Suboptimal stimulation by weak agonist epitope variants does not drive dysfunction of HIV-1-specific cytotoxic T lymphocyte clones.

OBJECTIVE: To assess whether weakly recognized epitope variants induce anergy in HIV-1-specific CD8 T lymphocyte (CTL) clones as a mechanism of dysfunction. DESIGN: HIV-1-specific CTL clones were exposed to suboptimally recognized epitope variants, and screened for anergy and other T-cell dysfunction markers, and subsequent capability to kill target cells bearing index epitope. METHODS: In addition to the optimally recognized index epitope, two suboptimally recognized epitope variants were selected based on titration curves for killing of peptide-labeled target cells by three HIV-1-specific CTL clones targeting the epitopes SLYNTVATL (Gag 77-85, A02-restricted), RPAEPVPLQL (Rev 66-75, B07-restricted), and KRWIIMGLNK (Gag 263-272, B27-restricted). Consequences of suboptimal stimulation were assessed by cytokine secretion, gene expression, and capacity to kill index epitope-labeled target cells upon rechallenge. RESULTS: Suboptimal recognition of epitope variants reduced cytokine production by CTL similarly to reduction in killing of target cells. Gene expression profiles after suboptimal stimulation demonstrated no patterns consistent with T-cell dysfunction due to anergy, exhaustion, or apoptosis. Preexposure of CTL to epitope variants had no discernable impact on their subsequent capacity to kill index epitope-bearing target cells. CONCLUSION: Our data explore the hypothesis that poorly recognized epitope variants not only facilitate HIV-1 evasion of CTL recognition, but also induce CTL dysfunction through suboptimal signaling causing anergy. However, the results do not suggest that suboptimal signaling induces anergy (or exhaustion or apoptosis), indicating that the major role of CTL epitope variation is likely viral escape.

Cross-Reactivity against Multiple HIV-1 Epitopes Is Characteristic of HIV-1-Specific Cytotoxic T Lymphocyte Clones.

Although a high level of promiscuity for heterologous epitopes is believed to exist for cellular immunity, limited data explore this issue for human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T lymphocyte (CTL) responses. Here, we found an unexpected degree of heterologous cross-reactivity against HIV-1 epitopes, in addition to the targeted index epitope. Most CTL clones screened cross-reacted against other known HIV-1 epitopes of the same major histocompatibility complex type I (MHC-I) restriction, up to 40% of tested nonindex epitopes in some cases. The observed cross-reactivity was universally lower avidity than recognition of the index epitope when examined for several A*02- and B*57-restricted CTL clones, demonstrating that the high concentrations of exogenous epitope typically used for screening of CTL responses are prone to detect such cross-reactivity spuriously. In agreement with this, we found that these cross-reactive responses do not appear to mediate CTL activity against HIV-1-infected cells. Overall, our data indicate that low-level cross-reactivity is remarkably common for HIV-1-specific CTLs. The role of this phenomenon is unclear, but low-avidity interactions have been shown to foster homeostatic proliferation of memory T cells.IMPORTANCE This study raises two issues related to HIV-1-specific CTL responses. These are key immune responses that retard disease progression in infected persons that are highly relevant to immunotherapies and vaccines for HIV-1. First, we make the novel observation that these responses are promiscuous and that CTLs targeting one epitope may cross-recognize other, completely distinct epitopes in the virus. While these are low-avidity interactions that do not appear to contribute directly to the antiviral activity of CTLs, this raises interesting biologic implications regarding the purpose of the phenomenon, such as providing a stimulus for these responses to persist long term. Second, the data raise a technical caveat to detection of CTL responses against particular epitopes, suggesting that some methodologies may unintentionally detect cross-reactivity and overestimate responses against an epitope.

Highly Attenuated Infection With a Vpr-Deleted Molecular Clone of Human Immunodeficiency Virus-1.

A 48-year-old woman was infected with a vpr-defective human immunodeficiency virus (HIV)-1 molecular clone. Seroconversion was markedly delayed, and without treatment she had durably suppressed viremia and normal T-cell levels. Neutralizing antibody and CD8+ T-cell immune responses against HIV-1 were unremarkable. Viral sequences confirmed the source but evolved defective nef, suggesting an unknown mechanistic link to vpr. There were subtle qualitative defects in T and B cells. To our knowledge, this is the only case of human infection with a characterized defective HIV-1 molecular clone, which furthermore recapitulated live-attenuated vaccination in macaque models of HIV-1 vaccine research.

