced by transient transfection and used to evaluate the antiviral profile of the combinations by drug susceptibility assays. Results: We found that the combination WG-am:ssON or VQ-am:ssON had low combination index values, suggesting strong antiviral synergism. Of the two combinations, WG-am:ssON (1 mM:1 μM) had high efficacy against all prototype or patient-derived HIV-1 isolates tested, independent of subtype including the HIV-1-A6 sub-subtype. In addition, the antiviral effect was independent of co-receptor usage in patient-derived strains. Conclusion: WG-am and ssON alone significantly inhibited HIV-1 replication regardless of viral subtype and co-receptor usage, and the combination WG-am:ssON (1 mM:1 μM) was even more effective due to synergism.

ABSTRACTIntroduction: Coronaviruses encode a helicase that is essential for viral replication and represents an excellent antiviral target. However, only a few coronavirus helicase inhibitors have been patented. These patents include drug-like compound SSYA10-001, aryl diketo acids (ADK), and dihydroxychromones. Additionally, adamantane-derived bananins, natural flavonoids, one acrylamide derivative [(E)-3-(furan-2-yl)-N-(4-sulfamoylphenyl)acrylamide], a purine derivative (7-ethyl-8-mercapto-3-methyl-3,7-dihydro-1 H-purine-2,6-dione), and a few bismuth complexes. The IC50of patented inhibitors ranges between 0.82 μM and 8.95 μM, depending upon the assays used. Considering the urgency of clinical interventions against Coronavirus Disease-19 (COVID-19), it is important to consider developing antiviral portfolios consisting of small molecules.Areas covered: This review examines coronavirus helicases as antiviral targets, and the potential of previously patented and experimental compounds to inhibit the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) helicase.Expert opinion: Small molecule coronavirus helicase inhibitors represent attractive pharmacological modalities for the treatment of coronaviruses such as SARS-CoV and SARS-CoV-2. Rightfully so, the current emphasis is focused upon the development of vaccines. However, vaccines may not work for everyone and broad-based adoption of vaccinations is an increasingly challenging societal endeavor. Therefore, it is important to develop additional pharmacological antivirals against the highly conserved coronavirus helicases to broadly protect against this and subsequent coronavirus epidemics.

Emerging and re-emerging infectious diseases due to RNA viruses cause major negative consequences for the quality of life, public health, and overall economic development. Most of the RNA viruses causing illnesses in humans are of zoonotic origin. Zoonotic viruses can directly be transferred from animals to humans through adaptation, followed by human-to-human transmission, such as in human immunodeficiency virus (HIV), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and, more recently, SARS coronavirus 2 (SARS-CoV-2), or they can be transferred through insects or vectors, as in the case of Crimean-Congo hemorrhagic fever virus (CCHFV), Zika virus (ZIKV), and dengue virus (DENV). At the present, there are no vaccines or antiviral compounds against most of these viruses. Because proteins possess a vast array of functions in all known biological systems, proteomics-based strategies can provide important insights into the investigation of disease pathogenesis and the identification of promising antiviral drug targets during an epidemic or pandemic. Mass spectrometry technology has provided the capacity required for the precise identification and the sensitive and high-throughput analysis of proteins on a large scale and has contributed greatly to unravelling key protein–protein interactions, discovering signaling networks, and understanding disease mechanisms. In this Review, we present an account of quantitative proteomics and its application in some prominent recent examples of emerging and re-emerging RNA virus diseases like HIV-1, CCHFV, ZIKV, and DENV, with more detail with respect to coronaviruses (MERS-CoV and SARS-CoV) as well as the recent SARS-CoV-2 pandemic.

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People with HIV-1 in low-income and middle-income countries increasingly need second-line regimens with boosted protease inhibitors. However, data are scarce for treatment response in patients with HIV-1 subtype C (HIV-1C), which is predominant in these regions. We aimed to examine factors associated with virological failure in patients in a standardised national health-care setting.

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The clinical outcome and disease severity in coronavirus disease 2019 (COVID-19) are heterogeneous, and the progression or fatality of the disease cannot be explained by a single factor like age or comorbidities. In this study, we used system-wide network-based system biology analysis using whole blood RNA sequencing, immunophenotyping by flow cytometry, plasma metabolomics, and single-cell-type metabolomics of monocytes to identify the potential determinants of COVID-19 severity at personalized and group levels. Digital cell quantification and immunophenotyping of the mononuclear phagocytes indicated a substantial role in coordinating the immune cells that mediate COVID-19 severity. Stratum-specific and personalized genome-scale metabolic modeling indicated monocarboxylate transporter family genes (e.g., SLC16A6), nucleoside transporter genes (e.g., SLC29A1), and metabolites such as α-ketoglutarate, succinate, malate, and butyrate could play a crucial role in COVID-19 severity. Metabolic perturbations targeting the central metabolic pathway (TCA cycle) can be an alternate treatment strategy in severe COVID-19.

Mucosal-associated invariant T (MAIT) cells are an evolutionarily conserved antimicrobial MR1-restricted T-cell subset. MAIT cells are CD161+, express a Vα7.2 TCR, are primarily CD8+ and numerous in blood and mucosal tissues. However, their role in HIV-1 infection is unknown. In this study, we found levels of MAIT cells to be severely reduced in circulation in patients with chronic HIV-1 infection. Residual MAIT cells were highly activated and functionally exhausted. Their decline was associated with time since diagnosis, activation levels, and the concomitant expansion of a subset of functionally impaired CD161− Vα7.2+ T cells. Such cells were generated in vitro by exposure of MAIT cells to Escherichia coli. Notably, whereas the function of residual MAIT cells was at least partly restored by effective antiretroviral therapy, levels of MAIT cells in peripheral blood were not restored. Interestingly, MAIT cells in rectal mucosa were relatively preserved, although some of the changes seen in blood were recapitulated in the mucosa. These findings are consistent with a model in which the MAIT-cell compartment, possibly as a result of persistent exposure to microbial material, is engaged, activated, exhausted, and progressively and persistently depleted during chronic HIV-1 infection.

