Nef Gene Research Articles

Primate lentiviral Nef proteins deregulate T-cell development by multiple mechanisms

BackgroundA nef gene is present in all primate lentiviral genomes and is important for high viral loads and progression to AIDS in human or experimental macaque hosts of HIV or SIV, respectively. In these hosts, infection of the thymus results in a decreased output of naive T cells that may contribute to the development of immunodeficiency. We have previously shown that HIV-1 subtype B Nef proteins can block human T-cell development. However, the underlying mechanism(s) and the conservation of this Nef function between different groups of HIV and SIV remained to be determined.ResultsWe investigated whether reduction of thymic output is a conserved function of highly divergent lentiviral Nef proteins including those from both types of human immunodeficiency viruses (HIV-1 and HIV-2), their direct simian counterparts (SIVcpz, SIVgor and SIVsmm, respectively), and some additional SIV strains. We found that expression of most of these nef alleles in thymocyte progenitors impaired T-cell development and reduced thymic output. For HIV-1 Nef, binding to active p21 protein (Cdc42/Rac)-activated kinase (PAK2) was a major determinant of this function. In contrast, selective disruption of PAK2 binding did not eliminate the effect on T-cell development of SIVmac239 Nef, as was shown by expressing mutants in a newly discovered PAK2 activating structural motif (PASM) constituted by residues I117, H121, T218 and Y221, as well as previously described mutants. Rather, down-modulation of cell surface CD3 was sufficient for reduced thymic output by SIVmac Nef, while other functions of SIV Nefs contributed.ConclusionsOur results indicate that primate lentiviral Nef proteins impair development of thymocyte precursors into T cells in multiple ways. The interaction of HIV-1 Nef with active PAK2 by HIV-1 seem to be most detrimental, and downregulation of CD3 by HIV-2 and most SIV Nef proteins sufficient for reduced thymic output. Since the reduction of thymic output by Nef is a conserved property of divergent lentiviruses, it is likely to be relevant for peripheral T-cell depletion in poorly adapted primate lentiviral infections.

HIV-1 evolution in patients undergoing immunotherapy with Tat, Rev, and Nef expressing dendritic cells followed by treatment interruption

This study aimed to evaluate HIV sequence evolution in whole genes and in CD8 T-cell epitope regions following immunotherapy and subsequent analytical treatment interruption (ATI). A second objective of this study was to analyze associations between vaccine-specific immune responses and epitope mutation rates. HIV-1-infected patients on combined antiretroviral therapy (cART) were subjected to immunotherapy by the administration of an autologous dendritic cell-based therapeutic vaccine expressing Tat, Rev, and Nef and subsequent ATI. HIV-1 genes were amplified and sequenced from plasma RNA obtained before initiation of cART as well as during ATI. Control sequences for virus evolution in untreated HIV-1-infected individuals were obtained from the HIV Sequence Database (Los Alamos). CD8 T-cell epitope regions were defined based on literature data and prediction models. HIV-1-specific immune responses were evaluated to analyze their impact on sequence evolution. Viral sequence evolution in the tat, rev, and nef genes of vaccinated patients was similar to that of controls. The number of mutations observed inside and outside CD8 T-cell epitopes was comparable for vaccine-targeted and nontargeted proteins. We found no evidence for an impact of vaccine-induced or enhanced immune responses on the number of mutations inside or outside epitopes. Therapeutic vaccination of HIV-1-infected patients with a dendritic cell-based vaccine targeting Tat, Rev, and Nef did not affect virus evolution at the whole gene level nor at the CD8 T-cell epitope level.

Structure of TCR and antigen complexes at an immunodominant CTL epitope in HIV-1 infection

We investigated the crystal structure of an HLA-A*2402-restricted CTL epitope in the HIV-1 nef gene (Nef134-10) before (pHLA) or after TCR docking. The wild type epitope and two escape mutants were included in the study. Y135F was an early-appearing major mutation, while F139L was a late-appearing mutation which was selected in the patients without Y135F. F139 was an eminent feature of the Nef134-10 epitope. Wild type-specific TCR was less fit to F139L mutant suggesting that F139L is an escape from the CTL against the wild type epitope. Although Y135F mutation disrupted the hydrogen bond to HLA-A*2402 His70, newly formed hydrogen bond between T138 and His70 kept the conformation of the epitope in the reconstituted pMHC. TCR from Y135F- or dually-specific CTL had unique mode of binding to the mutant epitope. Y135F has been reported as a processing mutant but CTL carrying structurally adequate TCR can be found in the patients.