Solution Structures of Engineered Vault Particles.

Prior crystal structures of the vault have provided clues of its structural variability but are non-conclusive due to crystal packing. Here, we obtained vaults by engineering at the N terminus of rat major vault protein (MVP) an HIV-1 Gag protein segment and determined their near-atomic resolution (∼4.8 Å) structures in a solution/non-crystalline environment. The barrel-shaped vaults in solution adopt two conformations, 1 and 2, both with D39 symmetry. From the N to C termini, each MVP monomer has three regions: body, shoulder, and cap. While conformation 1 is identical to one of the crystal structures, the shoulder in conformation 2 is translocated longitudinally up to 10 Å, resulting in an outward-projected cap. Our structures clarify the structural discrepancies in the body region in the prior crystallography models. The vault's drug-delivery potential is highlighted by the internal disposition and structural flexibility of its Gag-loaded N-terminal extension at the barrel waist of the engineered vault.

Acupuncture in the treatment of fatigue in Parkinson's disease: A pilot, randomized, controlled, study.

Introduction: Fatigue is a common and disabling problem in patients with Parkinson's disease (PD), and there is currently no satisfactory treatment. As acupuncture has been reported to be effective in fatigue related to other conditions, we sought to evaluate its efficacy in PD. Methods: This was a single center, randomized, sham-controlled study. Forty PD patients with moderately severe fatigue were randomized to receive 5 weeks of biweekly real or sham acupuncture. The primary outcome was change on the General Fatigue score of the Multidimensional Fatigue Inventory (MFI-GF) at 5 weeks. Secondary outcomes included MFI-Total score, Unified Parkinson's Disease Rating Scale Motor score (UPDRS Motor), Parkinson's Disease Questionnaire-39 (PDQ 39), Geriatrics Depression Scale (GDS), and Epworth Sleepiness Scale ESS). All outcome measures were evaluated at baseline, 5 and 9 weeks. Results: Both groups showed significant improvements in MFI-GF and MFI-Total scores at 5 and 9 weeks, but there were no significant between-group differences. There were no improvements from baseline for PDQ 39, GDS, and ESS. Although improvements were noted for the UPDRS Motor score in the real acupuncture group, no between-group difference could be demonstrated. Conclusions: Both real and sham acupuncture are equally effective in improving PD-related fatigue, and it is likely that this is due to nonspecific or placebo effects.

HIV-1 epitopes presented by MHC class I types associated with superior immune containment of viremia have highly constrained fitness landscapes.

Certain Major Histocompatibility-I (MHC-I) types are associated with superior immune containment of HIV-1 infection by CD8+ cytotoxic T lymphocytes (CTLs), but the mechanisms mediating this containment are difficult to elucidate in vivo. Here we provide controlled assessments of fitness landscapes and CTL-imposed constraints for immunodominant epitopes presented by two protective (B*57 and B*27) and one non-protective (A*02) MHC-I types. Libraries of HIV-1 with saturation mutagenesis of CTL epitopes are propagated with and without CTL selective pressure to define the fitness landscapes for epitope mutation and escape from CTLs via deep sequencing. Immunodominant B*57- and B*27- present epitopes are highly limited in options for fit mutations, with most viable variants recognizable by CTLs, whereas an immunodominant A*02 epitope-presented is highly permissive for mutation, with many options for CTL evasion without loss of viability. Generally, options for evasion overlap considerably between CTL clones despite highly distinct T cell receptors. Finally, patterns of variant recognition suggest population-wide CTL selection for the A*02-presented epitope. Overall, these findings indicate that these protective MHC-I types yield CTL targeting of highly constrained epitopes, and underscore the importance of blocking public escape pathways for CTL-based interventions against HIV-1.

HIV-1 Epitope Variability Is Associated with T Cell Receptor Repertoire Instability and Breadth.