Mucosal-associated invariant T (MAIT) cells are an evolutionarily conserved antimicrobial MR1-restricted T-cell subset. MAIT cells are CD161+, express a Vα7.2 TCR, are primarily CD8+and numerous in blood and mucosal tissues. However, their role in HIV-1 infection is unknown. In this study, we found levels of MAIT cells to be severely reduced in circulation in patients with chronic HIV-1 infection. Residual MAIT cells were highly activated and functionally exhausted. Their decline was associated with time since diagnosis, activation levels, and the concomitant expansion of a subset of functionally impaired CD161−Vα7.2+T cells. Such cells were generated in vitro by exposure of MAIT cells to Escherichia coli. Notably, whereas the function of residual MAIT cells was at least partly restored by effective antiretroviral therapy, levels of MAIT cells in peripheral blood were not restored. Interestingly, MAIT cells in rectal mucosa were relatively preserved, although some of the changes seen in blood were recapitulated in the mucosa. These findings are consistent with a model in which the MAIT-cell compartment, possibly as a result of persistent exposure to microbial material, is engaged, activated, exhausted, and progressively and persistently depleted during chronic HIV-1 infection.

Background The extreme flexibility of the HIV type-1 (HIV-1) genome makes it challenging to build the ideal antiretroviral treatment regimen. Interpretation of HIV-1 genotypic drug resistance is evolving from rule-based systems guided by expert opinion to data-driven engines developed through machine learning methods.Methods The aim of the study was to investigate linear and non-linear statistical learning models for classifying short-term virological outcome of antiretroviral treatment. To optimize the model, different feature selection methods were considered. Robust extra-sample error estimation and different loss functions were used to assess model performance. The results were compared with widely used rule-based genotypic interpretation systems (Stanford HIVdb, Rega and ANRS).Results A set of 3,143 treatment change episodes were extracted from the EuResist database. The dataset included patient demographics, treatment history and viral genotypes. A logistic regression model using high order interaction variables performed better than rule-based genotypic interpretation systems (accuracy 75.63% versus 71.74–73.89%, area under the receiver operating characteristic curve [AUC] 0.76 versus 0.68–0.70) and was equivalent to a random forest model (accuracy 76.16%, AUC 0.77). However, when rule-based genotypic interpretation systems were coupled with additional patient attributes, and the combination was provided as input to the logistic regression model, the performance increased significantly, becoming comparable to the fully data-driven methods.Conclusions Patient-derived supplementary features significantly improved the accuracy of the prediction of response to treatment, both with rule-based and data-driven interpretation systems. Fully data-driven models derived from large-scale data sources show promise as antiretroviral treatment decision support tools.

Background Inferring response to antiretroviral therapy from the viral genotype alone is challenging. The utility of an intermediate step of predicting in vitrodrug susceptibility is currently controversial. Here, we provide a retrospective comparison of approaches using either genotype or predicted phenotypes alone, or in combination.Methods Treatment change episodes were extracted from two large databases from the USA (Stanford-California) and Europe (EuResistDB) comprising data from 6,706 and 13,811 patients, respectively. Response to antiretroviral treatment was dichotomized according to two definitions. Using the viral sequence and the treatment regimen as input, three expert algorithms (ANRS, Rega and HIVdb) were used to generate genotype-based encodings and VircoTYPE™ 4.0 (Virco BVBA, Mechelen, Belgium) was used to generate a predicted phenotype-based encoding. Single drug classifications were combined into a treatment score via simple summation and statistical learning using random forests. Classification performance was studied on Stanford- California data using cross-validation and, in addition, on the independent EuResistDB data.Results In all experiments, predicted phenotype was among the most sensitive approaches. Combining single drug classifications by statistical learning was significantly superior to unweighted summation (P<2.2x10-16). Classification performance could be increased further by combining predicted phenotypes and expert encodings but not by combinations of expert encodings alone. These results were confirmed on an independent test set comprising data solely from EuResistDB.Conclusions This study demonstrates consistent performance advantages in utilizing predicted phenotype in most scenarios over methods based on genotype alone in inferring virological response. Moreover, all approaches under study benefit significantly from statistical learning for merging single drug classifications into treatment scores.

Although associations between older antiretroviral drug classes and cardiovascular disease in people living with HIV are well described, there is a paucity of data regarding a possible association with integrase strand-transfer inhibitors (INSTIs). We investigated whether exposure to INSTIs was associated with an increased incidence of cardiovascular disease.

Abstract: Objective: The aim of this study was to compare the level of adherence and motivation in two independent cross-sectional samples of HIV-infected patients conducted in 1998 and 2002, and to investigate the relationship between adherence and motivation.Method: Consecutive HIV-infected patients on treatment at a Swedish clinic were asked to complete an anonymous questionnaire. In 1998, 60 patients participated and in 2002, 53 participated. In 2002, the 9-item Morisky Medication Adherence Scale (MMAS) was added to the questionnaire set.Main outcome measure: Self-reported adherence and motivation.Results: In 1998, 28.1% of the respondents were considered adherent, while the corresponding proportion was 57.4% in 2002 (P = 0.002). The mean summary score for MMAS was 10.7 in 2002 (13 = perfect adherence). The proportion considered motivated were 22.4% in the 1998 survey and 41.3% in 2002 (P = 0.038). Of the respondents considered motivated in the 2002 survey, 46.7% scored the maximum summary score on the MMAS, while 8.7% of the non-motivated respondents did so (P = 0.016).Conclusion: The respondents in 2002 were more adherent and motivated than the respondents in 1998 and a relationship between motivation and adherence was found. The difference in adherence and motivation might be due to a new treatment model at the clinic.

