Greater diversity of HIV-1 quasispecies in HIV-infected individuals with active tuberculosis

OBJECTIVE: A continual increase in intrapatient HIV-1 heterogeneity is thought to contribute to evasion of host immune response and eventual progression to AIDS. Tuberculosis (TB) is diagnosed both early and late during the course of HIV-1 disease and may increase diversity of HIV-1 quasispecies by activating the HIV-1 immune response and increasing HIV-1 replication. We examined whether HIV-1 heterogeneity is altered in HIV-1-infected individuals with TB. METHODS: Blood samples were obtained from 7 HIV-1-infected patients with active TB (HIV/TB patients) and 9 HIV-1-infected patients (HIV patients) in Kampala, Uganda (CD4 counts of 0-650 cells/microl and HIV loads of 700-750,000 RNA copies/ml). The C2-C3 region of the HIV-1 envelope gene (env) was amplified by nested polymerase chain reaction (PCR) from lysed peripheral blood mononuclear cells (PBMCs) of each patient, and then subject to sequencing, clonal-quasispecies analysis and heteroduplex tracking analysis (HTA). RESULTS: HTA of env DNA fragments showed increased heterogeneity in the HIV/TB individuals compared with the HIV group. Further sequence and HTA analysis on ten individual env clones for each patient showed significantly greater HIV mutation frequencies in HIV/TB patients than in HIV patients. CONCLUSION: An increase in HIV-1 heterogeneity may be associated with a TB-mediated increase in HIV-1 replication. However, a diverse HIV-1 quasispecies population in HIV/TB patients as opposed to tight quasispecies clusters in HIV patients suggests a possible dissemination of lung-derived HIV-1 isolates from the TB-affected organ.     

Human immunodeficiency virus type-1 episomal cDNA in semen

Background

Episomal 2-long terminal repeat (LTR) HIV-1 cDNA, a by-product of HIV-1 infection, is used in clinical trials as a marker for ongoing viral replication. It would be useful to employ 2-LTR cDNA to monitor cryptic HIV-1 infection in the genital tract of men on antiretroviral therapy (ART) to predict the evolution of sexually transmissible drug-resistant HIV-1, but studies thus far have failed to detect this marker in semen. The objectives of this study were: 1) to use a technique that maximizes DNA recovery from HIV-1 infected white blood cells in semen to determine if episomal 2-LTR cDNA is detectable in semen of ART-naïve men with other evidence of genital tract HIV-1 infection, and 2) to compare levels of HIV-1 2-LTR cDNA, RNA, and proviral DNA in semen from HIV-1+ men on ART.

Results

Using a somatic cell DNA extraction technique, 2-LTR cDNA was detected by PCR/ELISA in 4 out of 8 semen samples from ART-naïve men selected for other signs of seminal HIV-1 infection (positive controls). Southern blot and DNA sequencing confirmed that the amplified sequences were HIV-1 2-LTR cDNA; copy numbers ranged from 55 to 504 copies/sample. Two semen samples from a cohort of 22 HIV-1-infected men on dual nucleoside therapy, one with and one without detectable seminal HIV-1 RNA, were 2-LTR cDNA positive (336 and 8,560 copies/sample). Following addition of indinavir to the therapy regimen, no semen samples from 21 men with controlled peripheral and seminal viral loads were 2-LTR cDNA positive at 1 and 6 month time points, despite the persistence of HIV-1 proviral DNA+ semen cells and seminal cytomegalovirus (CMV) shedding in some cases. However, one individual who failed indinavir therapy and later developed distinct protease inhibitor (PI) drug resistance mutations in semen, maintained elevated levels of HIV-1 RNA and 2-LTR cDNA in semen.

Conclusion

2-LTR HIV-1 cDNA is detectable in semen of HIV-1-infected men. Two men on ART had 2-LTR HIV-1 cDNA in semen, suggesting that this marker may prove to be useful to monitor HIV-1 infection in the genital tract of men on ART to predict the evolution of drug resistance mutations in semen.

