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1.  Long-Term Spontaneous Control of HIV-1 Is Related to Low Frequency of Infected Cells and Inefficient Viral Reactivation 
Journal of Virology  2016;90(13):6148-6158.
ABSTRACT
HIV establishes reservoirs of infected cells that persist despite effective antiretroviral therapy (ART). In most patients, the virus begins to replicate soon after treatment interruption. However, a low frequency of infected cells at the time of treatment interruption has been associated with delayed viral rebound. Likewise, individuals who control the infection spontaneously, so-called HIV-1 controllers (HICs), carry particularly low levels of infected cells. It is unclear, however, whether and how this small number of infected cells contributes to durable viral control. Here we compared 38 HICs with 12 patients on effective combined antiretroviral therapy (cART) and found that the low frequency of infected cells in the former subjects was associated both with less efficient viral reactivation in resting CD4+ T cells and with less efficient virion production ex vivo. We also found that a potent HIV-specific CD8+ T cell response was present only in those HICs whose CD4+ T cells produced virus ex vivo. Long-term spontaneous control of HIV infection in HICs thus appears to be sustained on the basis of the inefficient reactivation of viruses from a limited number of infected cells and the capacity of HICs to activate a potent HIV-specific CD8+ T cell response to counteract efficient viral reactivation events.
IMPORTANCE There is a strong scientific interest in developing strategies to eradicate the HIV-1 reservoir. Very rare HIV-1-infected patients are able to spontaneously control viremia for long periods of time (HIV-1 controllers [HICs]) and are put forward as a model of HIV-1 remission. Here, we show that the low viral reservoirs found in HICs are a critical part of the mechanisms underlying viral control and result in a lower probability of HIV-1 reactivation events, resulting in limited HIV-1 release and spread. We found that those HICs in whom viral reactivation and spread from CD4+ T cells in vitro were the most difficult were those with diminished CD8+ T cell responses. These results suggest that, in some settings, low HIV-1 reservoirs decisively contribute to at least the temporary control of infection without antiretroviral therapy. We believe that this work provides information of relevance in the context of the search for HIV-1 remission.
doi:10.1128/JVI.00419-16
PMCID: PMC4907242  PMID: 27122576
2.  Nonhuman TRIM5 Variants Enhance Recognition of HIV-1-Infected Cells by CD8+ T Cells 
Journal of Virology  2016;90(19):8552-8562.
ABSTRACT
Tripartite motif-containing protein 5 (TRIM5) restricts human immunodeficiency virus type 1 (HIV-1) in a species-specific manner by uncoating viral particles while activating early innate responses. Although the contribution of TRIM5 proteins to cellular immunity has not yet been studied, their interactions with the incoming viral capsid and the cellular proteasome led us to hypothesize a role for them. Here, we investigate whether the expression of two nonhuman TRIM5 orthologs, rhesus TRIM5α (RhT5) and TRIM-cyclophilin A (TCyp), both of which are potent restrictors of HIV-1, could enhance immune recognition of infected cells by CD8+ T cells. We illustrate how TRIM5 restriction improves CD8+ T-cell-mediated HIV-1 inhibition. Moreover, when TRIM5 activity was blocked by the nonimmunosuppressive analog of cyclosporine (CsA), sarcosine-3(4-methylbenzoate)–CsA (SmBz-CsA), we found a significant reduction in CD107a/MIP-1β expression in HIV-1-specific CD8+ T cells. This finding underscores the direct link between TRIM5 restriction and activation of CD8+ T-cell responses. Interestingly, cells expressing RhT5 induced stronger CD8+ T-cell responses through the specific recognition of the HIV-1 capsid by the immune system. The underlying mechanism of this process may involve TRIM5-specific capsid recruitment to cellular proteasomes and increase peptide availability for loading and presentation of HLA class I antigens. In summary, we identified a novel function for nonhuman TRIM5 variants in cellular immunity. We hypothesize that TRIM5 can couple innate viral sensing and CD8+ T-cell activation to increase species barriers against retrovirus infection.