Mutational escape of HIV-1 from HIV-1-specific CD8+ T lymphocytes (CTLs) is a major barrier for effective immune control. Each epitope typically is targeted by multiple clones with distinct T cell receptors (TCRs). While the clonal repertoire may be important for containing epitope variation, determinants of its composition are poorly understood. We investigate the clonal repertoire of 29 CTL responses against 23 HIV-1 epitopes longitudinally in nine chronically infected untreated subjects with plasma viremia of <3,000 RNA copies/ml over 17 to 179 weeks. The composition of TCRs targeting each epitope varied considerably in stability over time, although clonal stability (Sorensen index) was not significantly time dependent within this interval. However, TCR stability inversely correlated with epitope variability in the Los Alamos HIV-1 Sequence Database, consistent with TCR evolution being driven by epitope variation. Finally, a robust inverse correlation of TCR breadth against each epitope versus epitope variability further suggested that this variability drives TCR repertoire diversification. In the context of studies demonstrating rapidly shifting HIV-1 sequences in vivo, our findings support a variably dynamic process of shifting CTL clonality lagging in tandem with viral evolution and suggest that preventing escape of HIV-1 may require coordinated direction of the CTL clonal repertoire to simultaneously block escape pathways.IMPORTANCE Mutational escape of HIV-1 from HIV-1-specific CD8+ T lymphocytes (CTLs) is a major barrier to effective immune control. The number of distinct CTL clones targeting each epitope is proposed to be an important factor, but the determinants are poorly understood. Here, we demonstrate that the clonal stability and number of clones for the CTL response against an epitope are inversely associated with the general variability of the epitope. These results show that CTLs constantly lag epitope mutation, suggesting that preventing HIV-1 escape may require coordinated direction of the CTL clonal repertoire to simultaneously block escape pathways.

Structures and stabilization of kinetoplastid-specific split rRNAs revealed by comparing leishmanial and human ribosomes.

The recent success in ribosome structure determination by cryoEM has opened the door to defining structural differences between ribosomes of pathogenic organisms and humans and to understand ribosome-targeting antibiotics. Here, by direct electron-counting cryoEM, we have determined the structures of the Leishmania donovani and human ribosomes at 2.9 Å and 3.6 Å, respectively. Our structure of the leishmanial ribosome elucidates the organization of the six fragments of its large subunit rRNA (as opposed to a single 28S rRNA in most eukaryotes, including humans) and reveals atomic details of a unique 20 amino acid extension of the uL13 protein that pins down the ends of three of the rRNA fragments. The structure also fashions many large rRNA expansion segments. Direct comparison of our human and leishmanial ribosome structures at the decoding A-site sheds light on how the bacterial ribosome-targeting drug paromomycin selectively inhibits the eukaryotic L. donovani, but not human, ribosome.

HIV-1-Specific Chimeric Antigen Receptors Based on Broadly Neutralizing Antibodies.

UNLABELLED: Although the use of chimeric antigen receptors (CARs) based on single-chain antibodies for gene immunotherapy of cancers is increasing due to promising recent results, the earliest CAR therapeutic trials were done for HIV-1 infection in the late 1990s. This approach utilized a CAR based on human CD4 as a binding domain and was abandoned for a lack of efficacy. The growing number of HIV-1 broadly neutralizing antibodies (BNAbs) offers the opportunity to generate novel CARs that may be more active and revisit this modality for HIV-1 immunotherapy. We used sequences from seven well-defined BNAbs varying in binding sites and generated single-chain-antibody-based CARs. These CARs included 10E8, 3BNC117, PG9, PGT126, PGT128, VRC01, and X5. Each novel CAR exhibited conformationally relevant expression on the surface of transduced cells, mediated specific proliferation and killing in response to HIV-1-infected cells, and conferred potent antiviral activity (reduction of viral replication in log10 units) to transduced CD8(+) T lymphocytes. The antiviral activity of these CARs was reproducible but varied according to the strain of virus. These findings indicated that BNAbs are excellent candidates for developing novel CARs to consider for the immunotherapeutic treatment of HIV-1. IMPORTANCE: While chimeric antigen receptors (CARs) using single-chain antibodies as binding domains are growing in popularity for gene immunotherapy of cancers, the earliest human trials of CARs were done for HIV-1 infection. However, those trials failed, and the approach was abandoned for HIV-1. The only tested CAR against HIV-1 was based on the use of CD4 as the binding domain. The growing availability of HIV-1 broadly neutralizing antibodies (BNAbs) affords the opportunity to revisit gene immunotherapy for HIV-1 using novel CARs based on single-chain antibodies. Here we construct and test a panel of seven novel CARs based on diverse BNAb types and show that all these CARs are functional against HIV-1.

Clonal CD8+ T Cell Persistence and Variable Gene Usage Bias in a Human Transplanted Hand.