The non-nucleoside reverse transcriptase inhibitor efavirenz is mainly metabolised by the polymorphic cytochrome P450 enzyme CYP2B6. Genomic DNA from four subjects in a group of 51 patients being treated with efavirenz and having surprisingly high plasma concentrations were screened by direct sequencing for mutations in the CYP2B6gene. Four exonic single nucleotide polymorphisms (SNPs), 516G>T, 714G>A, 785A>G and 983T>C, and eight intronic SNPs were identified. Haplotype analysis revealed that 983T>C was linked with 785A>G defining a novel allele, CYP2B616.This allele was present in totally five of the patients. The CYP2B6.16 cDNA was expressed in yeast and HEK293 cells and significantly less protein was formed compared to the wild-type cDNA, in both heterologous systems. By contrast, the catalytic activity of the enzyme variant was not different from the CYP2B6.1 enzyme, using bupropion as a probe substrate. The CYP2B616allele was not found in Swedes, was present at 4 frequency among Turks, but was common among Africans. The steady-state level of efavirenz was significantly higher in the five carriers of CYP2B616, being of African origin, compared to the other patients. Higher efavirenz concentrations were also seen in carriers of 516G>T (CYP2B66and CYP2B69). In conclusion, a novel CYP2B616allele causing less expression of the corresponding enzyme was identified and found to influence the metabolism of efavirenz in vivo, a finding that is of potential impact for anti-HIV therapy in black populations.

The antiviral response of CD8 T cells involves the differentiation of naive T cells into distinct types of effector and memory cells, which may be distinguished by the level of CD7 expression. We have investigated CD8 T cells in adults and children infected with HIV-1 to determine the disease relevance of cell subsets defined by CD7. CD8 T cells from patients infected with HIV-1 displayed profound down-modulation of CD7 expression as compared with healthy subjects, with expansion of both CD7low and CD7negative effector subsets. Loss of CD7high cells correlated directly with HIV-1 load and was particularly pronounced in patients with rapid disease progression. CD8 T cells specific for HIV-1, as well as Epstein-Barr virus (EBV) and cytomegalovirus (CMV) were predominantly found in the CD7low effector cell subset. Furthermore, recovery of CD4 counts on antiretroviral therapy was associated with reversion of the skewed CD7 profile in CD8 T cells. Thus, effector CD8 T-cell subsets distinguished by lowered CD7 expression expand in a manner that correlates with the magnitude of HIV-1, EBV, and CMV antigenic challenge and contract in response to successful antiretroviral treatment. The results are discussed in relation to the dual roles of CD7 as a receptor of both costimulation and cell death.

The antiviral response of CD8 T cells involves the differentiation of naive T cells into distinct types of effector and memory cells, which may be distinguished by the level of CD7 expression. We have investigated CD8 T cells in adults and children infected with HIV-1 to determine the disease relevance of cell subsets defined by CD7. CD8 T cells from patients infected with HIV-1 displayed profound down-modulation of CD7 expression as compared with healthy subjects, with expansion of both CD7lowand CD7negativeeffector subsets. Loss of CD7highcells correlated directly with HIV-1 load and was particularly pronounced in patients with rapid disease progression. CD8 T cells specific for HIV-1, as well as Epstein-Barr virus (EBV) and cytomegalovirus (CMV) were predominantly found in the CD7loweffector cell subset. Furthermore, recovery of CD4 counts on antiretroviral therapy was associated with reversion of the skewed CD7 profile in CD8 T cells. Thus, effector CD8 T-cell subsets distinguished by lowered CD7 expression expand in a manner that correlates with the magnitude of HIV-1, EBV, and CMV antigenic challenge and contract in response to successful antiretroviral treatment. The results are discussed in relation to the dual roles of CD7 as a receptor of both costimulation and cell death.

Efavirenz is a drug subject to extensive metabolism, mainly by the cytochrome P-450 isoenzyme CYP2B6, known to exhibit extensive interindividual variability. The aim of the present study was 2-fold to investigate the relationship between plasma concentration and clinical effects of efavirenz and to investigate the extent of the inter- and intraindividual variability of the plasma concentration measurements. From an open clinic, 68 HIV-positive patients on efavirenz-containing treatment were recruited. From each patient 1 to 5 samples were collected; 43 had more than 1 sample taken. Most samples were taken 10–24 hours after the latest dose. Efavirenz was analyzed by high-performance liquid chromatography with UV detection. The data were analyzed by the variance component model analysis of variance. Efavirenz concentrations were reproducible, and intraindividual variability constituted only 16 of the total variance. Thus, 84 of the variance was attributed to interindividual variability. The incidence of primary treatment failure was related to low plasma concentrations with a geometric mean concentration of 6.1 μmol/L compared with 8.7 μmol/L in those responding to therapy (P< 0.05). If a cutoff of 7 μmol/L is used, 10 of 13 failing to respond were below this level compared with 15 of 45 in those responding. It is concluded that efavirenz plasma concentration measurement gives reproducible results predictive of primary treatment failure. A lower bound for the therapeutic level of 7 μmol/L is proposed, and data from other authors suggests that an upper level of 13 μmol/L may be applied.

Hypergammaglobulinemia and defective humoral immunity are hallmarks of HIV-1 infection. Naive B cells have been recently suggested as the major source of hypergammaglobulinemia in chronic viral infections. We recently reported that HIV-1–infected patients carry low levels of memory B cells. Here we studied whether defects in the naive and memory B cells in HIV-1–infected patients translated into hypergammaglobulinemia and defective humoral immunity against specific antigens. Naive B cells from HIV-1–infected patients exhibited abnormal expression of the activation/differentiation markers CD70 and leukocyte-associated Ig-like receptor (LAIR-1). Activated naive B cells from patients showed a significant increase in the intracellular immunoglobulin G (IgG) content ex vivo and this activated phenotype correlated to hypergammaglobulinemia and to the ability of naive B cells from patients to secrete IgG in vitro. We analyzed the levels of antibodies to tetanus toxoid, measles, and HIV-1 in relation to memory B cells and observed a significant reduction of antigen-specific antibodies in patients with low-memory B lymphocytes. Nevertheless, hypergammaglobulinemia and levels of polyspecific self-reactive antibodies were comparable in patients with normal and low memory B cells. We conclude that reduction of memory B lymphocytes in HIV-1 infection correlates with defective humoral immunity and that hyperactivated naive B cells may represent the source of abnormal IgG production in HIV-1 infection. Our results may be relevant to the design of HIV-1 therapeutical vaccines and to the clinical management of HIV-1–infected patients.