Genetic Architecture of HIV Type 1 Nef and Tat from HLA-B57-Typed Long-Term Survivors in an Indian Cohort of Perinatally HIV-Infected Children

Human immunodeficiency virus type 1 (HIV-1) viral genes nef and tat play an important role in disease progression. In this study we characterized the Nef and Tat proteins from a group of HLA-B57 typed pediatric perinatally infected long-term survivors (LTS) with ≥10 years of infection. We identified 19 therapy-naive LTS after screening 250 children from an Indian pediatric cohort. Nef and tat amplified from plasma virus showed that all the LTS harbored HIV-1 subtype C. The two B57(+) children showed mutations, deletions, and insertions in experimentally defined B57 epitopes in the virus that are likely to be escape mutants. Only GW12 (GPGVRYPLTFGW) and YY9 (YTPGPGIRY) were conserved, while the remaining 90% (18/20) of the epitopes showed some degree of mutations. The most variable epitopes were RR15, SE15, QP15, KF9, HW9, YT9, and GF15. To our knowledge this is the first study from India in which characterization of Nef and Tat from LTS has led to information on genetic alterations in these genes that are associated with slow disease progression, and can provide an important lead in future studies.

Frequent gross deletions in pol gene in 10 HIV-1 infected patients treated with Korean red ginseng for 3 years: dosage dependency

Background To my knowledge, there is only one report on gross deletions in the pol gene (gΔpol) [1] although there are many reports on gross deletions in the nef gene (gΔnef). It is known that Korean red ginseng (KRG) slows depletion of CD4 T cells in human immunodeficiency virus type 1-infected patients [2]. We reported an association between KRG intake and gΔnef [3] in 10 HIV-1 infected patients treated with KRG. Here, we investigated whether KRG intake also induces gΔpol.

Frequent genetic defects in long-term survivors for more than 26 years in the absence of antiretroviral therapy in Korea: its association with ginseng treatment

All LTNPs revealed at least one genetic defect in 4 genes, whereas controls revealed it in both 5’ LTR/gag and nef genes. Overall genetic defects were significantly higher in LTNPs (11.4%) than 4.9% in controls (P < 0.001). Among the 4 LTNPs, one patient who had taken the least amount of KRG revealed significantly higher proportion of premature stop codons (5.6%) than 1.6% in 3 LTNPs (P < 0.01). There was a significant difference in the frequency of genetic defects between on KRG (10.9%) and baseline prior to KRG intake (4.3%) (P < 0.01). There was a significant correlation between amount of KRG supplied and proportion of genetic defects (r=0.80, P < 0.05). At single gene level, only the nef gene showed a significantly higher frequency of genetic defects in LTNPs (11.5%) than 3.6% in controls (P < 0.01). Conclusion

No Evidence for Selection of HIV-1 with Enhanced Gag-Protease or Nef Function among Breakthrough Infections in the CAPRISA 004 Tenofovir Microbicide Trial

BackgroundUse of antiretroviral-based microbicides for HIV-1 prophylaxis could introduce a transmission barrier that inadvertently facilitates the selection of fitter viral variants among incident infections. To investigate this, we assessed the in vitro function of gag-protease and nef sequences from participants who acquired HIV-1 during the CAPRISA 004 1% tenofovir microbicide gel trial.Methods and ResultsWe isolated the earliest available gag-protease and nef gene sequences from 83 individuals and examined their in vitro function using recombinant viral replication capacity assays and surface protein downregulation assays, respectively. No major phylogenetic clustering and no significant differences in gag-protease or nef function were observed in participants who received tenofovir gel versus placebo gel prophylaxis.ConclusionResults indicate that the partial protective effects of 1% tenofovir gel use in the CAPRISA 004 trial were not offset by selection of transmitted/early HIV-1 variants with enhanced Gag-Protease or Nef fitness.

Formation of a Unique Cluster of G-Quadruplex Structures in the HIV-1 nef Coding Region: Implications for Antiviral Activity

G-quadruplexes are tetraplex structures of nucleic acids that can form in G-rich sequences. Their presence and functional role have been established in telomeres, oncogene promoters and coding regions of the human chromosome. In particular, they have been proposed to be directly involved in gene regulation at the level of transcription. Because the HIV-1 Nef protein is a fundamental factor for efficient viral replication, infectivity and pathogenesis in vitro and in vivo, we investigated G-quadruplex formation in the HIV-1 nef gene to assess the potential for viral inhibition through G-quadruplex stabilization. A comprehensive computational analysis of the nef coding region of available strains showed the presence of three conserved sequences that were uniquely clustered. Biophysical testing proved that G-quadruplex conformations were efficiently stabilized or induced by G-quadruplex ligands in all three sequences. Upon incubation with a G-quadruplex ligand, Nef expression was reduced in a reporter gene assay and Nef-dependent enhancement of HIV-1 infectivity was significantly repressed in an antiviral assay. These data constitute the first evidence of the possibility to regulate HIV-1 gene expression and infectivity through G-quadruplex targeting and therefore open a new avenue for viral treatment.

