Compartmentalized Replication of R5 T Cell-Tropic HIV-1 in the Central Nervous System Early in the Course of Infection

Compartmentalized HIV-1 replication within the central nervous system (CNS) likely provides a foundation for neurocognitive impairment and a potentially important tissue reservoir. The timing of emergence and character of this local CNS replication has not been defined in a population of subjects. We examined the frequency of elevated cerebrospinal fluid (CSF) HIV-1 RNA concentration, the nature of CSF viral populations compared to the blood, and the presence of a cellular inflammatory response (with the potential to bring infected cells into the CNS) using paired CSF and blood samples obtained over the first two years of infection from 72 ART-naïve subjects. Using single genome amplification (SGA) and phylodynamics analysis of full-length env sequences, we compared CSF and blood viral populations in 33 of the 72 subjects. Independent HIV-1 replication in the CNS (compartmentalization) was detected in 20% of sample pairs analyzed by SGA, or 7% of all sample pairs, and was exclusively observed after four months of infection. In subjects with longitudinal sampling, 30% showed evidence of CNS viral replication or pleocytosis/inflammation in at least one time point, and in approximately 16% of subjects we observed evolving CSF/CNS compartmentalized viral replication and/or a marked CSF inflammatory response at multiple time points suggesting an ongoing or recurrent impact of the infection in the CNS. Two subjects had one of two transmitted lineages (or their recombinant) largely sequestered within the CNS shortly after transmission, indicating an additional mechanism for establishing early CNS replication. Transmitted variants were R5 T cell-tropic. Overall, examination of the relationships between CSF viral populations, blood and CSF HIV-1 RNA concentrations, and inflammatory responses suggested four distinct states of viral population dynamics, with associated mechanisms of local viral replication and the early influx of virus into the CNS. This study considerably enhances the generalizability of our results and greatly expands our knowledge of the early interactions of HIV-1 in the CNS

Compartmentalization, Viral Evolution, and Viral Latency of HIV in the CNS

Human immunodeficiency virus type 1 (HIV-1) infection occurs throughout the body, and can have dramatic physical effects, such as neurocognitive impairment in the central nervous system (CNS). Furthermore, examining the virus that resides in the CNS is challenging due to its location and can only be done using samples collected either at autopsy, indirectly form the cerebral spinal fluid (CSF), or through the use of animal models. The unique milieu of the CNS fosters viral compartmentalization as well as evolution of viral sequences, allowing for new cell types, such as macrophages and microglia, to be infected. Treatment must also cross the blood brain barrier adding additional obstacles in eliminating viral populations in the CNS. These long-lived infected cell types and treatment barriers may affect functional cure strategies in people on highly active antiretroviral therapy (HAART)

Multivariable analysis to determine if HIV-1 Tat dicysteine motif is associated with neurodevelopmental delay in HIV-infected children in Malawi

Background HIV-1 Tat protein is implicated in HIV-neuropathogenesis. Tat C31S polymorphism (TatCS) has been associated with milder neuropathology in vitro and in animal models but this has not been addressed in a cohort of HIV-infected adults or children. Methods HIV viral load (VL) in plasma and cerebrospinal fluid (CSF) were determined and plasma HIV tat gene was sequenced. Neurodevelopmental assessment was performed using Bayley Scales of Infant Development III (BSID-III), with scores standardized to Malawian norms. The association between TatCS and BSID-III scores was evaluated using multivariate linear regression. Results Neurodevelopmental assessment and HIV tat genotyping were available for 33 children. Mean age was 19.4 (SD 7.1) months, mean log VL was 5.9 copies/mL (SD 0.1) in plasma and 3.9 copies/mL (SD 0.9) in CSF. The prevalence of TatCC was 27 %. Z-scores for BSID-III subtests ranged from −1.3 to −3.9. TatCC was not associated with higher BSID-III z-scores. Conclusions The hypothesis of milder neuropathology in individuals infected with HIV TatCS was not confirmed in this small cohort of Malawian children. Future studies of tat genotype and neurocognitive disorder should be performed using larger sample sizes and investigate if this finding is due to differences in HIV neuropathogenesis between children and adults

Subject population virologic, clinical and phylogenetic characteristics.