Hypergammaglobulinemia and defective humoral immunity are hallmarks of HIV-1 infection. Naive B cells have been recently suggested as the major source of hypergammaglobulinemia in chronic viral infections. We recently reported that HIV-1–infected patients carry low levels of memory B cells. Here we studied whether defects in the naive and memory B cells in HIV-1–infected patients translated into hypergammaglobulinemia and defective humoral immunity against specific antigens. Naive B cells from HIV-1–infected patients exhibited abnormal expression of the activation/differentiation markers CD70 and leukocyte-associated Ig-like receptor (LAIR-1). Activated naive B cells from patients showed a significant increase in the intracellular immunoglobulin G (IgG) content ex vivo and this activated phenotype correlated to hypergammaglobulinemia and to the ability of naive B cells from patients to secrete IgG in vitro. We analyzed the levels of antibodies to tetanus toxoid, measles, and HIV-1 in relation to memory B cells and observed a significant reduction of antigen-specific antibodies in patients with low-memory B lymphocytes. Nevertheless, hypergammaglobulinemia and levels of polyspecific self-reactive antibodies were comparable in patients with normal and low memory B cells. We conclude that reduction of memory B lymphocytes in HIV-1 infection correlates with defective humoral immunity and that hyperactivated naive B cells may represent the source of abnormal IgG production in HIV-1 infection. Our results may be relevant to the design of HIV-1 therapeutical vaccines and to the clinical management of HIV-1–infected patients.

To investigate whether it is appropriate to assume comparability of hepatitis virus C (HCV)‐RNA results across laboratories in multi‐centre studies, nine laboratories of the European Paediatric HCV Network participated in an international proficiency study of HCV‐RNA assays. A panel of 12 samples of different dilutions and genotypes was sent to each laboratory and tested with qualitative and/or quantitative HCV‐RNA assays according to local procedures. Commercial assays were used in seven laboratories and in‐house assays in two. All six laboratories in which a commercial qualitative assay was used were proficient, as were four of six runs (in five laboratories) in which a commercial quantitative assay was used. The proficiency of the laboratories where in‐house assays were used could not be assessed according to the VQC definition because of differences in the methods used. Overall, there were several false‐negative results, but only one false‐positive result with a quantitative assay and none with a qualitative assay. The false‐negative results may have implications for the diagnosis of infection, and highlight the need for an antibody test to be performed at 18 months to confirm the absence of infection. The results of qualitative assays were generally consistent across laboratories but it was difficult to evaluate and compare the results of quantitative assays. Multivariate analysis of data collected in multi‐centre studies should therefore allow for centre and/or assay used. J. Med. Virol. 69:195–201, 2003. © 2003 Wiley‐Liss, Inc.

To investigate whether it is appropriate to assume comparability of hepatitis virus C (HCV)-RNA results across laboratories in multi-centre studies, nine laboratories of the European Paediatric HCV Network participated in an international proficiency study of HCV-RNA assays. A panel of 12 samples of different dilutions and genotypes was sent to each laboratory and tested with qualitative and/or quantitative HCV-RNA assays according to local procedures. Commercial assays were used in seven laboratories and in-house assays in two. All six laboratories in which a commercial qualitative assay was used were proficient, as were four of six runs (in five laboratories) in which a commercial quantitative assay was used. The proficiency of the laboratories where in-house assays were used could not be assessed according to the VQC definition because of differences in the methods used. Overall, there were several false-negative results, but only one false-positive result with a quantitative assay and none with a qualitative assay. The false-negative results may have implications for the diagnosis of infection, and highlight the need for an antibody test to be performed at 18 months to confirm the absence of infection. The results of qualitative assays were generally consistent across laboratories but it was difficult to evaluate and compare the results of quantitative assays. Multivariate analysis of data collected in multi-centre studies should therefore allow for centre and/or assay used. J. Med. Virol. 69:195–201, 2003. © 2003 Wiley-Liss, Inc.

The prognosis of HIV-1-infected patients has dramatically improved but progression to liver failure occurs now frequently in subjects coinfected with hepatitis C virus (HCV). This has raised the issue of organ transplantation, but the knowledge about the effect of concomitant antiretroviral and immunosuppressive therapy is limited. The objective of the study was to describe viral and immunological events in antiretroviral-treated orthotopic liver transplant (OLT) recipients with primary (PHI) or chronic HIV-1 infection. Three HIV-1-infected patients with liver cirrhosis due to chronic HCV infection underwent OLT. A fourth patient developed PHI at OLT. Immunosuppressive drugs and combination antiretroviral therapy were given. The effects on HIV-1 load, viral diversity and divergence, and CD4+ T cell counts, were studied. One patient died after 3 months. Three subjects were alive after 9 months, 14 months, and 3 years, respectively. In the PHI patient, viral load decreased during the second week of illness despite immunosuppression. During the third week the viremia increased until antiretroviral treatment was initiated. In all four patients, the HIV-1 replication was effectively inhibited during follow-up by the treatment, as determined by undetectable plasma viremia, lack of viral sequence changes, and increase in CD4+ T cells. The pattern of viral dynamics may suggest that the innate immunity causes the earliest decline of viral load in PHI patients. A lack of adaptive immunity may thereafter lead to an increase in viremia in heavily immunosuppressed individuals. However, a specific HIV-1 immunity is not necessary to efficiently inhibit the viral replication when potent antiretroviral therapy is given in liver transplant recipients with primary or chronic HIV-1 infection.