     

Human immunodeficiency virus type 1 group m protease in cameroon: genetic diversity and protease inhibitor mutational features

To establish a baseline for monitoring resistance to protease inhibitors (PIs) and examining the efficacy of their use among persons in Cameroon infected with human immunodeficiency virus type 1 (HIV-1), we analyzed genetic variability and PI resistance-associated substitutions in PCR-amplified protease (PR) sequences in strains isolated from 110 HIV-1-infected, drug-na?ve Cameroonians. Of the 110 strains, 85 were classified into six HIV-1 PR subtypes, A (n = 1), B (n = 1), F (n = 4), G (n = 7), H (n = 1), and J (n = 7), and a circulating recombinant form, CRF02-AG (n = 64). PR genes from the remaining 25 (23%) specimens were unclassifiable, whereas 2% (7 of 301) unclassifiable PR sequences were reported for a global collection. Two major PI resistance-associated mutations, 20M and 24I, were detected in strains from only two specimens, whereas secondary mutations were found in strains from all samples except one strain of subtype B and two strains of CRF02-AG. The secondary mutations showed the typical PI resistance-associated pattern for non-subtype B viruses in both classifiable and unclassifiable PR genes, with 36I being the predominant (99%) mutation, followed by 63P (18%), 20R (15%), 77I (13%), and 10I or 10V (11%). Of these mutations, dual and triple PI resistance-associated substitutions were found in 38% of all the Cameroonian strains. Compared with classifiable PR sequences, unclassifiable sequences had significantly more dual and triple substitutions (64% versus 30%; P = 0.004). Phenotypic and clinical evaluations are needed to estimate whether PI resistance during antiretroviral drug treatment occurs more rapidly in individuals infected with HIV-1 strains harboring multiple PI resistance-associated substitutions. This information may be important for determination of appropriate drug therapies for HIV-1-infected persons in Cameroon, where more than one-third of HIV-1 strains were found to carry dual and triple minor PI resistance-associated mutations.     

Human immunodeficiency virus type 1 and hepatitis B virus transcription in peripheral blood lymphocytes from co-infected subjects

Summary We have investigated restriction fragment length polymorphism (RFLP) on the PrP gene and the frequencies of RFLP patterns in 35 healthy Suffolk sheep randomly collected. According to the combinations of PrP encoding DNA fragments generated by restriction enzymesEco RI andHind III, the RFLP patterns were classified into six types and designated as types I to VI. The frequencies of these types were as follows: I, 8.6%; II, 11.4%; III, 17.6%; IV, 11.4%; V, 28.6%; and VI, 22.9%. In 10 sheep diagnosed as having natural scrapie, RFLP types, I, III, IV, and V were determined. To examine the correlation between the RFLP type and the occurrence of scrapie, the frequencies of RFLP types in sheep infected with natural scrapie were compared with those in healthy sheep. It was found that the frequency of type I in the sheep with natural scrapie was 70%, about eight times higher than that in randomly collected healthy sheep. In the 13 experimentally infected sheep that had been used for other purposes, however, no relationship between the RFLP type and onset of scrapie was found.     

Hepatitis B virus genotype G: prevalence and impact in patients co-infected with human immunodeficiency virus

Relatively little is known about the role of hepatitis B virus (HBV) genotype G (HBV/G) in patients co‐infected with human immunodeficiency virus (HIV) and HBV. This study examined the prevalence and association of HBV/G to liver fibrosis in co‐infected patients. HBV genotypes were determined by direct sequencing of the HBV surface gene or Trugene® HBV 1.0 assay in 133 patients infected with HIV/HBV. Quantitative testing of HBV‐DNA, HBeAg, and anti‐HBe were performed using the Versant® HBV 3.0 (for DNA) and the ADVIA®Centaur assay. The non‐invasive biomarkers Fib‐4 and APRI were used to assess fibrosis stage. Genotype A was present in 103/133 (77%) of the cohort, genotype G in 18/133 (14%) with genotypes D in 8/133, (6%), F 2/133 (1.5%), and H 2/133 (1.5%). Genotype G was associated with hepatitis B e antigen‐positivity and high HBV‐DNA levels. Additionally, HBV/G (OR 8.25, 95% CI 2.3–29.6, P = 0.0012) was associated with advanced fibrosis score using Fib‐4, whereas, being black was not (OR 0.19, 95% CI 0.05–0.07, P = 0.01). HBV/G in this population exhibited a different phenotype than expected for pure G genotypes raising the question of recombination or mixed infections. The frequent finding of HBV/G in co‐infected patients and its association with more advanced fibrosis, suggests that this genotype leads to more rapid liver disease progression. Further studies are needed to understand why this genotype occurs more frequently and what impact it has on liver disease progression in patients with HBV/HIV. J. Med. Virol. 83:1551–1558, 2011. © 2011 Wiley‐Liss, Inc.     

