Effect of coinfections on neurocognitive functioning among people with clade C HIV infection in Zambia.

Despite the fact that many coinfections in people with HIV (PWH) are treatable or suppressible, they may still impact neurocognitive (NC) functioning. Here, we aim to evaluate the presence of latent/treated coinfections and their association with NC functioning in a cohort of PWH in Zambia. We carried out a cross-sectional, nested study involving 151 PWH with viral suppression, and a normative sample of 324 adults without HIV. Plasma samples from PWH who underwent a comprehensive NC assessment were evaluated for the presence of treated/latent coinfections that are common in Zambia. Information about treated pulmonary tuberculosis (TB) was obtained from participants' clinical charts. Overall, PWH differed significantly from the HIV seronegatives on all neuropsychological domains except for fine motor control. ANOVA comparisons of all 3 HIV + groups' demographically corrected mean NC T-scores showed that the HIV + /TB + group had the poorest NC functioning in the following domains: executive functioning (F = 4.23, p = 0.02), working memory (F = 5.05, p = 0.002), verbal fluency (F = 4.24, p = 0.006), learning (F = 11.26, p < 0.001), delayed recall (F = 4.56, p = 0.01), and speed of information processing (F = 5.16, p = 0.005); this group also was substantially worse on the total battery (global mean T-scores; F = 8.02, p < 0.001). In conclusion, treated TB coinfection in PWH was associated with worse NC performance compared to both those with antibodies against other coinfections and without. PWH with antibodies for other coinfections (HIV + /CI +) showed somewhat better NC performance compared to those without (HIV + /CI -), which was not expected, although comparisons with the HIV + /CI + group are limited by its lack of specificity regarding type of coinfection being represented.

Inconsistent reversal of HIV-1 latency ex vivo by antigens of HIV-1, CMV, and other infectious agents.

BACKGROUND: A reservoir of replication-competent but latent virus is the main obstacle to a cure for HIV-1 infection. Much of this reservoir resides in memory CD4 T cells. We hypothesized that these cells can be reactivated with antigens from HIV-1 and other common pathogens to reverse latency. RESULTS: We obtained mononuclear cells from the peripheral blood of antiretroviral-treated patients with suppressed viremia. We tested pools of peptides and proteins derived from HIV-1 and from other pathogens including CMV for their ability to reverse latency ex vivo by activation of memory responses. We assessed activation of the CD4 T cells by measuring the up-regulation of cell-surface CD69. We assessed HIV-1 expression using two assays: a real-time PCR assay for virion-associated viral RNA and a droplet digital PCR assay for cell-associated, multiply spliced viral mRNA. Reversal of latency occurred in a minority of cells from some participants, but no single antigen induced HIV-1 expression ex vivo consistently. When reversal of latency was induced by a specific peptide pool or protein, the extent was proportionally greater than that of T cell activation. CONCLUSIONS: In this group of patients in whom antiretroviral therapy was started during chronic infection, the latent reservoir does not appear to consistently reside in CD4 T cells of a predominant antigen-specificity. Peptide-antigens reversed HIV-1 latency ex vivo with modest and variable activity. When latency was reversed by specific peptides or proteins, it was proportionally greater than the extent of T cell activation, suggesting partial enrichment of the latent reservoir in cells of specific antigen-reactivity.

HIV persists throughout deep tissues with repopulation from multiple anatomical sources.

BACKGROUNDUnderstanding HIV dynamics across the human body is important for cure efforts. This goal has been hampered by technical difficulties and the challenge of obtaining fresh tissues.METHODSThis observational study evaluated 6 individuals with HIV (n = 4 with viral suppression using antiretroviral [ART] therapy; n = 2 with rebound viremia after stopping ART), who provided serial blood samples before death and their bodies for rapid autopsy. HIV reservoirs were characterized by digital droplet PCR, single-genome amplification, and sequencing of full-length (FL) envelope HIV. Phylogeographic methods were used to reconstruct HIV spread, and generalized linear models were tested for viral factors associated with dispersal.RESULTSAcross participants, HIV DNA levels varied from approximately 0 to 659 copies/106 cells (IQR: 22.9-126.5). A total of 605 intact FL env sequences were recovered in antemortem blood cells and across 28 tissues (IQR: 5-9). Sequence analysis showed (a) the emergence of large, identical, intact HIV RNA populations in blood after cessation of therapy, which repopulated tissues throughout the body; (b) that multiple sites acted as hubs for HIV dissemination but that blood and lymphoid tissues were the main source; (c) that viral exchanges occurred within brain areas and across the blood-brain barrier; and (d) that migration was associated with low HIV divergence between sites and greater diversity at the recipient site.CONCLUSIONHIV reservoirs persisted in all deep tissues, and blood was the main source of dispersal. This may explain why eliminating HIV susceptibility in circulating T cells via bone marrow transplants allowed some individuals with HIV to experience therapy-free remission, even though deeper tissue reservoirs were not targeted.TRIAL REGISTRATIONNot applicable.FUNDINGNIH grants P01 AI31385, P30 AI036214, AI131971-01, AI120009AI036214, HD094646, AI027763, AI134295, and AI68636.