Bone-derived mesenchymal stromal cells from HIV transgenic mice exhibit altered proliferation, differentiation capacity and paracrine functions along with impaired therapeutic potential in kidney injury

Mesenchymal stem cells (MSCs) secrete paracrine factors that could be cytoprotective and serve roles in immunoregulation during tissue injury. Although MSCs express HIV receptors, and co-receptors, and are susceptible to HIV infection, whether HIV-1 may affect biological properties of MSCs needs more study. We evaluated cellular proliferation, differentiation and paracrine functions of MSCs isolated from compact bones of healthy control mice and Tg26 HIV-1 transgenic mice. The ability of MSCs to protect against cisplatin toxicity was studied in cultured renal tubular cells as well as in intact mice. We successfully isolated MSCs from healthy mice and Tg26 HIV-1 transgenic mice and found the latter expressed viral Nef, Vpu, NL4-3 and Vif genes. The proliferation and differentiation of Tg26 HIV-1 MSCs was inferior to MSCs from healthy mice. Moreover, transplantation of Tg26 HIV-1 MSCs less effectively improved outcomes compared with healthy MSCs in mice with acute kidney injury. Also, Tg26 HIV-1 MSCs secreted multiple cytokines, but at significantly lower levels than healthy MSCs, which resulted in failure of conditioned medium from these MSCs to protect cultured renal tubular cells from cisplatin toxicity. Therefore, HIV-1 had adverse biological effects on MSCs extending to their proliferation, differentiation, function, and therapeutic potential. These findings will help in advancing mechanistical insight in renal injury and repair in the setting of HIV-1 infection.

ST111 Immune selection and vpu variability in a unique narrow source HIV-Positive village cohort

Background: HLA-class I associated selection pressure on HIV-1 evolution has been described in several cohorts and can leave characteristic “footprints” on HIV-1 strains (Moore et al, Science 2002). We had the opportunity to study a narrow source clade B’ HIV-1 infected Former Plasma Donors (FPD) village Cohort. We have previously reported that after 10 years of infection, clear HLA class I associated foot-prints are seen in the gag, pol and nef genes of bulk pro-viral DNA populations of untreated patients (Dong et al, Blood 2011). Here we present further studies from this cohort with the aim of defining and examining the selective pressures exerted on the vpu gene (which has important immune evasion functions). Methods: Samples were studied from 95 FDP in the village cohort using convenience sampling, collected an estimated 13-15 years after infection. We examined the sequences of HIV-1 vpu from plasma RNA, and assembled a consensus sequence, for which overlapping peptides were synthesised. The HLA class I and viral sequence data were analysed to determine the extent of HLA class I associations with sequence variation along the vpu sequence. Sites of strong class I associations were tested as possible HLA class I – restricted T-cell epitopes using overlapping peptides representing the cohort consensus vpu sequence in ELISpot assays, with cells from cohort members with the relevant HLA class I type. As antibody-dependent cellular cytotoxicity (ADCC) (Stratov et al, J Virology 2008) has been reported to select vpu variants a flow-cytometric ICS-based- assay was optimised for ADCC responses directed towards vpu. Results: Phylogenetic Analysis revealed the vpu sequences clustered together to reference B’ clade Chinese sequences. HLA association studies identified twenty-three mutations associated with vpu – amongst them, Position 27 and Position 38 of the vpu translated sequence were associated with HLA-B*13. Preliminary studies have demonstrated T-cell recognition of peptides containing these mutations in donors with appropriate HLA. Further studies underway will determine the optimal epitopes recognised, confirm the HLA restriction and demonstrate that the HLA-associated mutations represent T-cell escape mutations. Position A14 has been shown to be critical to vpu tetherin antagonism (Vigan et al, J Virology 2010), and our initial analysis reveals that there are a substantial proportion of A to V mutations in this position position A18 which may impact on tetherin binding. Further data on vpu T-cell and ADCC responses will be presented.