IMPORTANCE New therapeutics to tackle HIV-1 infection should aim to combine rapid innate viral sensing and cellular immune recognition. Such strategies could prevent seeding of the viral reservoir and the immune damage that occurs during acute infection. The nonhuman TRIM5 variants, rhesus TRIM5α (RhT5) and TRIM-cyclophilin A (TCyp), are attractive candidates owing to their potency in sensing HIV-1 and blocking its activity. Here, we show that expression of RhT5 and TCyp in HIV-1-infected cells improves CD8+ T-cell-mediated inhibition through the direct activation of HIV-1-specific CD8+ T-cell responses. We found that the potency in CD8+ activation was stronger for RhT5 variants and capsid-specific CD8+ T cells in a mechanism that relies on TRIM5-dependent particle recruitment to cellular proteasomes. This novel mechanism couples innate viral sensing with cellular immunity in a single protein and could be exploited to develop innovative therapeutics for control of HIV-1 infection.
doi:10.1128/JVI.00819-16
PMCID: PMC5021395  PMID: 27440884
3.  Virus Immune Evasion: New Mechanism and Implications in Disease Outcome 
Advances in Virology  2012;2012:490549.
doi:10.1155/2012/490549
PMCID: PMC3462381  PMID: 23049554
4.  SAMHD1 Limits HIV-1 Antigen Presentation by Monocyte-Derived Dendritic Cells 
Journal of Virology  2015;89(14):6994-7006.
ABSTRACT
Monocyte-derived dendritic cells (MDDC) stimulate CD8+ cytotoxic T lymphocytes (CTL) by presenting endogenous and exogenous viral peptides via major histocompatibility complex class I (MHC-I) molecules. MDDC are poorly susceptible to HIV-1, in part due to the presence of SAMHD1, a cellular enzyme that depletes intracellular deoxynucleoside triphosphates (dNTPs) and degrades viral RNA. Vpx, an HIV-2/SIVsm protein absent from HIV-1, antagonizes SAMHD1 by inducing its degradation. The impact of SAMHD1 on the adaptive cellular immune response remains poorly characterized. Here, we asked whether SAMHD1 modulates MHC-I-restricted HIV-1 antigen presentation. Untreated MDDC or MDDC pretreated with Vpx were exposed to HIV-1, and antigen presentation was examined by monitoring the activation of an HIV-1 Gag-specific CTL clone. SAMHD1 depletion strongly enhanced productive infection of MDDC as well as endogenous HIV-1 antigen presentation. Time-lapse microscopy analysis demonstrated that in the absence of SAMHD1, the CTL rapidly killed infected MDDC. We also report that various transmitted/founder (T/F) HIV-1 strains poorly infected MDDC and, as a consequence, did not stimulate CTL. Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyping of T/F alleviated a block in viral entry and induced antigen presentation only in the absence of SAMHD1. Furthermore, by using another CTL clone that mostly recognizes incoming HIV-1 antigens, we demonstrate that SAMHD1 does not influence exogenous viral antigen presentation. Altogether, our results demonstrate that the antiviral activity of SAMHD1 impacts antigen presentation by DC, highlighting the link that exists between restriction factors and adaptive immune responses.
IMPORTANCE Upon viral infection, DC may present antigens derived from incoming viral material in the absence of productive infection of DC or from newly synthesized viral proteins. In the case of HIV, productive infection of DC is blocked at an early postentry step. This is due to the presence of SAMHD1, a cellular enzyme that depletes intracellular levels of dNTPs and inhibits viral reverse transcription. We show that the depletion of SAMHD1 in DCs strongly stimulates the presentation of viral antigens derived from newly produced viral proteins, leading to the activation of HIV-1-specific cytotoxic T lymphocytes (CTL). We further show in real time that the enhanced activation of CTL leads to killing of infected DCs. Our results indicate that the antiviral activity of SAMHD1 not only impacts HIV replication but also impacts antigen presentation by DC. They highlight the link that exists between restriction factors and adaptive immune responses.
doi:10.1128/JVI.00069-15
PMCID: PMC4473575  PMID: 25926647
5.  Early Antigen Presentation of Protective HIV-1 KF11Gag and KK10Gag Epitopes from Incoming Viral Particles Facilitates Rapid Recognition of Infected Cells by Specific CD8+ T Cells 
Journal of Virology  2013;87(5):2628-2638.
CD8+ T cells are major players in antiviral immunity against human immunodeficiency virus type 1 (HIV-1) through recognition of viral epitopes presented on the surface of infected cells. However, the early events involving HIV-1 epitope presentation to CD8+ T cells remain poorly understood but are nonetheless crucial for the rapid clearance of virus-infected cells. Here, we comprehensively studied the kinetics of antigen presentation of two protective epitopes, KF11Gag and KK10Gag, restricted by HLA alleles B*57:01 and B*27:05, respectively, and compared these to KY9Pol and VL9Vpr epitopes in a single cycle of HIV-1 replication. We consistently demonstrate differences in epitope presentation kinetics, with very early presentation, within 3 h postinfection, for the protective KF11Gag, KK10Gag epitopes, and KY9Pol but only late presentation for VL9Vpr. We show that this early presentation relies on the antigen being presented from incoming viral particles and is correlated with rapid CD8+ T cell activation and clearance of virus-infected cells. Additionally, our data indicate a dose-response dependency between the levels of CD8+ T cell activation and the amount of virus inoculum. These data reflect a proof of principle emphasizing the importance of identifying early-presented viral epitopes for rapid elimination of HIV-1-infected cells.