Fifteen to 50% of AIDS-patients suffer from distal predominantly sensory neuropathy (DSP), which is commonly associated with painful symptoms. In the present study, we have focused on the function of fine calibre nerve channels, in 36 consecutive HIV-1-infected patients with painful (PPN) ( n=20; 54%) and non-painful (PN) ( n=16) sensory neuropathy, assessed by clinical, quantitative thermal testing (QTT) (31/36), and peripheral nerve conduction examination (32/36). Control QTT data were obtained from 49 healthy subjects with a corresponding age- and sex distribution. Demographics, antiviral treatment, immunological status, and nerve conduction examination did not differ between patients with and without painful symptoms. Hypoaesthesia to warmth, cold, and heat pain was observed in both neuropathy groups when compared to healthy controls. However, the perception threshold to warmth was more often impaired ( p<0.01) and the level of impairment was more pronounced ( p<0.001) in patients with painful neuropathy. Furthermore, increased pain sensitivity to cold was found only in patients with painful symptoms ( p<0.05). An abnormal outcome of any QTT parameter was found in all patients with pain, but only among 62% of patients without pain, p<0.01, and the cumulative frequency of abnormalities in any of the four thermal percepts (warmth, cold, heat pain, and cold pain) was higher in patients with painful symptoms, p<0.0001. This study demonstrates a more pronounced impairment of C-fibre-mediated innocuous warm perception in patients with painful neuropathy, which in the setting of impaired or absent heat pain perception suggests a more generalised loss of function in somatosensory C-fibre channels.

The objective was to study the persistence of drug resistance mutations detected earlier at virological failure during second or third line antiretroviral therapy. Therefore, in HIV‐1 infected patients, with a virological treatment failure, genotypic resistance testing was carried out before change of therapy and at the next treatment failure. The majority of primary and secondary resistance mutations persisted in both the reverse transcriptase (RT) and the protease genes. After changing from zidovudine‐ to stavudine‐containing regimens, the thymidine analogue mutations (especially M41L and T215Y/F) were found at new treatment failure in almost all patients. The M184V mutation disappeared in most (64%) non‐3TC treated patients, although it persisted in a few didanosine‐ and abacavir‐treated subjects. The primary protease inhibitor (PI) mutations reverted back to wild type in most patients who did not receive a new PI. In contrast, after changing from indinavir to saquinavir or nelfinavir, the M46I/L and/or V82A/F/ST disappeared in only 9 of 21 occasions at the new treatment failure. Most secondary mutations persisted with the exception of N88D. In patients with multiple treatment failures, most NRTI mutations thus persist frequently at new failures with modified treatment. A similar pattern is seen for protease inhibitors. The data suggest that clinical cross‐resistance may develop via common pathways within all categories of drugs in heavily treated patients. J. Med. Virol. 68:473–478, 2002. © 2002 Wiley‐Liss, Inc.

A cluster of HIV-1 infection has been identified in Libya in 1999, involving 402 children admitted to "ElFath" Children's Hospital in Benghazi (BCH) during 1998 and 19 of their mothers. Nosocomial transmission has been indicated as responsible for the spread of infection. Out of this group, 104 children and 19 adult women have been followed at the National Institute for Infectious Diseases L. Spallanzani in Rome during 1 year. At BCH, all children had received intravenous infusions but not blood or blood products. A single child receiving a blood transfusion in 1997 and the 17 infected mothers were never hospitalized in Benghazi. In addition, two nurses were diagnosed as HIV-1 infected. In 40 subjects out of this group HIV-1 gag, env, and pol fragments were amplified and sequenced. The phylogenetic analyses showed that a monophyletic recombinant HIV-1 form CRF02-AG was infecting all of the HIV-1-seropositive patients admitted at BCH with no close similarities to the other CRF02-AG reported to GenBank. A different strain was found in the child infected by blood transfusion. The data thus suggest a highly contagious nosocomial spread of HIV-1 infection and possibly transmission of the virus from child to mother during breastfeeding in connection with primary HIV-1 infection.

We have tested for combined anti-HIV-1 effects of a hammerhead ribozyme and antiretroviral drugs and the possibility of reducing the drug burden of patients on highly active antiretroviral therapy (HAART). The antiretroviral compounds used represent the three groups in HAART: nucleoside analogue reverse-transcriptase inhibitors, nonnucleoside reverse-transcriptase inhibitors, and protease inhibitors. A human T cell line (HUT78), stably expressing a hammerhead ribozyme targeted to nef (hhRz.nef9016–9029), was infected with HIV-1SF2in the presence of a single drug. The combined effects on HIV-1 replication were measured by p24 antigen determinations over a 2-week period. In the presence of the ribozyme, smaller amounts of antiretroviral drugs were required to reduce the HIV-1 p24 levels equally as much as when only drugs were present. The results support a strategy of combining ribozyme gene therapy with HAART to improve the long-term outcome of anti-HIV-1 therapy.Molecular Therapy (2001) 3, 531–535; doi: 10.1006/mthe.2001.0286

A latent pool of HIV-1 is established early in memory CD4+ T lymphocytes and persists during antiretroviral therapy. Also, viral replication may continue in subjects despite undetectable viremia. However, it remains unclear whether this residual replication results in any significant sequence evolution. We were therefore interested in studying the viral evolution and HIV-1 DNA dynamics in subjects with primary infection receiving or not receiving early potent antiretroviral therapy. In 16 subjects, HIV-1 DNA load was monitored from 1 to 23 days, up to 1253 days, after onset of symptoms. Extensive sequential cloning and sequence analysis of the V3 region was performed in four subjects. In the treated subjects a continuous decline in the proviral load was found, corresponding to a half-life of about 6 months. As expected in newly infected individuals the founder virus populations showed high intrasubject sequence similarity. Also, a limited increase in the viral divergence was detected during the first 6 months in three treated subjects. Thereafter, no significant sequence changes were found despite analysis of a large number of clones. Our data thus suggest that early and successful therapy in compliant subjects with primary HIV-1 infection results in a highly restricted viral evolution and a decline in the proviral load close to the decay rate of human memory T lymphocytes.