<p>^aTime point(s) beyond 2 years p.i. analyzed for subject 9018 and 9040 were not included in any overall population analyses.</p><p>^bEstimated.</p><p>^c,dVL, viral load; HIV-1 RNA (log₁₀ copies/ml).</p><p>^eCells/μl.</p><p>^fCSF white blood cell (WBC) count, cell/μl.</p><p>^gCSF/plasma albumin ratio.</p><p>^hThree statistical analyses of genetic compartmentalization between viral populations in the blood plasma and CSF: Slatkin-Maddison test (SM) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref018" target="_blank">18</a>], Wright’s measure of population subdivision (F_st) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref019" target="_blank">19</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref020" target="_blank">20</a>] and the Nearest-neighbor statistic (S_nn) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref021" target="_blank">21</a>]. <i>P</i> values <0.05 indicated statistically significant genetic compartmentalization.</p><p>ⁱHIV-1 population characteristics in the CSF compartment (compart). Eq, equilibrated blood plasma and CSF populations; Cp (compartmentalized), significant compartmentalization by three compartmentalization analyses; Ap, clonal amplification of ≥3 variants detected in the CSF.[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref018" target="_blank">18</a>]</p><p>^jTMRCA (Time to Most Recent Common Ancestor) of the entire viral population, analyzed by BEAST [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref015" target="_blank">15</a>]. An asterisk (*) indicates transmission of > 1 variant.</p><p>^kTMRCA for the compartmentalized (Comp) CSF population for compartmentalized subjects.</p><p>^lPatient 9018 diagnosed with neurosyphilis at indicated date.</p><p>^mSignificant compartmentalization scores were due to a compartmentalized lineage in the plasma. After removing this plasma lineage, the remaining plasma and CSF sequences were equilibrated.</p><p>Subject population virologic, clinical and phylogenetic characteristics.</p

Background demographic and clinical characteristics of study participants at baseline.

<p>Values shown are medians (interquartile ranges) except where noted</p><p>^aSubjects with sufficient viral RNA loads in the CSF (>1,000 copies of viral RNA/ml; to ensure adequate sampling) for further SGA analysis at one or more time points within the first two years.</p><p>Background demographic and clinical characteristics of study participants at baseline.</p

All viruses required high levels of CD4 for efficient entry, indicative of primarily being selected for replication in CD4+ T cells.

<p>Single-cycle infection of HIV-1 Env-pseudotyped reporter viruses on CD4^lowCCR5^high 293-Affinofile cells [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref028" target="_blank">28</a>]. Receptor expression was measured as follows: CD4^low = 972 receptors/cell, CD4^high = 72,041 receptors/cell, CCR5^high = 15,636 receptors/cell. The data are averaged from triplicate wells for each of 2 to 3 <i>env</i> clones that were generated per indicated amplicon. Amplicons were selected for cloning to represent different portions of the phylogenetic tree. Subjects with no evidence of CNS compartmentalization (i.e. equilibrated) are shown in panel (A); and subjects with CNS compartmentalization at one or more time points are in panel (B). Longitudinal time points are indicated (T1, T2, T3, etc.) and samples with marked pleocytosis are noted (*).</p

Central Nervous System Compartmentalization of HIV-1 Subtype C Variants Early and Late in Infection in Young Children

<div><p>HIV-1 subtype B replication in the CNS can occur in CD4+ T cells or macrophages/microglia in adults. However, little is known about CNS infection in children or the ability of subtype C HIV-1 to evolve macrophage-tropic variants. In this study, we examined HIV-1 variants in ART-naïve children aged three years or younger to determine viral genotypes and phenotypes associated with HIV-1 subtype C pediatric CNS infection. We examined HIV-1 subtype C populations in blood and CSF of 43 Malawian children with neurodevelopmental delay or acute neurological symptoms. Using single genome amplification (SGA) and phylogenetic analysis of the full-length <em>env</em> gene, we defined four states: equilibrated virus in blood and CSF (n = 20, 47%), intermediate compartmentalization (n = 11, 25%), and two distinct types of compartmentalized CSF virus (n = 12, 28%). Older age and a higher CSF/blood viral load ratio were associated with compartmentalization, consistent with independent replication in the CNS. Cell tropism was assessed using pseudotyped reporter viruses to enter a cell line on which CD4 and CCR5 receptor expression can be differentially induced. In a subset of compartmentalized cases (n = 2, 17%), the CNS virus was able to infect cells with low CD4 surface expression, a hallmark of macrophage-tropic viruses, and intermediate compartmentalization early was associated with an intermediate CD4 entry phenotype. Transmission of multiple variants was observed for 5 children; in several cases, one variant was sequestered within the CNS, consistent with early stochastic colonization of the CNS by virus. Thus we hypothesize two pathways to compartmentalization: early stochastic sequestration in the CNS of one of multiple variants transmitted from mother to child, and emergence of compartmentalized variants later in infection, on average at age 13.5 months, and becoming fully apparent in the CSF by age 18 months. Overall, compartmentalized viral replication in the CNS occurred in half of children by year three.</p> </div