doi:10.1128/JVI.02131-12
PMCID: PMC3571362  PMID: 23255798
6.  Discordant Impact of HLA on Viral Replicative Capacity and Disease Progression in Pediatric and Adult HIV Infection 
PLoS Pathogens  2015;11(6):e1004954.
HLA class I polymorphism has a major influence on adult HIV disease progression. An important mechanism mediating this effect is the impact on viral replicative capacity (VRC) of the escape mutations selected in response to HLA-restricted CD8+ T-cell responses. Factors that contribute to slow progression in pediatric HIV infection are less well understood. We here investigate the relationship between VRC and disease progression in pediatric infection, and the effect of HLA on VRC and on disease outcome in adult and pediatric infection. Studying a South African cohort of >350 ART-naïve, HIV-infected children and their mothers, we first observed that pediatric disease progression is significantly correlated with VRC. As expected, VRCs in mother-child pairs were strongly correlated (p = 0.004). The impact of the protective HLA alleles, HLA-B*57, HLA-B*58:01 and HLA-B*81:01, resulted in significantly lower VRCs in adults (p<0.0001), but not in children. Similarly, in adults, but not in children, VRCs were significantly higher in subjects expressing the disease-susceptible alleles HLA-B*18:01/45:01/58:02 (p = 0.007). Irrespective of the subject, VRCs were strongly correlated with the number of Gag CD8+ T-cell escape mutants driven by HLA-B*57/58:01/81:01 present in each virus (p = 0.0002). In contrast to the impact of VRC common to progression in adults and children, the HLA effects on disease outcome, that are substantial in adults, are small and statistically insignificant in infected children. These data further highlight the important role that VRC plays both in adult and pediatric progression, and demonstrate that HLA-independent factors, yet to be fully defined, are predominantly responsible for pediatric non-progression.
Author Summary
HLA plays a central role in determining disease outcome in adult HIV infection. A principal mechanism by which this HLA effect is mediated is via viral replicative capacity (VRC), protective HLA alleles such as HLA-B*57 driving the selection of viral escape mutants that reduce VRC. The factors contributing to the diverse disease progression rates observed in infected children, however, remain uncertain. We here address the role of HLA and VRC in pediatric disease progression in a large cohort in Kimberley, South Africa. The findings highlight the consistent and important role of VRC in both adult and pediatric progression. However, the impact of key HLA molecules in shaping disease outcome in adult infection is notably absent in pediatric infection. Further studies of pediatric infection therefore provide the potential to gain critical new insights into HLA-independent mechanisms of HIV disease non-progression that predominate in HIV-infected but healthy, ART-naive children. Understanding these mechanisms remains of direct relevance to the development of future interventions to minimize HIV disease.
doi:10.1371/journal.ppat.1004954
PMCID: PMC4468173  PMID: 26076345
7.  Novel Two-Round Phenotypic Assay for Protease Inhibitor Susceptibility Testing of Recombinant and Primary HIV-1 Isolates 
Journal of Clinical Microbiology  2012;50(12):3909-3916.
Antiretroviral drug susceptibility tests facilitate therapeutic management of HIV-1-infected patients. Although genotyping systems are affordable, inaccuracy in the interpretation of complex mutational patterns may limit their usefulness. Currently available HIV-1 phenotypic assays are based on the generation of recombinant viruses in which the specific viral gene of interest, derived from a patient plasma sample, is cloned into a susceptible genetic viral backbone prior to in vitro drug susceptibility evaluation. Nevertheless, in the case of protease inhibitors, not only are mutations in the HIV-1 protease-coding region involved in resistance, but the role of Gag in drug susceptibility has also recently been reported. In order to avoid the inherent limitations resulting from partial cloning of the viral genome, we designed and evaluated a new experimental strategy to test the in vitro susceptibility of primary viral isolates to protease inhibitors. Our protocol, which is based on a two-round infection protocol using the reporter TZM-bl cell line, showed a good correlation with genotypic resistance prediction and with the Antivirogram phenotypic assay, in both protease-recombinant viruses and primary viral isolates. The protocol is suitable for any HIV-1 subtype and enables rapid in-house measurement of protease inhibitor susceptibility, thus making it possible to evaluate the concomitant effects of both patient-derived gag and protease-coding regions.
doi:10.1128/JCM.01636-12
PMCID: PMC3503009  PMID: 23015664
8.  Sialyllactose in Viral Membrane Gangliosides Is a Novel Molecular Recognition Pattern for Mature Dendritic Cell Capture of HIV-1 
PLoS Biology  2012;10(4):e1001315.
