Maria Pernas

A dual superinfection and recombination within HIV-1 subtype B 12 years after primoinfection.

Summary: To analyze superinfection in an HIV-1-infected patient showing high-risk practices, viral quasispecies were analyzed in pol and env genes in several plasma samples. Phylogenetic analysis in the reverse transcriptase fragment in pol gene identified a single virus in the first 3 samples analyzed, but 12 years after primoinfection, 3 different viral strains were detected in the patient quasispecies. This result suggests a superinfection with 2 HIV-1 strains, one of which showed the T215Y + M184V resistance mutations. The analysis in the env gene confirmed the existence of 3 different strains in the viral population, one of them a recombinant. This study illustrates that events of superinfection and recombination contribute to the viral genetic variability observed in HIV-1-infected individuals.

Coinfection and Superinfection in Patients with Long-Term, Nonprogressive HIV-1 Disease

Human immunodeficiency virus 1 (HIV-1) dual infections are considered important because they have been related to AIDS progression. We identified dual infections in 2 patients with long-term, nonprogressive HIV-1 disease; the first patient was diagnosed as being already coinfected, on the basis of the first sample analyzed, but a previous superinfection could not be excluded; the second patient was diagnosed as having a superinfection, on the basis of the 9-year difference between the viral dating of the 2 strains. Dual infections occur in patients with long-term, nonprogressive disease, with no immediate clinical manifestations. Such occurrences could indicate a general phenomenon in natural HIV-1 infections.

Initial Fitness Recovery of HIV-1 Is Associated with Quasispecies Heterogeneity and Can Occur without Modifications in the Consensus Sequence

Background Fitness recovery of HIV-1 “in vitro” was studied using viral clones that had their fitness decreased as a result of plaque-to-plaque passages. Principal Findings After ten large population passages, the viral populations showed an average increase of fitness, although with wide variations among clones. While 5 clones showed significant fitness increases, 3 clones showed increases that were only marginally significant (p<0.1), and 4 clones did not show any change. Fitness recovery was not accompanied by an increase in p24 production, but was associated with an increase in viral titer. Few mutations (an average of 2 mutations per genome) were detected in the consensus nucleotide sequence of the entire genome in all viral populations. Five of the populations did not fix any mutation, and three of them displayed marginally significant fitness increases, illustrating that fitness recovery can occur without detectable alterations of the consensus genomic sequence. The investigation of other possible viral factors associated with the initial steps of fitness recovery, showed that viral quasispecies heterogeneity increased between the initial clones and the passaged populations. A direct statistical correlation between viral heterogeneity and viral fitness was obtained. Conclusions Thus, the initial fitness recovery of debilitated HIV-1 clones was mediated by an increase in quasispecies heterogeneity. This observation, together with the invariance of the consensus sequence despite fitness increases demonstrates the relevance of quasispecies heterogeneity in the evolution of HIV-1 in cell culture.

Viremic HIV Infected Individuals with High CD4 T Cells and Functional Envelope Proteins Show Anti-gp41 Antibodies with Unique Specificity and Function

Background CD4 T-cell decay is variable among HIV-infected individuals. In exceptional cases, CD4 T-cell counts remain stable despite high plasma viremia. HIV envelope glycoprotein (Env) properties, namely tropism, fusion or the ability to induce the NK ligand NKp44L, or host factors that modulate Env cytopathic mechanisms may be modified in such situation. Methods We identified untreated HIV-infected individuals showing non-cytopathic replication (VL>10,000 copies/mL and CD4 T-cell decay<50 cells/µL/year, Viremic Non Progressors, VNP) or rapid progression (CD4 T-cells<350 cells/µL within three years post-infection, RP). We isolated full-length Env clones and analyzed their functions (tropism, fusion activity and capacity to induce NKp44L expression on CD4 cells). Anti-Env humoral responses were also analyzed. Results Env clones isolated from VNP or RP individuals showed no major phenotypic differences. The percentage of functional clones was similar in both groups. All clones tested were CCR5-tropic and showed comparable expression and fusogenic activity. Moreover, no differences were observed in their capacity to induce NKp44L expression on CD4 T cells from healthy donors through the 3S epitope of gp41. In contrast, anti- Env antibodies showed clear functional differences: plasma from VNPs had significantly higher capacity than RPs to block NKp44L induction by autologous viruses. Consistently, CD4 T-cells isolated from VNPs showed undetectable NKp44L expression and specific antibodies against a variable region flanking the highly conserved 3S epitope were identified in plasma samples from these patients. Conversely, despite continuous antigen stimulation, VNPs were unable to mount a broad neutralizing response against HIV. Conclusions Env functions (fusion and induction of NKp44L) were similar in viremic patients with slow or rapid progression to AIDS. However, differences in humoral responses against gp41 epitopes nearby 3S sequence may contribute to the lack of CD4 T cell decay in VNPs by blocking the induction of NKp44L by gp41.

