Search

Abstract Background Long-term survival of HIV-1 infected individuals is usually achieved by continuous administration of combination antiretroviral therapy (ART). An exception to this scenario is represented by HIV-1 infected nonprogressors (NP) which maintain relatively high circulating CD4+ T cells without clinical symptoms for several years in the absence of ART. Several lines of evidence indicate an important role of the T-cell response in the modulation of HIV-1 infection during the acute and chronic phase of the disease. Results We analyzed the functional and the differentiation phenotype of Nef- and Tat-specific CD8+ T cells in a cohort of HIV-1 infected NP in comparison to progressors, ART-treated seropositive individuals and individuals undergoing a single cycle of ART interruption. We observed that a distinctive feature of NP is the presence of Nef-specific CD45RA+ CD8+ T cells secreting MIP-1beta but not IFN-gamma. This population was present in 7 out of 11 NP. CD45RA+ IFN-gammaneg MIP-1beta+ CD8+ T cells were not detected in HIV-1 infected individuals under ART or withdrawing from ART and experiencing a rebounding viral replication. In addition, we detected Nef-specific CD45RA+ IFN-gammaneg MIP-1beta+ CD8+ T cells in only 1 out of 10 HIV-1 infected individuals with untreated progressive disease. Conclusion The novel antigen-specific CD45RA+ IFN-gammaneg MIP-1beta+ CD8+ T cell population represents a new candidate marker of long-term natural control of HIV-1 disease progression and a relevant functional T-cell subset in the evaluation of the immune responses induced by candidate HIV-1 vaccines.

Vaccination strategies aimed at maturing broadly neutralizing antibodies (bnAbs) from naïve precursors are hindered by unusual features that characterize these Abs, including insertions and deletions (indels). Longitudinal studies of natural HIV infection cases shed light on the complex processes underlying bnAb development and have suggested a role for superinfection as a potential enhancer of neutralization breadth. Here we describe the development of a potent bnAb lineage that was elicited by two founder viruses to inform vaccine design. The V3-glycan targeting bnAb lineage (PC39-1) was isolated from subtype C-infected IAVI Protocol C elite neutralizer, donor PC39, and is defined by the presence of multiple independent insertions in CDRH1 that range from 1-11 amino acids in length. Memory B cell members of this lineage are predominantly atypical in phenotype yet also span the class-switched and antibody-secreting cell compartments. Development of neutralization breadth occurred concomitantly with extensive recombination between founder viruses before each virus separated into two distinct population "arms" that evolved independently to escape the PC39-1 lineage. Ab crystal structures show an extended CDRH1 that can help stabilize the CDRH3. Overall, these findings suggest that early exposure of the humoral system to multiple related Env molecules could promote the induction of bnAbs by focusing Ab responses to conserved epitopes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Joyce et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