A method for analysis of indinavir in serum, cerebrospinal fluid, and urine was developed. The method is based on liquid–liquid extraction followed by high performance liquid chromatography with UV detection. The method has a shorter analysis time than previously published methods, and it is sensitive enough to measure levels in all three fluids under routine clinical conditions. The method is linear up to 32 mol/L, the limit of detection is 0.01 mol/L, and recovery of the method is 86. The interassay coefficient of variation at 2.0 mol/L was 2.8, and no internal standard is needed. Over 700 clinical samples have been analyzed by this method, and concomitant antiviral drugs do not interfere with the assay. Paroxetin and dipyridamol are the only two compounds encountered to elute with retention times similar to that of indinavir. Examples of chromatograms and a pharmacokinetic curve are given. The method is well suited for routine therapeutic drug monitoring as well as for pharmacokinetic studies for research purposes.

Plasma levels of soluble Fas (sFas) are elevated in human immunodeficiency virus type 1 (HIV-1) infection, indicating dysregulation of the Fas apoptosis pathway and chronic immune activation. We performed a retrospective study to investigate the effects of HAART on plasma levels of sFas. A cross-sectional study of 27 drug-naive infected subjects and 49 patients under antiretroviral treatment showed that plasma levels of sFas were higher in HIV-1-infected subjects than in 52 HIV-1-negative controls, independently of the treatment status. In a longitudinal study of 69 patients undergoing HAART, we observed a minimal, but significant decrease in sFas plasma levels after 1 year of therapy. Levels of sFas, however, remained still higher than physiologic values. Patients undergoing HAART were further classified as nonresponders or responders on the basis of viremia suppression; no significant changes in plasma levels of sFas were observed between the two groups. These findings show that 1 year of HAART has a minor effect on the sFas levels in plasma. Long-term HAART may be required to normalize the dysregulation of the Fas apoptotic pathway and the persistent immune activation initiated by HIV-1.

To evaluate thermal and nociceptive function in a prospective, longitudinal study of 49 consecutive HIV-1–infected patients before and at 1, 4, and 8 months after initiation of highly active antiretroviral therapy.

Background & Aims:The extremely high rate of chronicity to hepatitis C virus (HVC) infection suggests an inefficient immune response. The humoral immune response to HCV was evaluated in 60 patients with chronic HCV infection and in 12 patients acutely infected with HCV. Methods:A number of recombinant HCV antigens including the core, envelope 2 (E2), nonstructural (NS) 3, NS4, and NS5 proteins, and NS4a and E2-HVR-1 peptides were used in enzyme-linked immunoassays. Results:Immunoglobulin (Ig) G antibody responses to these viral antigens, except for the HCV core, were highly restricted to the IgG1 isotype. The prevalence of antibodies of the IgG1 isotype specific for the HCV core, E2, E2-HVR1, NS3 (helicase domain), NS4, and NS5 antigens was 97%, 98%, 28%, 88%, 33%, and 68%, respectively. Antibodies of the IgG3 isotype specific for E2, E2-HVR-1, NS3, NS4, and NS5 were detected in a minority of serum samples. The IgG2 and IgG4 isotypes were rarely if ever detected. Furthermore, antibody responses to HCV viral antigens were of relatively low titer and, with the exception of anti-HCV core, were delayed in appearance until the chronic phase of infection. Conclusions:The IgG1 restriction, low titer, and delayed appearance of antibody responses elicited during HCV infection suggest that the immunogenicity of HCV proteins is limited in the context of natural infection. Inasmuch as recombinant HCV viral antigens perform as relatively normal immunogens in small animals, we suggest that the defective humoral immune responses during HCV infection may be attributable to an “immune avoidance” strategy.

Chemokines are pro-inflammatory cytokines that inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro. We studied the kinetics of the ß-chemokines, macrophage inhibitory protein (MIP)-1a, MIP-1ß, RANTES, and monocyte chemotactic protein (MCP)-1 in plasma during 12 months of antiretroviral therapy in 26 HIV-1-infected patients and in 11 untreated subjects. Eleven patients with moderate immunodeficiency had HIV-1 RNA levels <50 copies/ml after 1 year, whereas 12 out of 15 patients with severe immunodeficiency had detectable virus. At baseline, MCP-1 levels correlated positively with HIV-1 RNA and DNA levels and inversely with CD4 cell counts. A reverse pattern was seen for the MIP-1ß levels. No correlation was seen between MIP-1a or RANTES and any of the parameters. Also, there was a dichotomy between the different ß-chemokines in response to therapy. Decreases of MCP-1 and RANTES levels were found, but no durable changes of MIP-1a and MIP-1ß. The MCP-1 levels rebounded back to baseline after 1 year in the patients who responded virologically, which could possibly reflect an increased immune activation. The biological consequences of the changes in ß-chemokines levels during antiretroviral treatment are still unknown and deserve further studies.

The prevalence of pain in 211 HIV-infected patients with and without intravenous drug use was assessed and the prognostic information inherent in pain reporting was evaluated, using a questionnaire on pain and HIV-related symptoms combined with data on disease classification, route of HIV transmission, CD4+ lymphocyte counts in blood (CD4) and mortality rates at 15 months after completing the questionnaire. The pain prevalence was significantly higher among intravenous drug users (IDUs) compared with non-IDUs [76/89 (85%) vs87/122 (71%);p<0.05], especially among the patients classified as asymptomatic [43/53 (81%) vs35/59 (59%);p= 0.01]. No significant difference was found among AIDS patients. In non-IDUs, a strong correlation was found between HIV disease stages according to the Centers for Disease Control classification (CDC) and pain prevalence (CDC A: 59%vsB: 74%vsC: 96%, p<0.001), and between the number of concurrent pain sites and both the CD4 levels (no pains: CD4 0.26 × 109/l vs1–2 pain sites: CD4 0.22 vs>2 pain sites: CD4 0.09;r= 0.35, p<0.001), and the mortality rate [no pains: 2/35 (6%) vs1–2 pain sites: 8/45 (18%) vs> 2 pain sites: 12/42 (29%), p<0.01]. In IDUs, no such correlations were found. Our data demonstrates differences in the development, prevalence and prognostic value of pain among HIV-infected patients, with and without intravenous drug use, clearly indicating the need to differentiate risk groups in pain related studies.