Immune prophylaxis and treatment of transplanted tissue rejection act indiscriminately, risking serious infections and malignancies. Although animal data suggest that cellular immune responses causing rejection may be rather narrow and predictable based on genetic background, there are only limited data regarding the clonal breadth of anti-donor responses in humans after allogeneic organ transplantation. We evaluated the graft-infiltrating CD8+ T lymphocytes in skin punch biopsies of a transplanted hand over 178 days. Profiling of T cell receptor (TCR) variable gene usage and size distribution of the infiltrating cells revealed marked skewing of the TCR repertoire indicating oligoclonality, but relatively normal distributions in the blood. Although sampling limitation prevented complete assessment of the TCR repertoire, sequencing further identified 11 TCR clonal expansions that persisted through varying degrees of clinical rejection and immunosuppressive therapy. These 11 clones were limited to three TCR beta chain variable (BV) gene families. Overall, these data indicate significant oligoclonality and likely restricted BV gene usage of alloreactive CD8+ T lymphocytes, and suggest that changes in rejection status are more due to varying regulation of their activity or number rather than shifts in the clonal populations in the transplanted organ. Given that controlled animal models produce predictable BV usage in T lymphocytes mediating rejection, understanding the determinants of TCR gene usage associated with rejection in humans may have application in specifically targeted immunotherapy.

Ectopic expression of anti-HIV-1 shRNAs protects CD8(+) T cells modified with CD4ζ CAR from HIV-1 infection and alleviates impairment of cell proliferation.

Chimeric antigen receptors (CARs) are artificially engineered receptors that confer a desired specificity to immune effector T cells. As an HIV-1-specific CAR, CD4ζ CAR has been extensively tested in vitro as well as in clinical trials. T cells modified with this CAR mediated highly potent anti-HIV-1 activities in vitro and were well-tolerated in vivo, but exerted limited effects on viral load and reservoir size due to poor survival and/or functionality of the transduced cells in patients. We hypothesize that ectopic expression of CD4ζ on CD8(+) T cells renders them susceptible to HIV-1 infection, resulting in poor survival of those cells. To test this possibility, highly purified CD8(+) T cells were genetically modified with a CD4ζ-encoding lentiviral vector and infected with HIV-1. CD8(+) T cells were vulnerable to HIV-1 infection upon expression of CD4ζ as evidenced by elevated levels of p24(Gag) in cells and culture supernatants. Concurrently, the number of CD4ζ-modified CD8(+) T cells was reduced relative to control cells upon HIV-1 infection. To protect these cells from HIV-1 infection, we co-expressed two anti-HIV-1 shRNAs previously developed by our group together with CD4ζ. This combination vector was able to suppress HIV-1 infection without impairing HIV-1-dependent effector activities of CD4ζ. In addition, the number of CD4ζ-modified CD8(+) T cells maintained similar levels to that of the control even under HIV-1 infection. These results suggest that protecting CD4ζ-modified CD8(+) T cells from HIV-1 infection is required for prolonged HIV-1-specific immune surveillance.

Ineffectual targeting of HIV-1 Nef by cytotoxic T lymphocytes in acute infection results in no functional impairment or viremia reduction.

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.

Differential blood and mucosal immune responses against an HIV-1 vaccine administered via inguinal or deltoid injection.

UNLABELLED: Mucosal immunity is central to sexual transmission and overall pathogenesis of HIV-1 infection, but the ability of vaccines to induce immune responses in mucosal tissue compartments is poorly defined. Because macaque vaccine studies suggest that inguinal (versus limb) vaccination may better target sexually-exposed mucosa, we performed a randomized, double-blinded, placebo-controlled Phase I trial in HIV-1-uninfected volunteers, using the recombinant Canarypox (CP) vaccine vCP205 delivered by different routes. 12 persons received vaccine and 6 received placebo, divided evenly between deltoid-intramuscular (deltoid-IM) or inguinal-subcutaneous (inguinal-SC) injection routes. The most significant safety events were injection site reactions (Grade 3) in one inguinal vaccinee. CP-specific antibodies were detected in the blood of all 12 vaccinees by Day 24, while HIV-1-specific antibodies were observed in the blood and gut mucosa of 1/9 and 4/9 evaluated vaccinees respectively, with gut antibodies appearing earlier in inguinal vaccinees (24-180 versus 180-365 days). HIV-1-specific CD8(+) T lymphocytes (CTLs) were observed in 7/12 vaccinees, and blood and gut targeting were distinct. Within blood, both deltoid and inguinal responders had detectable CTL responses by 17-24 days; inguinal responders had early responses (within 10 days) while deltoid responders had later responses (24-180 days) in gut mucosa. Our results demonstrate relative safety of inguinal vaccination and qualitative or quantitative compartmentalization of immune responses between blood and gut mucosa, and highlight the importance of not only evaluating early blood responses to HIV-1 vaccines but also mucosal responses over time. TRIAL REGISTRATION: ClinicalTrials.gov NCT00076817.