HIV reservoirs persist in anatomic compartments despite antiretroviral therapy (ART). Characterizing archival HIV DNA in the central nervous system (CNS) and other tissues is crucial to inform cure strategies. We evaluated paired autopsy brain-frontal cortex (FC), occipital cortex (OCC), and basal ganglia (BG)-and peripheral lymphoid tissues from 63 people with HIV. Participants passed away while virally suppressed on ART at the last visit and without evidence of CNS opportunistic disease. We quantified total HIV DNA in all participants and obtained full-length HIV-envelope (FL HIV- env ) sequences from a subset of 14 participants. We detected HIV DNA ( gag ) in most brain (65.1%) and all lymphoid tissues. Lymphoid tissues had higher HIV DNA levels than the brain ( P  < 0.01). Levels of HIV gag between BG and FC were similar ( P  > 0.2), while OCC had the lowest levels ( P  = 0.01). Females had higher HIV DNA levels in tissues than males ( gag , P  = 0.03; 2-LTR, P  = 0.05), suggesting possible sex-associated mechanisms for HIV reservoir persistence. Most FL HIV- env sequences ( n  = 143) were intact, while 42 were defective. Clonal sequences were found in 8 out of 14 participants, and 1 participant had clonal defective sequences in the brain and spleen, suggestive of cell migration. From 10 donors with paired brain and lymphoid sequences, we observed evidence of compartmentalized sequences in 2 donors. Our data further the idea that the brain is a site for archival HIV DNA during ART where compartmentalized provirus may occur in a subset of people. Future studies assessing FL HIV-provirus and replication competence are needed to further evaluate the HIV reservoirs in tissues. IMPORTANCE HIV infection of the brain is associated with adverse neuropsychiatric outcomes, despite efficient antiretroviral treatment. HIV may persist in reservoirs in the brain and other tissues, which can seed virus replication if treatment is interrupted, representing a major challenge to cure HIV. We evaluated reservoirs and genetic features in postmortem brain and lymphoid tissues from people with HIV who passed away during suppressed HIV replication. We found a differential distribution of HIV reservoirs across brain regions which was lower than that in lymphoid tissues. We observed that most HIV reservoirs in tissues had intact envelope sequences, suggesting they could potentially generate replicative viruses. We found that women had higher HIV reservoir levels in brain and lymphoid tissues than men, suggesting possible sex-based mechanisms of maintenance of HIV reservoirs in tissues, warranting further investigation. Characterizing the archival HIV DNA in tissues is important to inform future HIV cure strategies., Competing Interests: The authors declare no conflict of interest.

A primary function of HIV-1 Nef is the enhancement of viral infectivity and replication. Whether counteraction of the antiretroviral proteins SERINC3 and SERINC5 is the cause of this positive influence on viral growth-rate and infectivity remains unclear. Here, we utilized CRISPR/Cas9 to knockout SERINC3 and SERINC5 in a leukemic CD4-positive T cell line (CEM) that displays nef-related infectivity and growth-rate phenotypes. Viral replication was attenuated in CEM cells infected with HIV-1 lacking Nef (HIV-1ΔNef). This attenuated growth-rate phenotype was observed regardless of whether the coding regions of the serinc3 or serinc5 genes were intact. Moreover, knockout of serinc5 alone or of both serinc5 and serinc3 together failed to restore the infectivity of HIV1ΔNef virions produced from infected CEM cells. Our results corroborate a similar study using another T-lymphoid cell line (MOLT-3) and indicate that the antagonism of SERINC3 and SERINC5 does not fully explain the virology of HIV-1 lacking Nef., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies that bind particular antigens 1 . In most mammals, antibodies are composed of heavy and light chains that are generated sequentially by recombination of V, D (for heavy chains), J and C gene segments. Each chain contains three complementarity-determining regions (CDR1-CDR3), which contribute to antigen specificity. Certain heavy and light chains are preferred for particular antigens 2-22 . Here we consider pairs of B cells that share the same heavy chain V gene and CDRH3 amino acid sequence and were isolated from different donors, also known as public clonotypes 23,24 . We show that for naive antibodies (those not yet adapted to antigens), the probability that they use the same light chain V gene is around 10%, whereas for memory (functional) antibodies, it is around 80%, even if only one cell per clonotype is used. This property of functional antibodies is a phenomenon that we call light chain coherence. We also observe this phenomenon when similar heavy chains recur within a donor. Thus, although naive antibodies seem to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain., (© 2022. The Author(s).)