Human immunodeficiency virus type 1 genetic evolution in patients with prolonged suppression of plasma viremia

Treatment of human immunodeficiency virus type 1 (HIV-1)-infected patients with combination drug regimens results in a reduction of plasma viral load to levels below the limit of detection. To investigate the genomic fluctuations in HIV-1 populations from long-term responders to antiviral therapies we analyzed the viral sequence evolution of env and pol genes from sequential peripheral blood mononuclear cell (PBMC) DNA samples of three infected patients. Analyses of sequences covering the V3 and flanking env regions obtained from blood samples at the beginning of the therapy and at 14 or 24 months from baseline revealed that HIV-1 quasispecies continue to evolve in the three patients following combination antiretroviral therapy. Minor drug-resistant mutant subpopulations were also searched for and found in one patient. Interestingly, no minor resistant subpopulations were found in the other two patients despite the fact that they showed evidence of ongoing viral replication. Finally, the genetic analysis of the env gene shows a reduction in PBMC env viral population diversity after long-term response to the therapy in all the patients analyzed.     

Human immunodeficiency virus type-2-A milder, kinder virus: an update

Human immunodeficiency virus type-2 (HIV-2) belongs to the family retroviridae which is phylogenetically clusters with SIV SM from sooty mangabeys. This virus is morphologically similar to human immunodeficiency virus type-1 (HIV-1) but has got only a 40% homology at the nucleotide level. There is a distinct geographical distribution of HIV-2 unlike HIV-1. There are currently eight subtypes/groups identified with subtype/group A responsible for the majority of infections. HIV-2 shows a considerable difference in the course of the disease. Clinical, haematological and immunological evaluation of individuals infected with HIV-2 has shown the virus to be less pathogenic than HIV-1 although the exact mechanism underlying this difference is not well defined. Similar to HIV-1, the HIV-2 isolates also showed distinct replicative and cytopathic characteristics. The transmission rate for HIV-2 compared to HIV-1 is very low both by heterosexual route and mother to child transmission. The clinical signs and symptoms of immunodeficiency associated with HIV-2 are similar to the ones seen among the HIV-1-infected individuals and they can also progress to AIDS. It is naturally resistant to NNRTI and hence the diagnosis become important as it affects the treatment strategy. Similar to HIV-1, HIV-2 strains of infected individuals also show mutations that can cause drug resistance. The current evidence suggests that there is no protective effective for HIV-2 against HIV-1.     

Human immunodeficiency virus type-1 infection impairs the formation of the immunological synapse

HIV-1-infected lymphocytes improperly respond to T cell antigen receptor (TCR) stimulation. To document this phenomenon, we studied the capacity of HIV-1-infected lymphocytes to form immunological synapses. We show here that HIV-1-infected T cells poorly conjugated with antigen-presenting cells, and when they formed conjugates, the synapses were abnormal. TCR and Lck accumulated in the recycling endosomal compartment, and their clustering at the synapse was severely reduced. These phenomena were, to a large extent, caused by Nef, a viral protein affecting intracellular trafficking and signaling pathways. Concomitantly, in HIV-infected cells, tyrosine phosphorylation at the synapse and the patterns of tyrosine phosphorylated proteins were disturbed in a Nef-dependent manner. These findings underscore the importance of Lck and TCR endosomal trafficking in synapse formation and early T cell signaling. Alteration of endocytic and signaling networks at the immunological synapse likely impacts the function and fate of HIV-1-infected cells.     