Enhanced rates of programmed cell death (apoptosis) have been detected in T cells and B cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals. To evaluate the possible relevance of this event to HIV pathogenesis and disease progression, apoptosis in CD4+T lymphocytes and CD19+B lymphocytes, viral load, and neutralizing antibody titers were assayed in HIV-1-infected slow progressors and progressors. A correlation was found between progressive disease and apoptosis of CD4+T cells. The extent of apoptosis in CD4+cells was similar in slow progressors and seronegative control subjects. By contrast, we found elevated levels of B-cell apoptosis in all HIV-1-infected individuals compared with seronegative control subjects, with a tendency toward increased levels of apoptosis with progressive disease. Apoptosis in CD4+T cells and CD19+B cells correlated with viral RNA levels in plasma. Furthermore, higher rates of B-cell apoptosis were observed in individuals with poor neutralizing activity against a panel of six clinical HIV-1 isolates. From these results we conclude that the extent of apoptosis in cultured CD4+cells and CD19+cells appears to parallel the decline in CD4 cell counts in infected individuals. The finding of a relation between apoptosis in B cells and poor neutralizing capacity suggests that apoptosis may be related to loss of immune function. A role for apoptosis in the pathogenesis of AIDS is also supported by the strong correlation between viral load and rates of apoptosis in CD4+T cells.

A region of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 has been claimed previously to be homologous to parts of snake venom neurotoxins and rabies virus glycoprotein (“the neurotoxic loop”). We have determined DNA sequences directly from a polymerase chain reaction amplified fragment corresponding to this region of HIV-1 gp120 and have translated these to protein sequences. This was performed with the prototype HIVSF2 isolate and several Swedish HIV-1 strains, which were precultivated from blood cells or cerebrospinal fluid (CSF) or were directly obtained from CSF cells of patients with and without neurological symptoms. The results show that there are sequence similarities between a short segment of gp120 of clinical HIV-1 strains and the neurotoxic loop. The strains of patients with neurological symptoms did not, however, show a genetic shift of their sequences towards a greater similarity to the sequences of snake venom neurotoxins and rabies virus glycoprotein as compared to the strains of asymptomatic individuals.

Phylogenetic tree analysis was performed on selected polymerase chain reaction (PCR)-amplified and sequenced regions of the gag and env reading frames of several Ethiopian and Swedish human immunodeficiency virus type 1 (HIV-1) strains. These regions are considered to be conserved parts of the HIV-1 genome and correspond to the p7 of the core (gag) and part of the carboxy terminal of the gp41 protein of env respectively. Comparisons were made with all available HIV-1 sequences.

The putative interferon sensitivity determining region (ISDR) in the NS5a region of the hepatitis C virus (HCV) was analyzed in 13 interferon alpha (IFN‐α) treated patients representing genotypes 1a, 1b, and 2b. These patients had previously been followed longitudinally during treatment with respect to viral load and to virus heterogeneity using the hypervariable region 1 (HVR1) sequence as a marker. In the present study, the NS5a region was analyzed for nonresponders and sustained responders using direct DNA sequencing. While the previous results of analyzing viral composition and load showed evidence of selection, no corresponding selection of specific NS5a ISDR sequences was observed in the nonresponders, and identical ISDR sequences were observed among both sustained responders and nonresponders. Thus, we cannot verify a correlation between ISDR sequence and the observed selection of IFN‐α‐resistant quasispecies demonstrated as a restriction of HVR1 heterogeneity. This indicates that the potential for using ISDR as a diagnostic or prognostic marker during IFN‐α treatment is limited. J. Med. Virol. 56:33–38, 1998. © 1998 Wiley‐Liss, Inc.

A retrospective study of hepatitis C virus (HCV) transmission by transfusion was conducted in Örebro county. Out of the 7900 active, registered blood donors, 21 repeatedly anti-HCV reactive (RIBA 2 positive or indeterminate) donors were diagnosed. Their 84 recipients from January 1990 through June 1992 were identified and 41 (49%) were alive in December 1992. A total of 13 anti-HCV reactive (RIBA 2 positive or indeterminate) were diagnosed in 39 investigated recipients. Of these 11 were previously undiagnosed, and seven were HCV RNA-positive. In the donor population 1.03% were anti-HCV-positive by ELISA, but only 0.09% were RIBA and HCV RNA-positive. In 1990, 0.06% of the blood components came from the HCV RNA-positive donors, and none during the first 6 months of 1992. In order to identify transfusion-transmitted HCV infections that took place before the introduction of tests for anti-HCV antibodies, patients with a history of transfusion and symptoms and signs of liver dysfunction or damage should be thoroughly tested.

A total of 42 Swedish patients with biopsy-proven chronic hepatitis C virus (HCV) infection were treated with a natural human leucocyte alpha-interferon (HuIFN-alpha-Le), Alfanative® (BioNative AB, Umeå, Sweden) in an open uncontrolled study. Two patients were withdrawn from treatment within 2 weeks due to non-compliance and were omitted from further analysis, and 40 patients (17 females), mean age 39 years (range 24-71) completed the study. All patients were HCV RNA-positive in serum prior to treatment, with raised alanine aminotransferase (ALT) levels >1.5 times the upper normal limit known for more than 6 months. Interferon was given at a dose of 3 MU t.i.w. for an intended 24 weeks and follow-up was a further 24 weeks after treatment. Biochemical non-responders were withdrawn from treatment within 12-16 weeks but continued follow-up. Overall 21/40 (52.5%) patients had a complete biochemical response with normal ALT levels at the end of treatment. Sustained response during follow-up was seen in 8 (20%) whereas 13 (32.5%) had a non-sustained response. At the end of treatment 23 (58%) patients had undetectable serum HCV RNA and 9 (23%) at follow-up. Patients with sustained, non-sustained and non-response had a mean pretreatment HCV RNA level of 3.2 × 105, 2.5 × 106 and 3.2 × 106 genomes/ml, respectively, differences that did not reach statistical significance. Of the patients 3, 9, 10 and 14 had genotype 1b, 3a, 1a, and 2b, respectively, and 4 had mixed genotypes. Of the 23 patients with genotype 2b or 3a, 7 had a sustained response vs. none of the 13 patients with genotype 1a or 1b (p=0.03). No patients with cirrhosis had a sustained response whereas 4/18 with chronic persistent and 4/18 with chronic active hepatitis had such a response. It is concluded that some 50% of patients treated with HuIFN-alpha-Le responded with normalisation of ALT levels but that only 20% had a durable response 24 weeks post-treatment, and that patients with genotypes 3a or 2b seem to respond better than patients with other genotypes.