Purpose: To evaluate the diagnostic performance of layer-specific cardiac MRI feature-tracking (FT) strain analysis in patients with acute myocarditis., Materials and Methods: Seventy patients (mean age, 43 years ± 19 [SD]; 46 men) with clinically defined acute myocarditis and 42 healthy controls who underwent cardiac MRI from March 2014 to November 2018 were retrospectively analyzed. FT-based left ventricular peak systolic global longitudinal strain (GLS) and global circumferential strain (GCS) were assessed at subendocardial, midmyocardial, and subepicardial layers. The 2018 Lake Louise criteria (LLC) were assessed. Patients with myocarditis were dichotomized into two groups: those with preserved and those with reduced ejection fraction. For statistical analysis, unpaired t test, one-way analysis of variance, Pearson correlation, and receiver operating characteristic analysis were used., Results: GLS and GCS values of all layers (eg, midmyocardial GCS: -21.3% ± 5.5 vs -28.0% ± 4.3; P < .001) were impaired in patients with myocarditis compared with controls. Only subepicardial GLS (-20.0% ± 3.3 vs -17.5% ± 3.3; P < .001) and midmyocardial GCS values (-28.0% ± 4.3 vs -23.1% ± 4.3; P < .001) could differentiate between controls and patients with preserved ejection fraction. Midmyocardial GCS correlated with inflammatory myocardial parameters (eg, late gadolinium enhancement percentage, r = 0.48, P < .001). Midmyocardial GCS (area under the receiver operating characteristic curve [AUC], 0.82) and subepicardial GLS (AUC, 0.77) had the highest diagnostic performance for acute myocarditis diagnosis ( P < .05 against all other strain parameters). The diagnostic performance of the 2018 LLC was significantly improved by inclusion of these two strain parameters (AUC, 0.92 vs 0.97; P = .04)., Conclusion: Diagnostic performance of cardiac MRI FT strain was different between myocardial layers in acute myocarditis, with midmyocardial GCS and subepicardial GLS providing the highest diagnostic performance. Keywords: MRI, Cardiac, Heart, Left Ventricle, Inflammation, Tissue Characterization, MR-Functional Imaging, Feature-Tracking Strain, Acute Myocarditis Supplemental material is available for this article. © RSNA, 2022., Competing Interests: Disclosures of conflicts of interest: A.I. Funded by BONFOR-Forschungskommission der Medizinischen Fakultät Bonn and by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy, EXC2151–390873048; funders had no influence on study conceptualization and design, collection and analysis of the data, manuscript preparation, or the decision to publish. D. Kravenchko No relevant relationships. N.M. No relevant relationships. C.E. No relevant relationships. L.M.B. No relevant relationships. T.V. No relevant relationships. D.T. No relevant relationships. D.D. No relevant relationships. S.Z. No relevant relationships. U.A. No relevant relationships. D. Kuetting No relevant relationships. J.A.L. Received payments for lectures from Philips Healthcare and for activities related to the scientific advisory board for Bayer HealthCare., (© 2022 by the Radiological Society of North America, Inc.)

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 broadly neutralizing antibodies (bnAbs) can suppress viremia and protect against HIV infection. However, their elicitation is made difficult by low frequencies of appropriate precursor B cell receptors and the complex maturation pathways required to generate bnAbs from these precursors. Antibody genes can be engineered into B cells for expression as both a functional antigen receptor on cell surfaces and as secreted antibody. Here, we show that HIV bnAb-engineered primary mouse B cells can be adoptively transferred and vaccinated in immunocompetent mice resulting in the expansion of durable bnAb memory and long-lived plasma cells. Somatic hypermutation after immunization indicates that engineered cells have the capacity to respond to an evolving pathogen. These results encourage further exploration of engineered B cell vaccines as a strategy for durable elicitation of HIV bnAbs to protect against infection and as a contributor to a functional HIV cure.