An accessible sialyllactose moiety on viral membrane gangliosides is shown to be essential for HIV-1 uptake into mature dendritic cells, thereby promoting viral transfer and infection of bystander CD4+ T lymphocytes.
HIV-1 is internalized into mature dendritic cells (mDCs) via an as yet undefined mechanism with subsequent transfer of stored, infectious virus to CD4+ T lymphocytes. Thus, HIV-1 subverts a DC antigen capture mechanism to promote viral spread. Here, we show that gangliosides in the HIV-1 membrane are the key molecules for mDC uptake. HIV-1 virus-like particles and liposomes mimicking the HIV-1 lipid composition were shown to use a common internalization pathway and the same trafficking route within mDCs. Hence, these results demonstrate that gangliosides can act as viral attachment factors, in addition to their well known function as cellular receptors for certain viruses. Furthermore, the sialyllactose molecule present in specific gangliosides was identified as the determinant moiety for mDC HIV-1 uptake. Thus, sialyllactose represents a novel molecular recognition pattern for mDC capture, and may be crucial both for antigen presentation leading to immunity against pathogens and for succumbing to subversion by HIV-1.
Author Summary
Antigen-presenting cells such as dendritic cells (DCs) are required to combat infections, but viruses including HIV have evolved strategies to evade their anti-viral activity. HIV can enter DCs via a non-infectious endocytic mechanism and trick them into passing infectious virus on to bystander CD4+ T cells. Immature DC (iDCs) are characterized by high endocytic activity and low T-cell activation potential. Interestingly, several groups have shown that DCs that have undergone “‘maturation’” (mDCs), a process that occurs on contact with a presentable antigen, capture higher numbers of HIV-1 particles than iDCs when they are matured in the presence of lipopolysaccharide. mDCs move to the lymph nodes where they have more opportunity to interact with T cells than iDCs, and thus to pass on infectious virus. But the molecular mechanism underlying HIV-1 uptake by mDCs has until now been elusive. Here we show that gangliosides, basic components of the host cell's plasma membrane, have an important role in this process. Gangliosides are known to be incorporated into the viral envelope membrane during the process of viral particle budding and here we show that they serve as viral attachment factors: they are recognized and enable HIV-1 uptake by mDCs. Thus, in addition to the well-known function of gangliosides as host cell receptors that mediate virus (e.g., polyoma and SV40) attachment and transport from the plasma membrane to the ER, we now demonstrate that they can also act as determinants for capture by mDCs. Furthermore, we identify a moiety composed of sialyllactose on HIV-1 membrane gangliosides as the specific domain recognized by mDCs. We propose that this novel recognition moiety might be crucial for inducing immune responses, but also critical to disseminate HIV-1 and other ganglioside-containing viruses.
doi:10.1371/journal.pbio.1001315
PMCID: PMC3335875  PMID: 22545022
9.  In–Depth Characterization of Viral Isolates from Plasma and Cells Compared with Plasma Circulating Quasispecies in Early HIV-1 Infection 
PLoS ONE  2012;7(2):e32714.
Background
The use of in vitro models to unravel the phenotypic characteristics of circulating viral variants is key to understanding HIV-1 pathogenesis but limited by the availability of primary viral isolates from biological samples. However, overall in vivo genetic variability of HIV-1 within a subject may not be reflected in the viable viral population obtained after isolation. Although several studies have tried to determine whether viral populations expanded in vitro are representative of in vivo findings, the answer remains unclear due to the reduced number of clonal sequences analyzed or samples compared. In order to overcome previous experimental limitations, here we applied Deep Pyrosequencing (DPS) technology in combination with phenotypic experiments to analyze and compare with unprecedented detail the composition of viral isolates and in vivo quasispecies.