Low-replicating viruses and strong anti-viral immune response associated with prolonged disease control in a superinfected HIV-1 LTNP elite controller.

Objective To study the causes for the lack of clinical progression in a superinfected HIV-1 LTNP elite controller patient. Methodology and Principal Findings We studied host genetic, virological and immunological factors associated with viral control in a SI long term non progressor elite controller (LTNP-EC). The individual contained both viruses and maintained undetectable viral loads for >20 years and he did not express any of the described host genetic polymorphisms associated with viral control. None of four full-length gp160 recombinants derived from the LTNP-EC replicated in heterologous peripheral blood mononuclear cells. CTL responses after SI were maintained in two samples separated by 9 years and they were higher in breadth and magnitude than responses seen in most of 250 treatment naïve patients and also 25 controller subjects. The LTNP-EC showed a neutralization response, against 4 of the 6 viruses analyzed, superior to other ECs. Conclusions The study demonstrated that a strong and sustained cellular and humoral immune response and low replicating viruses are associated with viral control in the superinfected LTNP-EC.

Identification of a cluster of HIV-1 controllers infected with low replicating viruses.

Long term non-progressor patients (LTNPs) are characterized by the natural control of HIV-1 infection. This control is related to host genetic, immunological and virological factors. In this work, phylogenetic analysis of the proviral nucleotide sequences in env gene from a Spanish HIV-1 LTNPs cohort identified a cluster of 6 HIV-1 controllers infected with closely-related viruses. The patients of the cluster showed common clinical and epidemiological features: drug user practices, infection in the same city (Madrid, Spain) and at the same time (late 70’s-early 80’s). All cluster patients displayed distinct host alleles associated with HIV control. Analysis of the virus envelope nucleotide sequences showed ancestral characteristic, lack of evolution and presence of rare amino-acids. Biological characterization of recombinant viruses with the envelope proteins from the cluster viruses showed very low replicative capacity in TZMbl and U87-CD4/CCR5 cells. The lack of clinical progression in the viral cluster patients with distinct combinations of protective host genotypes, but infected by low replicating viruses, indicate the important role of the virus in the non-progressor phenotype in these patients.

Factors Leading to the Loss of Natural Elite Control of HIV-1 Infection

ABSTRACT HIV-1 elite controllers (EC) maintain undetectable viral loads (VL) in the absence of antiretroviral treatment. However, these subjects have heterogeneous clinical outcomes, including a proportion that loses HIV-1 control over time. In this work, we compared, in a longitudinal design, transient EC, analyzed before and after the loss of virological control, with persistent EC. The aim was to identify factors leading to the loss of natural virological control of HIV-1 infection with a longitudinal retrospective study design. Gag-specific T-cell responses were assessed by in vitro intracellular polycytokine production quantified by flow cytometry. Viral diversity determinations and sequence dating were performed in proviral DNA by PCR amplification at limiting dilution of env and gag genes. The expression profile of 70 serum cytokines and chemokines was assessed by multiplex immunoassays. We identified transient EC as subjects with low Gag-specific T-cell polyfunctionality, high viral diversity, and high proinflammatory cytokine levels before the loss of control. Gag-specific T-cell polyfunctionality was inversely associated with viral diversity in transient controllers before the loss of control (r = −0.8; P = 0.02). RANTES was a potential biomarker of transient control. This study identified virological and immunological factors, including inflammatory biomarkers associated with two different phenotypes within EC. These results may allow a more accurate definition of EC, which could help in better clinical management of these individuals and in the development of future curative approaches. IMPORTANCE There is a rare group of HIV-infected patients who have the extraordinary capacity to maintain undetectable viral load levels in the absence of antiretroviral treatment, the so-called HIV-1 elite controllers (EC). However, there is a proportion within these subjects that eventually loses this capability. In this work, we found differences in virological and immune factors, including soluble inflammatory biomarkers, between subjects with persistent control of viral replication and EC that will lose virological control. The identification of these factors could be a key point for a right medical care of those EC who are going to lose natural control of viral replication and for the design of future immunotherapeutic strategies using as a model the natural persistent control of HIV infection.