Higher Anti-Cytomegalovirus Immunoglobulin G Concentrations Are Associated With Worse Neurocognitive Performance During Suppressive Antiretroviral Therapy.

Background: Cytomegalovirus (CMV) has been linked to higher risk of cardiovascular disease and mortality. We aimed to determine if CMV is associated with neurocognitive performance in adults infected with human immunodeficiency virus (HIV). Methods: In this cross-sectional analysis, anti-CMV immunoglobulin G (IgG) concentrations in blood and CMV DNA copies in blood and cerebrospinal fluid (CSF) were measured in stored specimens of 80 HIV-infected adults who were previously assessed with a comprehensive neurocognitive test battery. Thirty-eight were taking suppressive antiretroviral therapy (ART) and 42 were not taking ART. A panel of 7 soluble biomarkers was measured by immunoassay in CSF. Results: Anti-CMV IgG concentrations ranged from 5.2 to 46.1 IU/mL. CMV DNA was detected in 7 (8.8%) plasma specimens but in no CSF specimens. Higher anti-CMV IgG levels were associated with older age (P = .0017), lower nadir CD4+ T-cell count (P < .001), AIDS (P < .001), and higher soluble CD163 (P = .009). Higher anti-CMV IgG levels trended toward an association with worse neurocognitive performance overall (P = .059). This correlation was only present in those taking suppressive ART (P = .0049). Worse neurocognitive performance remained associated with higher anti-CMV IgG levels after accounting for other covariates in multivariate models (model P = .0038). Detectable plasma CMV DNA was associated with AIDS (P = .05) but not with neurocognitive performance. Conclusions: CMV may influence neurocognitive performance in HIV-infected adults taking suppressive ART. Future clinical trials of anti-CMV therapy should help to determine whether the observed relationships are causal.

(1→3)-ß-d-Glucan: A Biomarker for Microbial Translocation in Individuals with Acute or Early HIV Infection?

BACKGROUND: The extent of gut microbial translocation, which plays roles in HIV disease progression and non-AIDS comorbidities, appears to vary with the composition of the gut microbiome, particularly the presence of Lactobacillales, which reduce mucosal injury. While low proportions of Lactobacillales in the distal gut microbiome are a very promising indicator of microbial translocation, measurement is expensive and complicated and not feasible for clinical routine. (1→3)-ß-d-Glucan (BDG) is a component of most fungal cell walls and might be a surrogate marker for Lactobacillales proportion in the gut and a useful indicator of HIV-associated gut injury. This study evaluated BDG as a biomarker of gut integrity in adults with acute or early HIV infection (AEH). METHODS: Study samples were collected longitudinally during study visits at weeks 0, 12, and 24 in a cohort of 11 HIV-infected men starting antiretroviral therapy during AEH. Blood plasma levels of BDG, soluble cluster of differentiation 14 (sCD14) and lipopolysaccharide (LPS) were measured and then correlated with the proportion of Lactobacillales in the distal gut microbiome, as measured by 16s rDNA sequencing by using mixed-effects models with random intercepts. RESULTS: Mean BDG and sCD14 levels across subjects were associated with Lactobacillales after controlling for time effects and within-subjects correlations (p-values < 0.05), while LPS levels were not. Specifically, each point increase in mean BDG and sCD14 levels across participants was associated with 0.31 ± 0.14 and 0.03 ± 0.01 percent decrease in mean Lactobacillales proportions, respectively. CONCLUSION: BDG and sCD14 may be indicators of low Lactobacillales in the gut in adults with acute or early HIV infection, and serve as biomarkers of gut integrity and microbial translocation in HIV infection. Larger studies are needed to confirm our findings.

(1→3)-ß-D-Glucan Levels Correlate With Neurocognitive Functioning in HIV-Infected Persons on Suppressive Antiretroviral Therapy: A Cohort Study.