Methodology/Principal Findings
We amplified by DPS HIV-1 genomic regions covering gag, protease, integrase and env-V3 to characterize paired isolates from plasma and peripheral blood mononuclear cells and compare them with total plasma viral RNA in four recently HIV-1 infected subjects. Our study demonstrated the presence of unique haplotypes scattered between sample types with conservation of major variants. In addition, no differences in intra- and inter-population encoded protein variability were found between the different types of isolates or when these were compared to plasma viral RNA within subjects. Additionally, in vitro experiments demonstrated phenotypic similarities in terms of replicative capacity and co-receptor usage between viral isolates and plasma viral RNA.
Conclusion
This study is the first in-depth comparison and characterization of viral isolates from different sources and plasma circulating quasispecies using DPS in recently HIV-1 infected subjects. Our data supports the use of primary isolates regardless of their plasma or cellular origin to define genetic variability and biological traits of circulating HIV-1 quasispecies.
doi:10.1371/journal.pone.0032714
PMCID: PMC3290612  PMID: 22393441
10.  Adaptation of HIV-1 to human leukocyte antigen class I 
Nature  2009;458(7238):641-645.
The rapid and extensive spread of the human immunodeficiency virus (HIV) epidemic provides a rare opportunity to witness host–pathogen co-evolution involving humans. A focal point is the interaction between genes encoding human leukocyte antigen (HLA) and those encoding HIV proteins. HLA molecules present fragments (epitopes) of HIV proteins on the surface of infected cells to enable immune recognition and killing by CD8+ T cells; particular HLA molecules, such as HLA-B*57, HLA-B*27 and HLA-B*51, are more likely to mediate successful control of HIV infection1. Mutation within these epitopes can allow viral escape from CD8+ T-cell recognition. Here we analysed viral sequences and HLA alleles from >2,800 subjects, drawn from 9 distinct study cohorts spanning 5 continents. Initial analysis of the HLA-B*51-restricted epitope, TAFTIPSI (reverse transcriptase residues 128–135), showed a strong correlation between the frequency of the escape mutation I135X and HLA-B*51 prevalence in the 9 study cohorts (P = 0.0001). Extending these analyses to incorporate other well-defined CD8+ T-cell epitopes, including those restricted by HLA-B*57 and HLA-B*27, showed that the frequency of these epitope variants (n = 14) was consistently correlated with the prevalence of the restricting HLA allele in the different cohorts (together, P < 0.0001), demonstrating strong evidence of HIV adaptation to HLA at a population level. This process of viral adaptation may dismantle the well-established HLA associations with control of HIV infection that are linked to the availability of key epitopes, and highlights the challenge for a vaccine to keep pace with the changing immunological landscape presented by HIV.
doi:10.1038/nature07746
PMCID: PMC3148020  PMID: 19242411
11.  HLA-Cw*03-Restricted CD8+ T-Cell Responses Targeting the HIV-1 Gag Major Homology Region Drive Virus Immune Escape and Fitness Constraints Compensated for by Intracodon Variation▿ †  
Journal of Virology  2010;84(21):11279-11288.
The potential importance of HLA-C-restricted CD8+ cytotoxic T lymphocytes (CTL) in HIV infection remains undetermined. We studied the dominant HLA-Cw*03-restricted CTL response to YVDRFFKTL296-304 (YL9), within the conserved major homology region (MHR) of the Gag protein, in 80 HLA-Cw*03-positive individuals with chronic HIV infection to better define the efficacy of the YL9 HLA-C-restricted response. The HLA-Cw*03 allele is strongly associated with HIV sequence changes from Thr-303 to Val, Ile, or Ala at position 8 within the YL9 epitope (P = 1.62 × 10−10). In vitro studies revealed that introduction of the changes T303I and T303A into the YL9 epitope both significantly reduced CTL recognition and substantially reduced the viral replicative capacity. However, subsequent selection of the Val-303 variant, via intracodon variation from Ile-303 (I303V) or Ala-303 (A303V), restored both viral fitness and CTL recognition, as supported by our in vivo data. These results illustrate that HLA-C-restricted CTL responses are capable of driving viral immune escape within Gag, but in contrast to what was previously described for HLA-B-restricted Gag escape mutants, the common Cw*03-Gag-303V variant selected resulted in no detectable benefit to the host.
doi:10.1128/JVI.01144-10
PMCID: PMC2953179  PMID: 20739527
12.  Efficacious Early Antiviral Activity of HIV Gag- and Pol-Specific HLA-B*2705-Restricted CD8+ T Cells ▿  
Journal of Virology  2010;84(20):10543-10557.