HIV-1 Nef sequence and functional compartmentalization in the gut is not due to differential cytotoxic T lymphocyte selective pressure.

The gut is the largest lymphoid organ in the body and a site of active HIV-1 replication and immune surveillance. The gut is a reservoir of persistent infection in some individuals with fully suppressed plasma viremia on combination antiretroviral therapy (cART) although the cause of this persistence is unknown. The HIV-1 accessory protein Nef contributes to persistence through multiple functions including immune evasion and increasing infectivity. Previous studies showed that Nef's function is shaped by cytotoxic T lymphocyte (CTL) responses and that there are distinct populations of Nef within tissue compartments. We asked whether Nef's sequence and/or function are compartmentalized in the gut and how compartmentalization relates to local CTL immune responses. Primary nef quasispecies from paired plasma and sigmoid colon biopsies from chronically infected subjects not on therapy were sequenced and cloned into Env(-) Vpu(-) pseudotyped reporter viruses. CTL responses were mapped by IFN-γ ELISpot using expanded CD8+ cells from blood and gut with pools of overlapping peptides covering the entire HIV proteome. CD4 and MHC Class I Nef-mediated downregulation was measured by flow cytometry. Multiple tests indicated compartmentalization of nef sequences in 5 of 8 subjects. There was also compartmentalization of function with MHC Class I downregulation relatively well preserved, but significant loss of CD4 downregulation specifically by gut quasispecies in 5 of 7 subjects. There was no compartmentalization of CTL responses in 6 of 8 subjects, and the selective pressure on quasispecies correlated with the magnitude CTL response regardless of location. These results demonstrate that Nef adapts via diverse pathways to local selective pressures within gut mucosa, which may be predominated by factors other than CTL responses such as target cell availability. The finding of a functionally distinct population within gut mucosa offers some insight into how HIV-1 may persist in the gut despite fully suppressed plasma viremia on cART.

HIV-1 gag cytotoxic T lymphocyte epitopes vary in presentation kinetics relative to HLA class I downregulation.

Although CD8(+) cytotoxic T lymphocytes (CTLs) are protective in HIV-1 infection, the factors determining their antiviral efficiency are poorly defined. It is proposed that Gag targeting is superior because of very early Gag epitope presentation, allowing early killing of infected cells before Nef-mediated downregulation of human leukocyte antigen class I (HLA-I). To study Gag epitope presentation kinetics, three epitopes (SL977-85, KF11162-172, and TW10240-249) were genetically translocated from their endogenous location in the Rev-dependent (late) gag gene into the Rev-independent (early) nef gene with concomitant mutation of the corresponding endogenous epitopes to nonrecognized sequences. These viruses were compared to the index virus for CTL-mediated suppression of replication and the susceptibility of this antiviral activity to Nef-mediated HLA-I downregulation. SL9-specific CTLs gained activity after SL9 translocation to Nef, going from Nef sensitive to Nef insensitive, indicating that translocation accelerated infected cell recognition from after to before HLA-I downregulation. KF11-specific CTL antiviral activity was unchanged and insensitive to HLA-I downregulation before and after KF11 translocation, suggesting that already rapid recognition of infected cells was not accelerated. However, TW10-specific CTLs that were insensitive to Nef at the baseline became sensitive with reduced antiviral activity after translocation, indicating that translocation retarded epitope expression. Cytosolic peptide processing assays suggested that TW10 was inefficiently generated after translocation to Nef, compared to SL9 and KF11. As a whole, these data demonstrate that epitope presentation kinetics play an important role in CTL antiviral efficiency, that Gag epitopes are not uniformly presented early, and that the epitope context can play a major role in presentation kinetics.

Early HLA-B*57-restricted CD8+ T lymphocyte responses predict HIV-1 disease progression.