Microbial translocation from the gut is associated with immune dysfunction, persistent inflammation, and likely plays a role in the pathogenesis of neurocognitive dysfunction during HIV infection. (1→3)-ß-D-Glucan (BDG) is a component of most fungal cell walls and might be a useful indicator of gut mucosal barrier impairment. The objective of this study was to evaluate whether higher blood BDG levels correlate with impaired neurocognitive functioning in a cohort of HIV-infected adults with suppressed levels of HIV RNA in blood plasma. In this cross-sectional cohort study, we measured levels of BDG in blood plasma and cerebrospinal fluid (CSF) supernatant samples in a cohort of adults with acute/early HIV infection, who initiated antiretroviral therapy (ART) during the earliest phase of infection and achieved suppressed levels of HIV RNA in blood plasma (<50 copies/mL) thereafter. We compared BDG with established biomarkers of microbial translocation, immune activation, and cognitive dysfunction (evaluated by global deficit score). We found that higher blood BDG levels were significantly related to higher global deficit scores, reflecting worse neurocognitive performance (Spearman r = 0.47; P = 0.042) among HIV-infected adults with suppressed viral loads who initiated ART early in infection. Two CSF samples presented elevated BDG levels. Interestingly, these 2 samples originated from the 2 subjects with the highest global deficit scores of the cohort. BDG may be a promising independent biomarker associated with neurocognitive functioning in virologically suppressed HIV-infected individuals.

Circulating HIV DNA Correlates With Neurocognitive Impairment in Older HIV-infected Adults on Suppressive ART.

Older HIV-infected adults have a higher risk of neurocognitive impairment, but the underlying mechanisms are poorly understood. Here, we investigated the associations between levels of HIV DNA in peripheral blood, soluble markers of inflammation and cellular trafficking in blood and cerebrospinal fluid (CSF) and neurocognitive functioning among 18 younger (22-40 years) and 26 older (50-71 years) HIV-infected subjects, who were administered a comprehensive neurocognitive battery. Older HIV-infected individuals presented higher levels of inflammation in CSF and blood compared to younger individuals, but no difference was observed in HIV DNA levels. Among older participants, higher HIV DNA levels were significantly associated with more severe neurocognitive impairment (p = 0.005), particularly in the Executive Functions domain (p = 0.004). No association was observed between HIV DNA and neurocognition among younger individuals. Despite significantly increased inflammation observed in the older group, none of the inflammatory markers were associated with neurocognitive impairment among older HIV+ individuals (p > 0.05). Our study supports the involvement of peripheral HIV DNA reservoir in the pathogenesis of neurocognitive disorder during suppressive ART. Correlates of neurocognitive impairment might differ between younger and older adults, suggesting that future treatment and prevention strategies for HIV-associated neurocognitive disorders likely need to be tailored based on age.

Comparative Analysis of Cell-Associated HIV DNA Levels in Cerebrospinal Fluid and Peripheral Blood by Droplet Digital PCR.

BACKGROUND: Measurement of HIV DNA-bearing cells in cerebrospinal fluid (CSF) is challenging because few cells are present. We present a novel application of the sensitive droplet digital (dd)PCR in this context. METHODS: We analyzed CSF cell pellets and paired peripheral blood mononuclear cells (PBMC) from 28 subjects, 19 of whom had undetectable HIV RNA (<48 copies/mL) in both compartments. We extracted DNA from PBMC using silica-based columns and used direct lysis on CSF cells. HIV DNA and the host housekeeping gene (RPP30) were measured in CSF and PBMC by (dd)PCR. We compared HIV DNA levels in virally-suppressed and-unsuppressed subgroups and calculated correlations between HIV DNA and RNA levels in both compartments using non-parametric tests. RESULTS: HIV DNA was detected in 18/28 (64%) CSF cell pellets, including 10/19 (53%) samples with undetectable HIV RNA. HIV DNA levels in CSF cell pellets were not correlated with RPP30 (p = 0.3), but correlated positively with HIV RNA in CSF (p = 0.04) and HIV DNA in PBMC (p = 0.03). Cellular HIV DNA in CSF was detected in comparable levels in HIV RNA-suppressed and unsuppressed subjects (p = 0.14). In contrast, HIV DNA levels in PBMC were significantly lower in HIV RNA-suppressed than in unsuppressed subjects (p = 0.014). Among subjects with detectable HIV DNA in both compartments, HIV DNA levels in CSF were significantly higher than in PBMC (p<0.001). CONCLUSIONS: Despite low mononuclear cell numbers in CSF, HIV DNA was detected in most virally suppressed individuals. In contrast to PBMC, suppressive ART was not associated with lower HIV DNA levels in CSF cells, compared to no ART, perhaps due to poorer ART penetration, slower decay of HIV DNA, or enrichment of HIV DNA-bearing mononuclear cells into the CSF, compared to blood. Future studies should determine what fraction of HIV DNA is replication-competent in CSF leukocytes, compared to PBMC.