Sera from 39 out of 40 patients with chronic hepatitis C virus (HCV) infection who had been treated for 60 weeks with interferon alfa-2b proved initially HCV RNA positive by reversed transcriptase polymerase chain reaction (PCR). These patients were analysed for genotype and quantitatively for HCV RNA levels prior to treatment by using a competitive PCR method with colorimetric detection of the amplified products. HCV RNA levels were correlated to outcome of treatment, mode of acquisition, histology and HCV genotype. The median pretreatment HCV RNA level in sustained responders (n = 15) with eradication of the viremia and normalization of serum ALT levels lasting 24 weeks post treatment was significantly lower than that in the combined group of non-sustained responders (n = 9) and non-responders (n = 15), 2.52 × 105 vs 8.90 × 105 genome equivalents per ml serum, p < 0.0125, respectively. 10 out of 17 patients with HCV RNA levels lower than the median level (5.64 × 105 genome equivalents per ml serum) had a sustained response to interferon treatment versus only 5/22 with levels equal to or higher than the median level, p = 0.04. No significant pretreatment differences in median HCV RNA levels according to mode of acquisition, genotype, or liver histology prior to treatment were seen. It is concluded that a low pretreatment HCV RNA level seems to be indicative of a sustained response to interferon alfa-2b treatment, whereas a high level seems to be indicative of a non-sustained or non-response. In the individual patient, however, the levels varied widely irrespective of response category.

Serum RNA was extracted from 5 Swedish patients infected with hepatitis C virus (HCV). The N-terminal part of the genomic region coding for the gp70 (E2/NS1), including the hypervariable domain of the E2 protein, was reverse transcribed and amplified by the polymerase chain reaction (PCR) using biotinylated primers. The amplicon was immobilized on magnetic polystyrene beads coated with streptavidine. Solid-state sequencing was carried out on the bound single-stranded DNA, after denaturation. The results of phylogenetic sequence analysis and calculated ratios of transition and transversion mutations showed that 4 of the strains clustered together with the USA prototype strains HCV-1 and HCV-H (genotype I), while 1 strain was close to the Japanese isolate HCV-J, and particularly to isolate HCV-BK (genotype II). Possible antigenic epitopes in the Swedish strains were mapped in the HVR region.

We have tested the sequence UUC CAG UCA GAC CU, at position 9016–9029 within the HIV-1SF2nefopen reading frame, for accessibility to antisense and hammerhead ribozyme attack. The accessibility was first studied using a phosphorothioate-modified 14-nt DNA oligo (complementary to thenef9016–9029site). A dose-dependent repression of HIV-1SF2growth was observed in human peripheral blood mononuclear cells after exogenous administration of the oligo to the cell culture medium. A hammerhead ribozyme with a 6+7-nt antisense specificity for thenef9016–9029site (hhRz.nef9016–9029) was constructed and transfected into the human T-cell line HUT78. Again, a dose-dependent repression of virus growth was observed when different individual clones expressing hhRz.nef9016–9029were infected with HIV-1SF2. A complete abrogation of virus production was observed after infection with a low (0.5 TCID50) HIV-1 titer. Increasing doses (2.5 and 12.5 TCID50) of HIV-1 virus yielded a low production (103-fold reduced) of virus particles in most cases; but a complete, or close to complete, abrogation was observed even in individual cultures infected with the highest dose. Presence of proviralpolandgagsequences in hhRz.nef9016–9029-expressing HUT78 clones was assayed, using PCR. Interestingly, since nopolandgagPCR products could be detected, the results strongly indicated that the hammerhead ribozyme was already acting on the infecting HIV RNA before its reverse transcription and integration as proviral DNA. In summary, the results obtained in this study support thenef9016–9029site as a strong new candidate for ribozymal gene therapy against HIV-1 infection.

The mean plasma content of malondialdehyde (MDA) in 30 patients in different stages of HIV infection was found to be about 30% higher than that in controls. The phenomenon was not correlated to the degree of immunodeficiency and was noted early in the course of the disease. This indicates a higher degree of basal lipid peroxidation, which might contribute to the tissue damage seen in these patients. A new reverse phase liquid chromatography method was used for quantitative measurements of MDA in plasma after reaction of this compound with thiobarbituric acid.

Viruses hijack host metabolic pathways for their replicative advantage. In this study, using patient-derived multiomics data and in vitroinfection assays, we aimed to understand the role of key metabolic pathways that can regulate severe acute respiratory syndrome coronavirus-2 reproduction and their association with disease severity. We used multiomics platforms (targeted and untargeted proteomics and untargeted metabolomics) on patient samples and cell-line models along with immune phenotyping of metabolite transporters in patient blood cells to understand viral-induced metabolic modulations. We also modulated key metabolic pathways that were identified using multiomics data to regulate the viral reproduction in vitro. Coronavirus disease 2019 disease severity was characterized by increased plasma glucose and mannose levels. Immune phenotyping identified altered expression patterns of carbohydrate transporter, glucose transporter 1, in CD8+T cells, intermediate and nonclassical monocytes, and amino acid transporter, xCT, in classical, intermediate, and nonclassical monocytes. In in vitrolung epithelial cell (Calu-3) infection model, we found that glycolysis and glutaminolysis are essential for virus replication, and blocking these metabolic pathways caused significant reduction in virus production. Taken together, we therefore hypothesized that severe acute respiratory syndrome coronavirus-2 utilizes and rewires pathways governing central carbon metabolism leading to the efflux of toxic metabolites and associated with disease severity. Thus, the host metabolic perturbation could be an attractive strategy to limit the viral replication and disease severity.