Prevalence of HIV-1 dual infection in long-term nonprogressor-elite controllers

Introduction:Human immunodeficiency virus type 1 (HIV-1) dual infection (DI) in long-term nonprogressor–elite controller patients (LTNP-EC) has been described only in sporadic cases and then, consequences in disease progression are not clearly established. To fill-up this limited knowledge, we analyzed, for the first time, the prevalence, host genetic polymorphisms, and clinical consequences of HIV-1 DI in a group of LTNP-EC. Methods:For DI detection, nucleotide sequences in env gene from viruses from 20 LTNP-EC were analyzed by maximum likelihood. Epidemiological and clinical parameters and host factors of patients were also studied. Results:DI was detected in 4 (20%) of the 20 LTNP-EC, of which 3 maintained the elite controller status. CD4+ T-cell counts were not different between single and DI patients although higher CD8+ T-cell counts were observed in DI patients, and, consequently, the CD4+/CD8+ ratios were lower in LTNP-EC DI patients. Conclusions:Prevalence of HIV-1 DIs in LTNP-EC is similar to other groups of HIV-1 patients; in addition, DI was not associated with loss of disease control in the patients. These DI LTNP-EC patients showed, in comparison with single infected patients, higher numbers of CD8+ T cells and lower CD4+/CD8+ ratios.

HIV-1 Dual Infected LTNP-EC Patients Developed an Unexpected Antibody Cross-Neutralizing Activity.

This study evaluated the neutralization breadth in dually infected (DI) HIV-1 long-term non-progressor elite controller patients (LTNP-EC) using a representative minipanel of 6 viruses from 5 different subtypes. Our results showed an improved neutralization breadth in DI LTNP-EC patients when compared with matched LTNP single-infected patients. The role of viral diversity in neutralization was estimated with the Shannon Entropy and the p-distance in viral quasispecies. We found a positive correlation between neutralization breadth and diversity within the viral quasispecies. This correlation could explain why a group of LTNP-EC patients developed a broad neutralizing response despite having undetectable levels of viremia.

Viral Characteristics Associated with the Clinical Nonprogressor Phenotype Are Inherited by Viruses from a Cluster of HIV-1 Elite Controllers

ABSTRACT A small group of HIV-1-infected individuals, called long-term nonprogressors (LTNPs), and in particular a subgroup of LTNPs, elite controllers (LTNP-ECs), display permanent control of viral replication and lack of clinical progression. This control is the result of a complex interaction of host, immune, and viral factors. We identified, by phylogenetic analysis, a cluster of LTNP-ECs infected with very similar low-replication HIV-1 viruses, suggesting the contribution of common viral features to the clinical LTNP-EC phenotype. HIV-1 envelope (Env) glycoprotein mediates signaling and promotes HIV-1 fusion, entry, and infection, being a key factor of viral fitness in vitro, cytopathicity, and infection progression in vivo. Therefore, we isolated full-length env genes from viruses of these patients and from chronically infected control individuals. Functional characterization of the initial events of the viral infection showed that Envs from the LTNP-ECs were ineffective in the binding to CD4 and in the key triggering of actin/tubulin-cytoskeleton modifications compared to Envs from chronic patients. The viral properties of the cluster viruses result in a defective viral fusion, entry, and infection, and these properties were inherited by every virus of the cluster. Therefore, inefficient HIV-1 Env functions and signaling defects may contribute to the low viral replication capacity and transmissibility of the cluster viruses, suggesting a direct role in the LTNP-EC phenotype of these individuals. These results highlight the important role of viral characteristics in the LTNP-EC clinical phenotype. These Env viral properties were common to all the cluster viruses and thus support the heritability of the viral characteristics. IMPORTANCE HIV-1 long-term nonprogressor elite controller patients, due to their permanent control of viral replication, have been the object of numerous studies to identify the factors responsible for this clinical phenotype. In this work, we analyzed the viral characteristics of the envelopes of viruses from a phylogenetic cluster of LTNP-EC patients. These envelopes showed ineffective binding to CD4 and the subsequent signaling activity to modify actin/tubulin cytoskeletons, which result in low fusion and deficient entry and infection capacities. These Env viral characteristics could explain the nonprogressor clinical phenotype of these patients. In addition, these inefficient env viral properties were present in all viruses of the cluster, supporting the heritability of the viral phenotype. IMPORTANCE HIV-1 long-term nonprogressor elite controller patients, due to their permanent control of viral replication, have been the object of numerous studies to identify the factors responsible for this clinical phenotype. In this work, we analyzed the viral characteristics of the envelopes of viruses from a phylogenetic cluster of LTNP-EC patients. These envelopes showed ineffective binding to CD4 and the subsequent signaling activity to modify actin/tubulin cytoskeletons, which result in low fusion and deficient entry and infection capacities. These Env viral characteristics could explain the nonprogressor clinical phenotype of these patients. In addition, these inefficient env viral properties were present in all viruses of the cluster, supporting the heritability of the viral phenotype.