The host protein SERINC5 inhibits the infectivity of HIV-1 virions in an Env-dependent manner and is counteracted by Nef. The conformation of the Env trimer reportedly correlates with sensitivity to SERINC5. Here, we tested the hypothesis that the "open" conformation of the Env trimer revealed by sensitivity to the V3-loop specific antibody 447-52D directly correlates with sensitivity to SERINC5. Of five Envs tested, SF162 was the most sensitive to neutralization by 447-52D, but it was not the most sensitive to SERINC5; instead the Env of LAI was substantially more sensitive to SERINC5 than all the other Envs. Mutational opening of the trimer by substitution of two tyrosines that mediate interaction between the V2 and V3 loops sensitized the Envs of JRFL and LAI to 447-52D as previously reported, but only BaL was sensitized to SERINC5. These data suggest that trimer "openness" is not sufficient for sensitivity to SERINC5., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The membrane-proximal external region (MPER) of HIV-1 envelope glycoprotein (Env) can be targeted by neutralizing antibodies of exceptional breadth. MPER antibodies usually have long, hydrophobic CDRH3s, lack activity as inferred germline precursors, are often from the minor IgG3 subclass, and some are polyreactive, such as 4E10. Here we describe an MPER broadly neutralizing antibody from the major IgG1 subclass, PGZL1, which shares germline V/D-region genes with 4E10, has a shorter CDRH3, and is less polyreactive. A recombinant sublineage variant pan-neutralizes a 130-isolate panel at 1.4 μg/ml (IC 50 ). Notably, a germline revertant with mature CDR3s neutralizes 12% of viruses and still binds MPER after DJ reversion. Crystal structures of lipid-bound PGZL1 variants and cryo-EM reconstruction of an Env-PGZL1 complex reveal how these antibodies recognize MPER and viral membrane. Discovery of common genetic and structural elements among MPER antibodies from different patients suggests that such antibodies could be elicited using carefully designed immunogens.

Long-read next-generation amplicon sequencing shows promise for studying complete genes or genomes from complex and diverse populations. Current long-read sequencing technologies have challenging error profiles, hindering data processing and incorporation into downstream analyses. Here we consider the problem of how to reconstruct, free of sequencing error, the true sequence variants and their associated frequencies from PacBio reads. Called 'amplicon denoising', this problem has been extensively studied for short-read sequencing technologies, but current solutions do not always successfully generalize to long reads with high indel error rates. We introduce two methods: one that runs nearly instantly and is very accurate for medium length reads and high template coverage, and another, slower method that is more robust when reads are very long or coverage is lower. On two Mock Virus Community datasets with ground truth, each sequenced on a different PacBio instrument, and on a number of simulated datasets, we compare our two approaches to each other and to existing algorithms. We outperform all tested methods in accuracy, with competitive run times even for our slower method, successfully discriminating templates that differ by a just single nucleotide. Julia implementations of Fast Amplicon Denoising (FAD) and Robust Amplicon Denoising (RAD), and a webserver interface, are freely available., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The plasma membrane is a site of conflict between host defenses and many viruses. One aspect of this conflict is the host's attempt to eliminate infected cells using innate and adaptive cell-mediated immune mechanisms that recognize features of the plasma membrane characteristic of viral infection. Another is the expression of plasma membrane-associated proteins, so-called restriction factors, which inhibit enveloped virions directly. HIV-1 encodes two countermeasures to these host defenses: The membrane-associated accessory proteins Vpu and Nef. In addition to inhibiting cell-mediated immune-surveillance, Vpu and Nef counteract membrane-associated restriction factors. These include BST-2, which traps newly formed virions at the plasma membrane unless counteracted by Vpu, and SERINC5, which decreases the infectivity of virions unless counteracted by Nef. Here we review key features of these two antiviral proteins, and we review Vpu and Nef, which deplete them from the plasma membrane by co-opting specific cellular proteins and pathways of membrane trafficking and protein-degradation. We also discuss other plasma membrane proteins modulated by HIV-1, particularly CD4, which, if not opposed in infected cells by Vpu and Nef, inhibits viral infectivity and increases the sensitivity of the viral envelope glycoprotein to host immunity., Competing Interests: The authors declare no conflict of interest.