Although HLA-B*57 (B57) is associated with slow progression to disease following HIV-1 infection, B57 heterozygotes display a wide spectrum of outcomes, including rapid progression, viremic slow progression, and elite control. Efforts to identify differences between B57-positive (B57(+)) slow progressors and B57(+) rapid progressors have largely focused on cytotoxic T lymphocyte (CTL) phenotypes and specificities during chronic stages of infection. Although CTL responses in the early months of infection are likely to be the most important for the long-term rate of HIV-1 disease progression, few data on the early CTL responses of eventual slow progressors have been available. Utilizing the Multicenter AIDS Cohort Study (MACS), we retrospectively examined the early HIV-1-specific CTL responses of 14 B57(+) individuals whose time to development of disease ranged from 3.5 years to longer than 25 years after infection. In general, a greater breadth of targeting of epitopes from structural proteins, especially Gag, as well as of highly conserved epitopes from any HIV-1 protein, correlated with longer times until disease. The single elite controller in the cohort was an outlier on several correlations of CTL targeting and time until disease, consistent with reports that elite control is typically not achieved solely by protective HLA-mediated CTLs. When targeting of individual epitopes was analyzed, we found that early CTL responses to the IW9 (ISPRTLNAW) epitope of Gag, while generally subdominant, correlated with delayed progression to disease. This is the first study to identify early CTL responses to IW9 as a correlate of protection in persons with HLA-B*57.

Epitope targeting and viral inoculum are determinants of Nef-mediated immune evasion of HIV-1 from cytotoxic T lymphocytes.

The impact of HIV-1 Nef-mediated HLA-I down-regulation on CD8(+) cytotoxic T lymphocytes (CTLs) varies by epitope, but the determining factors have not been elucidated. In the present study, we investigated the impact of Nef on the antiviral efficiency of HIV-1-specific CTLs targeting 17 different epitopes to define properties that determine susceptibility to Nef. The impact of Nef was not correlated with the presenting HLA-I type or functional avidity of CTLs, but instead was related directly to the kinetics of infected cell clearance. Whereas Gag-specific CTLs generally were less susceptible to Nef than those targeting other proteins, this was determined by the ability to eliminate infected cells before de novo synthesis of viral proteins, which was also observed for CTLs targeting a Nef epitope. This very early clearance of infected cells depended on virus inoculum, and the required inoculum varied by epitope. These results suggest that whereas Gag-specific CTLs are more likely to recognize infected cells before Nef-mediated HLA-I down-regulation, this varies depending on the specific epitope and virus inoculum. Reduced susceptibility to Nef therefore may contribute to the overall association of Gag-specific CTL responses to better immune control if a sufficient multiplicity of infection is attained in vivo, but this property is not unique to Gag.

Immune selection in vitro reveals human immunodeficiency virus type 1 Nef sequence motifs important for its immune evasion function in vivo.

Human immunodeficiency virus type 1 (HIV-1) Nef downregulates major histocompatibility complex class I (MHC-I), impairing the clearance of infected cells by CD8(+) cytotoxic T lymphocytes (CTLs). While sequence motifs mediating this function have been determined by in vitro mutagenesis studies of laboratory-adapted HIV-1 molecular clones, it is unclear whether the highly variable Nef sequences of primary isolates in vivo rely on the same sequence motifs. To address this issue, nef quasispecies from nine chronically HIV-1-infected persons were examined for sequence evolution and altered MHC-I downregulatory function under Gag-specific CTL immune pressure in vitro. This selection resulted in decreased nef diversity and strong purifying selection. Site-by-site analysis identified 13 codons undergoing purifying selection and 1 undergoing positive selection. Of the former, only 6 have been reported to have roles in Nef function, including 4 associated with MHC-I downregulation. Functional testing of naturally occurring in vivo polymorphisms at the 7 sites with no previously known functional role revealed 3 mutations (A84D, Y135F, and G140R) that ablated MHC-I downregulation and 3 (N52A, S169I, and V180E) that partially impaired MHC-I downregulation. Globally, the CTL pressure in vitro selected functional Nef from the in vivo quasispecies mixtures that predominately lacked MHC-I downregulatory function at the baseline. Overall, these data demonstrate that CTL pressure exerts a strong purifying selective pressure for MHC-I downregulation and identifies novel functional motifs present in Nef sequences in vivo.