The association between HLA-B*2705 and the immune control of human immunodeficiency virus type 1 (HIV-1) has previously been linked to the targeting of the HLA-B*2705-restricted Gag epitope KRWIILGLNK (KK10) by CD8+ T cells. In order to better define the mechanisms of the HLA-B*2705 immune control of HIV, we first characterized the CD8+ T-cell responses of nine highly active antiretroviral therapy (HAART)-naïve B*2705-positive subjects. Unexpectedly, we observed a strong response to an HLA-B*2705-restricted Pol epitope, KRKGGIGGY (KY9), in 8/9 subjects. The magnitude of the KY9 response was only marginally lower than that of the KK10-specific response (median, 695 versus 867 spot-forming cells [SFC]/million peripheral blood mononuclear cells [PBMCs]; not significant [NS]), and viral escape mutants were observed in both KY9 and KK10, resulting from selection pressure driven by the respective CD8+ T-cell response. By comparing inhibitions of viral replication by CD8+ T cells specific for the Gag KK10, Pol KY9, and Vpr VL9 HLA-B*2705-restricted epitopes, we observed a consistent hierarchy of antiviral efficacy (Gag KK10 > Pol KY9 > Vpr VL9). This hierarchy was associated with early recognition of HIV-1-infected cells, within 6 h of infection, by KK10- and KY9-specific CD8+ T cells but not until 18 h postinfection by VL9-specific CD8+ T cells. There was no association between antiviral efficacy and proliferative capacity, cytotoxicity, polyfunctionality, or T-cell receptor (TCR) avidity. These data are consistent with previous studies indicating an important role for the B*2705-Gag KK10 response in the control of HIV but also suggest a previously unrecognized role played by the subdominant Pol-specific KY9 response in HLA-B*2705-mediated control of HIV and that the recognition of HIV-infected cells by CD8+ T cells early in the viral life cycle may be important for viral containment in HIV-infected individuals.
doi:10.1128/JVI.00793-10
PMCID: PMC2950555  PMID: 20686036
13.  Replicative Capacity of Human Immunodeficiency Virus Type 1 Transmitted from Mother to Child Is Associated with Pediatric Disease Progression Rate▿  
Journal of Virology  2009;84(1):492-502.
Human immunodeficiency virus (HIV)-infected infants in the developing world typically progress to AIDS or death within the first 2 years of life. However, a minority progress relatively slowly. This study addresses the potential contribution of viral factors to HIV disease progression in eight infants selected from a well-characterized cohort of C clade HIV-infected infants, monitored prospectively from birth in Durban, South Africa. Three infants were defined as “progressors,” and five were defined as “slow progressors.” We observed that slow-progressor infants carry HIV isolates with significantly lower replicative capacity compared to virus from progressors. Furthermore, our data suggest a link between the attenuated viral phenotype and HLA-B* 57/5801 epitope-specific Gag mutational patterns of the transmitted virus and not to coreceptor usage or to the presence of Nef deletions or insertions. These data underline the importance of virus-host interactions and highlight the contribution of viral attenuation through Gag-specific CD8+ T-cell escape mutations, among other factors, in the control of pediatric HIV infection.
doi:10.1128/JVI.01743-09
PMCID: PMC2798410  PMID: 19828603
14.  Evolution of HLA-B*5703 HIV-1 escape mutations in HLA-B*5703–positive individuals and their transmission recipients 
HLA-B*57 is the class I allele most consistently associated with control of human immunodeficiency virus (HIV) replication, which may be linked to the specific HIV peptides that this allele presents to cytotoxic T lymphocytes (CTLs), and the resulting efficacy of these cellular immune responses. In two HIV C clade–infected populations in South Africa and Zambia, we sought to elucidate the role of HLA-B*5703 in HIV disease outcome. HLA-B*5703–restricted CTL responses select for escape mutations in three Gag p24 epitopes, in a predictable order. We show that the accumulation of these mutations sequentially reduces viral replicative capacity in vitro. Despite this, in vivo data demonstrate that there is ultimately an increase in viral load concomitant with evasion of all three HLA-B*5703–restricted CTL responses. In HLA-B*5703–mismatched recipients, the previously described early benefit of transmitted HLA-B*5703–associated escape mutations is abrogated by the increase in viral load coincident with reversion. Rapid disease progression is observed in HLA-matched recipients to whom mutated virus is transmitted. These data demonstrate that, although costly escape from CTL responses can progressively attenuate the virus, high viral loads develop in the absence of adequate, continued CTL responses. These data underline the need for a CTL vaccine against multiple conserved epitopes.
doi:10.1084/jem.20081984
PMCID: PMC2715113  PMID: 19307327
15.  Functional Consequences of Human Immunodeficiency Virus Escape from an HLA-B*13-Restricted CD8+ T-Cell Epitope in p1 Gag Protein▿  
Journal of Virology  2008;83(2):1018-1025.