The VH1-2 restricted VRC01-class of antibodies targeting the HIV envelope CD4 binding site are a major focus of HIV vaccine strategies. However, a detailed analysis of VRC01-class antibody development has been limited by the rare nature of these responses during natural infection and the lack of longitudinal sampling of such responses. To inform vaccine strategies, we mapped the development of a VRC01-class antibody lineage (PCIN63) in the subtype C infected IAVI Protocol C neutralizer PC063. PCIN63 monoclonal antibodies had the hallmark VRC01-class features and demonstrated neutralization breadth similar to the prototype VRC01 antibody, but were 2- to 3-fold less mutated. Maturation occurred rapidly within ∼24 months of emergence of the lineage and somatic hypermutations accumulated at key contact residues. This longitudinal study of broadly neutralizing VRC01-class antibody lineage reveals early binding to the N276-glycan during affinity maturation, which may have implications for vaccine design., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite the major role of Gag in establishing resistance of HIV-1 to protease inhibitors (PIs), very limited data are available on the total contribution of Gag residues to resistance to PIs. To identify in detail Gag residues and structural interfaces associated with the development of HIV-1 resistance to PIs, we traced viral evolution under the pressure of PIs using Gag-protease single genome sequencing and coevolution analysis of protein sequences in 4 patients treated with PIs over a 9-year period. We identified a total of 38 Gag residues correlated with the protease, 32 of which were outside Gag cleavage sites. These residues were distributed in 23 Gag-protease groups of coevolution, with the viral matrix and the capsid represented in 87% and 52% of the groups. In addition, we uncovered the distribution of Gag correlated residues in specific protein surfaces of the inner face of the viral matrix and at the Cyclophilin A binding loop of the capsid. In summary, our findings suggest a tight interdependency between Gag structural proteins and the protease during the development of resistance of HIV-1 to PIs.