Correlation of (1→3)-ß-D-glucan with other inflammation markers in chronically HIV infected persons on suppressive antiretroviral therapy.

We evaluated associations between levels of BDG and other biomarkers of inflammation in blood from 41 virologically suppressed persons with chronic HIV-infection. We found a significant correlation between BDG and neopterin levels (r=0.68), and trends to significance for correlations with other inflammation markers (tumor-necrosis-factor-α: r=0.30; interleukin-8: r=0.30; interleukin-6: r=0.28). In conclusion, BDG levels correlated with inflammation markers in a cohort of virologically suppressed individuals with chronic HIV infection. Future studies are needed to evaluate whether BDG may be a marker for morbidity in chronic HIV infection.

Aprimoramento da plataforma de expressão na região intergênica E/NS1 do vírus da febre amarela 17D / Improvement of the platform of expression in intergênica region E/NS1 of the virus of the yellow fever 17D

A Febre Amarela (FA) é uma doença viral da África e América do Sul e é transmitida por mosquitos. O vírus da FA é o protótipo do gênero Flavivírus e caracteriza-se por ser um vírus envelopado com genoma de RNA fita simples positiva. O controle da FA foi possível após o desenvolvimento de uma vacina atenauda e, graças às suas boas características, tem sido amplamente utilizada. Também tem sido avaliada como vetor de expressão para antígenos exógenos, tendo em vista a obtenção de novas vacinas. Como o genoma de FA é pequeno e compacto, inserções poderiam provocar um efeito deletério sobre a viabilidade viral e, assim, deve-se respeitar os motivos funcionais importantes para o processamento viral. Foi desenvolvida em nosso laboratório, uma estratégia de expressão de preteínas exógenas, por meio da inserção do gene da proteína repórter EGFP na região intergênica E/NS1 do genoma do vírus vacinal FA 17D. Esta abordagem considerou os motivos funcionais desta região, que foram duplicados e utilizados para flanquear a EGFP, permitindo o correto processamento da poliproteína viral. Foram construídos dois vírus que diferiam entre si por conter a região completa ou truncada da Haste-âncora de FA (HA FA) fusionada à EGFP. Eles foram caracterizados quanto à capacidade de replicação e imunogenicidade em comparação ao vacinal FA 17DD e quanto à expressão e estabilidade da inserção heteróloga no genoma viral (Bonaldo et al., 2007; Mello, 2007). Os resultados obtidos apontaram para a necessidade de aprimoramento da estabilidade das inserções no genoma. Portanto, nesta dissertação, apresentamos uma variação desta estratégia com a finalidade de melhoramento da estabilidade das inserções no genoma dos vírus recombinantes. Uma modificação realizada foi a utilização da variante pG1/2 na construção destes novos vírus, a fim de torná-los fenotipicamente mais próximos do vacinal.

A outra modificação foi a substituição dos domínios HA completo ou truncado de FA, que foram fusionados ao gene exógeno, pelas seqüências homólogas dos elementos HA Den4. As características de replicação, expressão e estabilidade da inserção heteróloga no genoma viral foram determinadas e comparadas com as da primeira geração de vírus construídos por esta estratégia, após o cultivo em monocamadas celulares. Sugerimos aqui, que estes novos vírus apresentaram um perfil mais replicativo do que os da primeira geração, porém menos replicativo que o vacinal. A EGFP foi eficientemente expressa, porém manteve-se retida no Retículo Endoplasmático (RE), confirmando-se como uma característica comum dos vírus construídos por esta tecnologia. Observou-se um aprimoramento da estabilidade genética da inserção contendo a HA Den4 truncada, indicando que a diminuição da identidade seqüencial entre este domínio e aquele natural do vírus FA foi capaz de melhorar a estabilidade da inserção, mas somente quando omitimos os elementos H1 e CS, sugerindo uma interferência deletéria do acúmulo destes motivos no RE.