The observed association between HLA-B*13 and control of human immunodeficiency virus type 1 (HIV-1) infection has been linked to the number of Gag-specific HLA-B*13-restricted cytotoxic T-cell (CTL) responses identified. To date, the Gag escape mutations described that result in an in vitro fitness cost to the virus have been located within structural protein p24 only. Here we investigated the hypothesis that CTL escape mutations within other regions of HIV Gag may also reduce viral fitness and contribute to immune control. We analyzed an HLA-B*13-restricted CTL response toward an epitope in p1 Gag, RQANFLGKI429-437 (RI9), where amino acid variation at Gag residues 436 and 437 is associated with HLA-B*13 expression. In this work, we assessed the impact of amino acid substitutions at these positions on CTL recognition and on HIV-1 fitness. We demonstrated that substitutions I437L and I437M largely abrogate CTL recognition and reduce viral fitness while variants K436R and I437V have only a marginal effect on recognition and fitness. Examination of the patterns of protein synthesis indicated that the loss of fitness in the I437L and I437M mutants is associated with the accumulation of unprocessed Gag precursors. A significant reduction in ribosomal frameshifting efficiency was observed with I437M, suggesting that this mechanism contributes to the observed reduced fitness of this virus. These studies illustrate the apparent trade-off available to the virus between evasion of CTL recognition in p1 Gag and the functional consequences for viral fitness.
doi:10.1128/JVI.01882-08
PMCID: PMC2612351  PMID: 18945768
17.  Compensatory Mutation Partially Restores Fitness and Delays Reversion of Escape Mutation within the Immunodominant HLA-B*5703-Restricted Gag Epitope in Chronic Human Immunodeficiency Virus Type 1 Infection▿  
Journal of Virology  2007;81(15):8346-8351.
HLA-B*5703 is associated with effective immune control in human immunodeficiency virus type 1 (HIV-1) infection. Here we describe an escape mutation within the immunodominant HLA-B*5703-restricted epitope in chronic HIV-1 infection, KAFSPEVIPMF (Gag 162-172), and demonstrate that this mutation reduces viral replicative capacity. Reversion of this mutation following transmission to HLA-B*5703-negative recipients was delayed by the compensatory mutation S165N within the same epitope. These data may help explain the observed association between HLA-B*5703 and long-term control of viremia.
doi:10.1128/JVI.00465-07
PMCID: PMC1951305  PMID: 17507468
18.  Relative Fitness and Replication Capacity of a Multinucleoside Analogue-Resistant Clinical Human Immunodeficiency Virus Type 1 Isolate with a Deletion of Codon 69 in the Reverse Transcriptase Coding Region▿  
Journal of Virology  2007;81(9):4713-4721.
Deletions, insertions, and amino acid substitutions in the β3-β4 hairpin loop-coding region of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been associated with resistance to nucleoside RT inhibitors when appearing in combination with other mutations in the RT-coding region. In this work, we have measured the in vivo fitness of HIV-1 variants containing a deletion of 3 nucleotides affecting codon 69 (Δ69) of the viral RT as well as the replication capacity (RC) ex vivo of a series of recombinant HIV-1 variants carrying an RT bearing the Δ69 deletion or the T69A mutation in a multidrug-resistant (MDR) sequence background, including the Q151M complex and substitutions M184V, K103N, Y181C, and G190A. Patient-derived viral clones having RTs with Δ69 together with S163I showed increased RCs under drug pressure. These data were consistent with the viral population dynamics observed in a long-term-treated HIV-1-infected patient. In the absence of drugs, viral clones containing T69A replicated more efficiently than those having Δ69, but only when patient-derived sequences corresponding to RT residues 248 to 527 were present. These effects could be attributed to a functional interaction between the C-terminal domain of the p66 subunit (RNase H domain) and the DNA polymerase domain of the RT. Finally, recombinant HIV-1 clones bearing RTs with MDR-associated mutations, including deletions at codon 69, showed increased susceptibilities to protease inhibitors in phenotypic assays. These effects correlated with impaired Gag cleavage and could be attributed to delayed maturation and decreased production of active protease in those variants.
doi:10.1128/JVI.02135-06
PMCID: PMC1900151  PMID: 17314158
19.  Fitness Cost of Escape Mutations in p24 Gag in Association with Control of Human Immunodeficiency Virus Type 1 
Journal of Virology  2006;80(7):3617-3623.