Oligo(2′-O-methylribonucleotides) and their derivatives: IV. Conjugates of oligo(2′-O-methylribonucleotides) with minor groove binders and intercalators: Synthesis, properties, and application

Conjugates of pyrimidine triplex forming 3'-protected oligo(2'-O-methylribonucleotides) with minor groove binders (MGB) and triplex specific intercalator benzoindoloquinoline (BIQ) at 5'-terminus were synthesized. The conjugates formed stable complexes with target dsDNA by simultaneous binding both in its minor and major grooves and BIQ intercalation. The dissociation constants and thermal stability of the conjugate complexes with model dsDNA corresponding to polypurine tract (PPT) of genes nef and pol from HIV proviral genome were determined. Conjugation of oligo(2'-O-methylribonucleotides) with MGB and intercalator increased the stability of the triple complexes with dsDNA at pH 7.2 and 37 degrees C. Intercalator introduction accelerates the process of complex formation. Dose-dependent arrest of the in vitro transcription was demonstrated when a 780 b.p. DNA fragment containing the polypurine tract was transcribed under the control of T7 promoter in the presence of different concentrations of conjugates of oligo(2'-O-methylribonucleotides) containing MGB and BIQ intercalator.

Safety and Immunogenicity of DNA and MVA HIV-1 Subtype C Vaccine Prime-Boost Regimens: A Phase I Randomised Trial in HIV-Uninfected Indian Volunteers

Study DesignA randomized, double-blind, placebo controlled phase I trial.MethodsThe trial was conducted in 32 HIV-uninfected healthy volunteers to assess the safety and immunogenicity of prime-boost vaccination regimens with either 2 doses of ADVAX, a DNA vaccine containing Chinese HIV-1 subtype C env gp160, gag, pol and nef/tat genes, as a prime and 2 doses of TBC-M4, a recombinant MVA encoding Indian HIV-1 subtype C env gp160, gag, RT, rev, tat, and nef genes, as a boost in Group A or 3 doses of TBC-M4 alone in Group B participants. Out of 16 participants in each group, 12 received vaccine candidates and 4 received placebos.ResultsBoth vaccine regimens were found to be generally safe and well tolerated. The breadth of anti-HIV binding antibodies and the titres of anti-HIV neutralizing antibodies were significantly higher (p<0.05) in Group B volunteers at 14 days post last vaccination. Neutralizing antibodies were detected mainly against Tier-1 subtype B and C viruses. HIV-specific IFN-γ ELISPOT responses were directed mostly to Env and Gag proteins. Although the IFN-γ ELISPOT responses were infrequent after ADVAX vaccinations, the response rate was significantly higher in group A after 1st and 2nd MVA doses as compared to the responses in group B volunteers. However, the priming effect was short lasting leading to no difference in the frequency, breadth and magnitude of IFN-γELISPOT responses between the groups at 3, 6 and 9 months post-last vaccination.ConclusionsAlthough DNA priming resulted in enhancement of immune responses after 1st MVA boosting, the overall DNA prime MVA boost was not found to be immunologically superior to homologous MVA boosting.Trial RegistrationClinical Trial Registry CTRI/2009/091/000051

Mutations in the nef and vif genes associated with progression to AIDS in elite controller and slow‐progressor Patients

Progression towards AIDS can vary from 5 to 10 years from the establishment of the primary infection by HIV-1 to more than 10 years in the complete absence of antiretroviral therapy. Several factors can contribute to the outcome of HIV infection, including host genetic and viral replicating characteristics. Historically, nef-deleted viral genomes have been associated with disease progression. Therefore, the lentiviral Nef protein is regarded as a progression factor. The objective of this work was to characterize the nef gene from a group of treatment naive patients infected with HIV-1 for more than 10 years. These patients were classified as long-term non-progressors, elite controller, and slow-progressors according to clinical and laboratorial data. A premature stop codon within the nef gene leading to the expression of a truncated peptide was observed on samples from the elite controller patient. For the slow-progressor patients, several degrees of deletions at the C-terminal of Nef were observed predicting a loss of function of this protein. The vif gene was characterized for these patients and a rare mutation that predicts a miss localization of the Vif protein to the nucleus of infected cells that could prevent its function as an APOBEC neutralization factor was also observed. These data indicate the importance of the HIV accessory proteins as factors that contribute to the outcome of AIDS.