Reproduction and fertility in human immunodeficiency virus type-1 infection

Human immunodeficiency virus type-1 (HIV-1) affects mostly men and women in their reproductive years. For those who have access to highly active antiretroviral therapy (HAART), the course of HIV-1 infection has shifted from a lethal to a chronic disease. As a result of this, many patients with HIV-1 consider having offspring, as do other patients of reproductive age with chronic illnesses. This article summarizes the current knowledge on the presence of HIV in the male and female genital tract, the effects of HIV-1 infection and HAART on male and female fertility and the results of various assisted reproduction techniques (ART) in HIV-1-infected men and women who wish to have offspring.     

Long-term evolution in liver fibrosis and immune profile after direct-acting antivirals therapy in hepatitis C virus-human immunodeficiency virus co-infected patients

ObjectiveHepatitis C virus (HCV) therapy with direct-acting antivirals (DAA) achieves high rates of sustained virological response in people living with human immunodeficiency virus (HIV) (PLWH). Information on its long-term clinical impact is scarce. The aim of this study was to analyse liver fibrosis and immune response evolution after DAA treatment.MethodsRetrospective, single centre cohort study of HIV-HCV co-infected patients treated with DAA between June 2013 and June 2018. We analysed the changes during follow up in liver fibrosis (assessed by transient elastography (TE), aspartate aminotransferase to platelet ratio index (APRI) and FIB-4 scores) and immunity (CD4 and CD8 cells counts and CD4/CD8 ratio).ResultsWe included 410 patients; 75% (308/407) men with a mean age of 50 years (SD 8); 78% (318/410) had long chronic HCV infection (median 21 years, interquartile range (IQR) 6–27 years) and 27% (107/393) had liver cirrhosis. Liver fibrosis improvement based on the decrease in TE value compared with the baseline occurred in 43% (131/302) of patients and 31% of patients based on biological scores (APRI: 124/398; FIB-4: 104/398) (p < 0.0001), being more frequent in those with advanced baseline fibrosis (83/144). The higher decrease was observed at 6 months after DAA therapy (–0.23; 95% CI –0.29 to –0.18), but a continuum in fibrosis regression of at least 30% from baseline value of TE was observed along the follow up (32% of patients at month 6, 51% at month 24 and 55% at month 48). Regarding the immunological profile, there was a significant decrease in CD8 counts at month 48 (–62.38; 95% CI –106.77 to –17.99; p 0.0001) and a progressive rise in the CD4/CD8 ratio after 24 months of follow up reaching an increment of +0.07 (95% CI 0.03–0.10, p 0.0001) at month 48.ConclusionsHCV treatment with DAA in PLWH is associated with significant progressive improvement in liver fibrosis and recovery of the immune system with an increase in the CD4/CD8 ratio in long-term follow up.     

T cell line passage can select for pre-existing neutralization-sensitive variants from the quasispecies of primary human immunodeficiency virus type-1 isolates

Primary human immunodeficiency type 1 viruses (HIV-1) resist antibody neutralization but become sensitive after passage through T cell lines. We and others previously reported an increased neutralization sensitivity of HIV-1 after prolonged culture on primary peripheral blood mononuclear cells (PBMC). Hence we hypothesized that adaptation to growth in T cell lines is in fact selection of a pre-existing neutralization-sensitive HIV-1 variant from the quasispecies in the PBMC culture. Indeed, increased neutralization sensitivity was associated with largely identical synonymous and non-synonymous mutations between progeny of parallel H9 passages from the same split inoculum from 2 of 3 viruses. H9 T cell line adaptation of molecular cloned HIV-1 was less successful and associated with only a few de novo mutations that varied between parallel H9-adapted progeny from the same split inoculum. We conclude that T cell line adaptation of HIV-1 can indeed select for a pre-existing variant but that this most likely depends on the viral diversity in the inoculum.     