Unlabelled: Molecular evolutionary arms races between viruses and their hosts are important drivers of adaptation. These Red Queen dynamics have been frequently observed in primate retroviruses and their antagonists, host restriction factor genes, such as APOBEC3F/G, TRIM5-α, SAMHD1, and BST-2. Host restriction factors have experienced some of the most intense and pervasive adaptive evolution documented in primates. Recently, two novel host factors, SERINC3 and SERINC5, were identified as the targets of HIV-1 Nef, a protein crucial for the optimal infectivity of virus particles. Here, we compared the evolutionary fingerprints of SERINC3 and SERINC5 to those of other primate restriction factors and to a set of other genes with diverse functions. SERINC genes evolved in a manner distinct from the canonical arms race dynamics seen in the other restriction factors. Despite their antiviral activity against HIV-1 and other retroviruses, SERINC3 and SERINC5 have a relatively uneventful evolutionary history in primates., Importance: Restriction factors are host proteins that block viral infection and replication. Many viruses, like HIV-1 and related retroviruses, evolved accessory proteins to counteract these restriction factors. The importance of these interactions is evidenced by the intense adaptive selection pressures that dominate the evolutionary histories of both the host and viral genes involved in this so-called arms race. The dynamics of these arms races can point to mechanisms by which these viral infections can be prevented. Two human genes, SERINC3 and SERINC5, were recently identified as targets of an HIV-1 accessory protein important for viral infectivity. Unexpectedly, we found that these SERINC genes, unlike other host restriction factor genes, show no evidence of a recent evolutionary arms race with viral pathogens., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: HIV-1 Vpu decreases the exposure of epitopes within the viral envelope glycoprotein (Env) on the surface of infected cells by downregulating both BST2 and CD4. To test the hypothesis that inhibiting Vpu activity would increase the exposure of these epitopes and sensitize infected cells to antibody-dependent cellular cytotoxicity (ADCC), we treated cells with the Nedd8 activation enzyme (NAE) inhibitor MLN4924, which inhibits the cullin1-based ubiquitin ligase complex coopted by Vpu to degrade cellular targets. Treatment of HeLa cells with MLN4924 or expression of a dominant negative mutant of cullin1 inhibited the Vpu-mediated downregulation of CD4 but not the downregulation of BST2. NAE inhibition also increased the surface exposure of CD4-induced epitopes within Env on HEK293 cells containing an inducible HIV genome, on infected CEM T cells, and on infected primary T cells. In contrast, the Vpu-mediated downregulation of BST2 was substantially inhibited by MLN4924 only when T cells were treated with alpha interferon (IFN-α) to induce high levels of BST2 expression. As reported previously, the absence of vpu or nef and even more so the combined absence of these two genes sensitized infected cells to ADCC. However, NAE inhibition affected ADCC minimally. Paradoxically, even in infected, IFN-treated cells in which NAE inhibition substantially rescued the surface level of BST2, the surface level of Env detected with an antibody recognizing a CD4-independent epitope (2G12) was minimally increased. Mutation of the C-terminal Vpu residue W76, which supports the ability of Vpu to stimulate virion release by displacing BST2 from assembly sites on the plasma membrane by a cullin1-independent mechanism, increased the exposure of Env detected by 2G12 on infected T cells. Thus, inhibiting the displacement function of Vpu together with its ability to degrade CD4 and BST2 may be required to sensitize infected cells to ADCC., Importance: Pathogenic viruses encode gene products that enable evasion of host immune surveillance mechanisms. One such mechanism is antibody-dependent cellular cytotoxicity (ADCC), whereby host antibodies bind envelope glycoproteins of the virus that are inserted into the cellular membrane and direct the destruction of infected cells. Targeting pharmacologically the activity of HIV-1 Vpu, which contributes to evasion of ADCC, could potentially sensitize infected cells to this immune surveillance mechanism, an outcome that would have therapeutic implications with respect to the goal of curing HIV-1 infection. The Nedd8 activation enzyme inhibitor MLN4924 blocks the activity of the host ubiquitin ligase that Vpu coopts to direct the degradation of CD4 and BST2. We observed that while MLN4924 partially reverses the activity of Vpu and could become part of a therapeutic approach by virtue of CD4-induced epitope exposure, sufficient Vpu activity as an antagonist of BST2 persists despite this drug to allow escape from ADCC., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Methods for identifying physiologically relevant CD8 T-cell epitopes are critically important not only for the development of T-cell-based vaccines but also for understanding host-pathogen interactions. As experimentally mapping an optimal CD8 T-cell epitope is a tedious procedure, many bioinformatic tools have been developed that predict which peptides bind to a given MHC molecule. We assessed the ability of the CD8 T-cell epitope prediction tools syfpeithi, ctlpred and iedb to foretell nine experimentally mapped optimal HIV-specific epitopes. Randomly - for any of the subjects' HLA type and with any matching score - the optimal epitope was predicted in seven of nine epitopes using syfpeithi, in three of nine epitopes using ctlpred and in all nine of nine epitopes using iedb. The optimal epitope within the three highest ranks was given in four of nine epitopes applying syfpeithi, in two of nine epitopes applying ctlpred and in seven of nine epitopes applying iedb when screening for all of the subjects' HLA types. Knowing the HLA restriction of the peptide of interest improved the ranking of the optimal epitope within the predicted results. Epitopes restricted by common HLA alleles were more likely to be predicted than those restricted by uncommon HLA alleles. Epitopes with aberrant lengths compared with the usual HLA-class I nonamers were most likely not predicted. Application of epitope prediction tools together with literature searches for already described optimal epitopes narrows down the possibilities of optimal epitopes within a screening peptide of interest. However, in our opinion, the actual fine-mapping of a CD8 T-cell epitope cannot yet be replaced., (© 2014 John Wiley & Sons Ltd.)