Mutational escape by human immunodeficiency virus (HIV) from cytotoxic T-lymphocyte (CTL) recognition is a major challenge for vaccine design. However, recent studies suggest that CTL escape may carry a sufficient cost to viral replicative capacity to facilitate subsequent immune control of a now attenuated virus. In order to examine how limitations can be imposed on viral escape, the epitope TSTLQEQIGW (TW10 [Gag residues 240 to 249]), presented by two HLA alleles associated with effective control of HIV, HLA-B*57 and -B*5801, was investigated. The in vitro experiments described here demonstrate that the dominant TW10 escape mutation, T242N, reduces viral replicative capacity. Structural analysis reveals that T242 plays a critical role in defining the start point and in stabilizing helix 6 within p24 Gag, ensuring that escape occurs at a significant cost. A very similar role is played by Thr-180, which is also an escape residue, but within a second p24 Gag epitope associated with immune control. Analysis of HIV type 1 gag in 206 B*57/5801-positive subjects reveals three principle alternative TW10-associated variants, and each is strongly linked to concomitant additional variants within p24 Gag, suggesting that functional constraints operate against their occurrence alone. The extreme conservation of p24 Gag and the predictable nature of escape variation resulting from these tight functional constraints indicate that p24 Gag may be a critical immunogen in vaccine design and suggest novel vaccination strategies to limit viral escape options from such epitopes.
doi:10.1128/JVI.80.7.3617-3623.2006
PMCID: PMC1440414  PMID: 16537629
20.  Immune Selection for Altered Antigen Processing Leads to Cytotoxic T Lymphocyte Escape in Chronic HIV-1 Infection 
Mutations within cytotoxic T lymphocyte (CTL) epitopes impair T cell recognition, but escape mutations arising in flanking regions that alter antigen processing have not been defined in natural human infections. In human histocompatibility leukocyte antigen (HLA)-B57+ HIV-infected persons, immune selection pressure leads to a mutation from alanine to proline at Gag residue 146 immediately preceding the NH2 terminus of a dominant HLA-B57–restricted epitope, ISPRTLNAW. Although N-extended wild-type or mutant peptides remained well-recognized, mutant virus–infected CD4 T cells failed to be recognized by the same CTL clones. The A146P mutation prevented NH2-terminal trimming of the optimal epitope by the endoplasmic reticulum aminopeptidase I. These results demonstrate that allele-associated sequence variation within the flanking region of CTL epitopes can alter antigen processing. Identifying such mutations is of major relevance in the construction of vaccine sequences.
doi:10.1084/jem.20031982
PMCID: PMC2211885  PMID: 15067030
CD8 T cell responses; viral evolution; immune evasion; antigen presentation
21.  Lack of Longitudinal Intrapatient Correlation between p24 Antigenemia and Levels of Human Immunodeficiency Virus (HIV) Type 1 RNA in Patients with Chronic HIV Infection during Structured Treatment Interruptions 
Journal of Clinical Microbiology  2004;42(4):1620-1625.
Structured treatment interruptions (STIs) have been proposed as a potential treatment strategy during human immunodeficiency virus type 1 (HIV-1) antiretroviral therapy. This still-experimental intervention requires a close monitoring of patients' plasma viremia and CD4+-T-cell counts during the treatment interruption phase. By using signal amplification of a heat-dissociated p24 antigen (p24Ag) assay, we compared p24Ag levels with levels of HIV RNA in plasma. One hundred seventy-four plasma samples were obtained from 51 chronically HIV-infected patients: 117 from patients who underwent STIs and 57 from patients who did not. Partial immune complex dissociation and clearance of those complexes by the erythrocytes were also investigated. A significant association between the two assays was observed (β = 0.23, 95% confidence interval = 0.18, 0.28; P < 0.0001), but the association was smaller in the subset of samples from patients undergoing STIs. Moreover, discordant results and lack of longitudinal intrapatient correlation between levels of p24Ag and HIV-1 RNA were higher in this group. Incomplete immune complex dissociation and binding of those complexes to erythrocytes could be contributing factors involved in the diminished detection of p24Ag. Therefore, signal amplification of a heat-dissociated p24Ag had a positive association with current HIV RNA assays in a population-based analysis. However, it might not be sensitive enough to monitor longitudinal intrapatient viremia during STIs in patients with high CD4+-T-cell counts potentially due to the production of high-affinity anti-p24 antibodies and clearance of immune complexes by erythrocytes.
doi:10.1128/JCM.42.4.1620-1625.2004
PMCID: PMC387543  PMID: 15071015

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