Variability and immunogenicity of human immunodeficiency virus type 1 p24 gene quasispecies

Despite the conserved nature of the human immunodeficiency virus type 1 (HIV-1) gag gene, multiple quasispecies of the p24 gene coexist in HIV-1-infected patients. We cloned and sequenced 31 p24 genes from four HIV-1-infected patients. The intrapatient homology between the p24 genes ranged from 97.1 to 99.1%, whereas the interpatient homology ranged from 91.5 to 93.8%, suggesting a host-specific evolution. Synonymous and nonsynonymous nucleotide changes were evenly distributed in the p24 gene, with 27 and 28%, respectively, located within host human leukocyte antigen class I recognition sites. This would suggest only a minor influence from the host cytotoxic T-cell response on the evolution of the p24 gene. The importance of minor variations within p24 was analyzed by designing DNA-based immunogens from two distinct p24 quasispecies genes simultaneously derived from one patient. In plasmid-immunized H-2(b), H-2(d), and H-2(k) haplotype mice, a clear influence from the host major histocompatibility complex was noted on the immune responses, fully consistent with those noted when a recombinant p24 protein is used as the immunogen. The two p24 DNA immunogens did not differ in their immunogenicity, indicating that the limited genetic variability (<1%) had little influence on the immune responses.     

Differing patterns of liver disease progression and hepatitis C virus (HCV) quasispecies evolution in children vertically coinfected with HCV and human immunodeficiency virus type 1

Hepatitis C virus (HCV) quasispeciation was studied in two children vertically coinfected with HCV and human immunodeficiency virus type 1 (HIV-1). HCV quasispecies diversification and liver injury were more significant in patient C1, who was immunocompetent with anti-HIV therapy, than in patient C2, who was immunosuppressed, in consistency with modulation of HCV quasispeciation and liver injury by immunocompetence in coinfected children.     

Impact of highly active antiretroviral therapy on hepatitis C virus protease quasispecies diversity in HIV co‐infected patients

Many hepatitis C virus (HCV)‐infected patients are also infected with HIV, and undergo antiretroviral (ARV) treatment for their human immunodeficiency virus (HIV) infection. Due to changes in HIV burden and immunologic status, HIV ARV treatment may have indirect effects on the HCV population, which could impact the effectiveness of subsequent HCV protease inhibitor (PI) treatment. The genetic variability of the protease‐encoding HCV NS3 gene was evaluated in 10 co‐infected patients initiating ARVs (both before and after ARV initiation), and compared to the genetic variability in 10 patients on stable ARV therapy. After RT‐PCR of plasma‐derived HCV RNA, a mean of 20 clones per patient time‐point were sequenced and analyzed for changes in the HCV quasispecies population. No significant differences in sequence diversity or complexity at the nucleic acid or amino acid levels were seen at baseline between groups or between the two time points in either group. HCV protease diversity in the pre‐ and post‐ARV treatment samples was not significantly different than samples from patients on stable ARV therapy. There was no significant development of amino acid substitutions known to confer HCV PI resistance in either group. Initiation of ARV for HIV infection does not significantly alter the genetic diversity or complexity of the HCV NS3 gene or result in increased number of HCV PI‐associated amino acid changes. These results suggest ARV treatment for HIV would not affect the efficacy of HCV PI treatment. J. Med. Virol. 82: 791–798, 2010. © 2010 Wiley‐Liss, Inc.     

Detection of circulating platelet-monocyte complexes in persons infected with human immunodeficiency virus type-1

Activated platelets form transient aggregates with monocytes in circulation and have a half-life of approximately 30-60 min. These complexes are increased in various inflammatory conditions and are an early marker of myocardial infarction. HIV-1 infection is associated with chronic inflammation, and increased CD16? inflammatory monocytes have been observed in these individuals, probably as a result of increased interaction with platelets. However, narrow detection period and platelet activation during sample processing pose significant problems in detecting platelet-monocyte complexes (PMCs). A method was standardized addressing these difficulties, to enumerate PMCs involving CD16? or CD16? monocytes in whole blood using flow cytometry. Blood collected from healthy individuals was treated with either collagen (for platelet activation) or LPS (for monocyte activation) and subsequently used to study effect of these treatments on PMC formation. This method was also validated for the ex vivo quantitation of PMCs in blood obtained from persons infected with HIV. The in vitro results demonstrated that platelet activation, but not monocyte activation, resulted in significant increase in PMC formation. There was a significant increase in CD16? PMCs and platelet activation, in samples obtained from persons infected with HIV as compared to those without HIV infection. Furthermore, PMC percentages correlated positively with platelet activation. These findings improve the ability to detect PMCs and shed light on HIV pathogenesis.     