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef is heavily targeted by CD8(+) T lymphocytes (CTLs) during acute infection and therefore is included in many candidate vaccines. We investigated whether CTL targeting of Nef during acute infection contributes to immune control by disrupting the function of Nef. The sequence and function of Nef in parallel with CTL responses were assessed longitudinally from peak viremia until the viremia set point in a cohort of six subjects with acute infection. All but one individual had a single founder strain. Nef-specific CTL responses were detected in all subjects and declined in magnitude over time. These responses were associated with mutations, but none of the mutations were detected in important functional motifs. Nef-mediated downregulation of CD4 and major histocompatibility complex (MHC) class I molecules was better preserved in acute infection than in chronic infection. Finally, Nef-specific CTL responses were not associated with a reduction in viremia from its acute-phase peak. Our results indicate that CTLs targeting Nef epitopes outside critical functional domains have little effect on the pathogenic functions of Nef, rendering these responses ineffective in acute infection. Importance: These data indicate that using the whole Nef protein as a vaccine immunogen likely allows immunodominance that leads to targeting of CTL responses that are rapidly escaped with little effect on Nef-mediated pathogenic functions. Pursuing vaccination approaches that can more precisely direct responses to vulnerable areas would maximize efficacy. Until vaccine-induced targeting can be optimized, other approaches, such as the use of Nef function inhibitors or the pursuit of immunotherapies such as T cell receptor gene therapy or adoptive transfer, may be more likely to result in successful control of viremia., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

HIV evades CD8 T cell mediated pressure by viral escape mutations in targeted CD8 T cell epitopes. A viral escape mutation can lead to a decline of the respective CD8 T cell response. Our question was what happened after the decline of a CD8 T cell response and - in the case of viral escape - if a new CD8 T cell response towards the mutated antigen could be generated in a population not selected for certain HLA alleles. We studied 19 antiretroviral-naïve HIV-1 infected individuals with different disease courses longitudinally. A median number of 12 (range 2-24) CD8 T cell responses towards Gag and Nef were detected per study subject. A total of 30 declining CD8 T cell responses were studied in detail and viral sequence analyses showed amino acid changes in 25 (83%) of these. Peptide titration assays and definition of optimal CD8 T cell epitopes revealed 12 viral escape mutations with one de-novo response (8%). The de-novo response, however, showed less effector functions than the original CD8 T cell response. In addition we identified 4 shifts in immunodominance. For one further shift in immunodominance, the mutations occurred outside the optimal epitope and might represent processing changes. Interestingly, four adaptations to the virus (the de-novo response and 3 shifts in immunodominance) occurred in the group of chronically infected progressors. None of the subjects with adaptation to the changing virus carried the HLA alleles B57, B*58:01 or B27. Our results show that CD8 T cell responses adapt to the mutations of HIV. However it was limited to only 20% (5 out of 25) of the epitopes with viral sequence changes in a cohort not expressing protective HLA alleles.

Pulmonary expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) is critically important for normal functional maturation of alveolar macrophages. We found previously that lung GM-CSF is dramatically suppressed in mice exposed to hyperoxia. Alveolar epithelial cells (AEC) are a major source of GM-CSF in the peripheral lung, and in vivo hyperoxia resulted in greatly reduced expression of GM-CSF protein by AEC ex vivo. We now explore the mechanisms responsible for this effect, using primary cultures of murine AEC exposed to hyperoxia in vitro. Exposure of AEC to 80% oxygen/5% CO(2) for 48 h did not induce overt toxicity, but resulted in significantly decreased GM-CSF protein and mRNA expression compared with cells in normoxia. Similar effects were seen when AEC were stressed with serum deprivation, an alternative inducer of oxidative stress. The effects in AEC were opposite those in a murine lung epithelial cell line (MLE-12 cells), in which hyperoxia induced GM-CSF expression. Both hyperoxia and serum deprivation resulted in increased intracellular reactive oxygen species (ROS) in AEC. Hyperoxia and serum deprivation induced significantly accelerated turnover of GM-CSF mRNA. Treatment of AEC with catalase during oxidative stress preserved GM-CSF protein and mRNA and was associated with stabilization of GM-CSF mRNA. We conclude that hyperoxia-induced suppression of AEC GM-CSF expression is a function of ROS-induced destabilization of GM-CSF mRNA. We speculate that AEC oxidative stress results in significantly impaired pulmonary innate immune defense due to effects on local GM-CSF expression in the lung.