Treatment of tuberculosis in patients with advanced human immunodeficiency virus infection

BACKGROUND AND METHODS. Infection with the human immunodeficiency virus (HIV) increases the risk of tuberculosis and may interfere with the effectiveness of antituberculosis chemotherapy. To examine the outcomes in patients with both diagnoses, we conducted a retrospective study of all 132 patients listed in both the acquired immunodeficiency syndrome (AIDS) and tuberculosis case registries in San Francisco from 1981 through 1988. RESULTS. At the time of the diagnosis of tuberculosis, 78 patients (59 percent) did not yet have a diagnosis of AIDS, 18 patients (14 percent) were given a concomitant diagnosis of AIDS (as determined by the presence of an AIDS-defining disease other than tuberculosis), and the remaining 36 patients (27 percent) already had AIDS. The manifestations of tuberculosis were entirely pulmonary in 50 patients (38 percent), entirely extrapulmonary in 40 patients (30 percent), and both pulmonary and extrapulmonary in 42 patients (32 percent). The treatment regimens were as follows: isoniazid and rifampin supplemented by ethambutol for the first two months, 52 patients; isoniazid and rifampin supplemented by pyrazinamide and ethambutol for the first two months, 39 patients; isoniazid and rifampin, 13 patients; isoniazid and rifampin supplemented by pyrazinamide for the first two months, 4 patients; and other drug regimens, 17 patients. The intended duration of treatment for patients whose regimen included pyrazinamide was six months, and for patients who did not receive pyrazinamide, nine months. Seven patients received no treatment because tuberculosis was first diagnosed after death. Sputum samples became clear of acid-fast organisms after a median of 10 weeks of therapy. Abnormalities on all chest radiographs taken after three months of treatment were stable or improved except for those of patients who had new nontuberculous infections. The only treatment failure occurred in a man infected with multiple drug-resistant organisms who did not comply with therapy. Adverse drug reactions occurred in 23 patients (18 percent). For all 125 treated patients, median survival was 16 months from the diagnosis of tuberculosis. Tuberculosis was a major contributor to death in 5 of the 7 untreated patients and 8 of the 125 treated patients. Three of 58 patients who completed therapy had a relapse (5 percent); compliance was poor in all 3. CONCLUSIONS. Tuberculosis causes substantial mortality in patients with advanced HIV infection. In patients who comply with the regimen, conventional therapy results in rapid sterilization of sputum, radiographic improvement, and low rates of relapse.     

Human pegivirus (HPgV) infection in Ghanaians co-infected with human immunodeficiency virus (HIV) and hepatitis B virus (HBV)

Human pegivirus (HPgV) is a positive single-stranded RNA virus in the Flaviviridae family. Phylogenetic analysis reveals the presence of multiple HPgV genotypes with distinct geographic locations. HPgV is of interest because of its potential beneficial impact on HIV disease progression. Despite this, the effects of HPgV in the context of other viral infections, such as hepatitis B virus (HBV), are poorly understood, and data from resource-limited settings are scarce. Therefore, we conducted a cross-sectional analysis of HPgV in HIV/HBV co-infected patients in Ghana. Sera from 100 HIV/HBV co-infected individuals were evaluated for HPgV RNA, and the genotype determined by sequencing the 5′ untranslated region. HPgV RNA was detected in 27 samples (27%). Of these, 26 were genotyped successfully with 23 belonging to HPgV genotype 1 and 3 belonging to HPgV genotype 2. The presence of HPgV RNA had no statistically significant impact on CD4 cell count or HBV DNA titers in the HIV/HBV co-infected patients. However, there was a trend towards decreased HBV DNA levels in HPgV RNA-positive patients with CD4 cell count?<?200 (p?=?0.0626). HPgV co-infection is common in Ghana. The effect of HPgV on HIV or HBV disease among HIV/HBV co-infected patients was minimal. However, decreased HBV DNA levels in HPgV RNA-positive patients with low CD4 cell counts highlight the need for prospective studies of HPgV in HIV and hepatitis co-infected patients, especially in those with advanced HIV disease, to study further the effects of HPgV on liver disease.