Characterization of HIV-1 gag and nef in Cameroon: further evidence of extreme diversity at the origin of the HIV-1 group M epidemic

BackgroundCameroon, in west central Africa, has an extraordinary degree of HIV diversity, presenting a major challenge for the development of an effective HIV vaccine. Given the continuing need to closely monitor the emergence of new HIV variants in the country, we analyzed HIV-1 genetic diversity in 59 plasma samples from HIV-infected Cameroonian blood donors. Full length HIV gag and nef sequences were generated and phylogenetic analyses were performed.FindingsAll gag and nef sequences clustered within HIV-1M. Circulating recombinant form CRF02_AG predominated, accounting for 50% of the studied infections, followed by clade G (11%), clade D and CRF37_cpx (4% each), and clades A, F, CRF01_AE and CRF36_cpx (2% each). In addition, 22% of the studied viruses apparently had nef and gag genes from viruses belonging to different clades, with the majority (8/10) having either a nef or gag gene derived from CRF02_AG. Interestingly, five gag sequences (10%) and three (5%) nef sequences were neither obviously recombinant nor easily classifiable into any of the known HIV-1M clades.ConclusionThis suggests the widespread existence of highly divergent HIV lineages in Cameroon. While the genetic complexity of the Cameroonian HIV-1 epidemic has potentially serious implications for the design of biomedical interventions, detailed analyses of divergent Cameroonian HIV-1M lineages could be crucial for dissecting the earliest evolutionary steps in the emergence of HIV-1M.

Novel factors interfering with human immunodeficiency virus‐type 1 replication in vivo and in vitro

The strategy of all retroviral infections is based on establishing an equilibrium between virus replication and proviral latency in the infected host. The human immunodeficiency virus-type 1 (HIV-1), belonging to the subfamily of lentiviridae, adds an additional level of sophistication to this general rule by encoding two regulatory genes (tat and rev) and four accessory genes (nef, vif, vpr and vpu); HIV-2, structurally similar to HIV-1 but characterized by lower pathogenicity in vivo, encodes another accessory gene, vpx. The function of these accessory genes has become clear in recent years: they serve as countermeasures to host-cell restriction factors that prevent or curtail the capacity of HIV to productively infect its target cells (typically, CD4+ T lymphocytes, macrophages and dendritic cells). Some of the best characterized restriction factors for HIV-1 are Tripartite Motif-5α (TRIM5α), preventing infection of nonhuman primates, although not being effective in humans, and apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (APOBEC 3G), counteracted by the viral accessory protein Vif. In addition, several other molecules are under scrutiny for their mechanism of action and potential exploitation as novel anti-HIV agents. This review will summarize the recently emerging knowledge on these novel factors and their potential relevance for the discovery of new anti-HIV agents targeting not only the replicative, but also the latent state of HIV infection.

Phylogenetic Analysis of Near Full-Length HIV Type 1 Genomic Sequences from 21 Korean Individuals

The Korean subclade of subtype B (KSB) is the most prevalent HIV-1 strain found in Korea. To date, only two near full-length HIV-1 sequences from Korean patients have been reported. Here, we analyzed a total of 24 near full-length genomes of HIV-1 strains that were isolated from 17 antiretroviral therapy (ART)-naive patients and four ART-exposed patients. Proviral DNA from peripheral blood mononuclear cells was PCR amplified and directly sequenced. Phylogenetic analyses were used to classify viruses from 19 patients as KSB, from one patient as subtype B, from one patient as subtype D, and three viruses from one patient as CRF02_AG. All KSB viruses demonstrated TAAAA instead of TATAA at the TATA box in the LTR. Of the 19 KSB patients, their sequence identities at the nucleotide level ranged from 89.8% to 97.1% from the lowest env gene to the highest pol gene. Other than the CRF02_AG viruses, no recombination events were noted in any of the 19 KSB patients, which is consistent with our previous studies on the pol, vif, and nef genes. Except for one strain, all of the strains were classified as non-syncytium-inducing strains. This is the first report to describe near full-length KSB.

Overexpression of recombinant HIV-1 Subtype C Tat and Nef in a Salmonella vaccine vector

Tat and Nef are very important regulatory proteins of HIV-1. They enhance viral replication and down-regulate expression of MHC Class I molecules, respectively. The antigens are now considered to be targets for HIV vaccine development. The expression of Tat and Nef in Salmonella vaccines has not previously been investigated. In this study, HIV-1 Subtype C tat and nef genes were cloned into an expression plasmid and their expression investigated in Salmonella. Very high-level expression of the two HIV-1 antigens was demonstrated in the recombinant Salmonella. The antigens were also successfully purified in bulk from the bacterium.Salmonella can therefore potentially be used to overexpress HIV-1 antigens and used as a possible delivery system in HIV-1 vaccine development.

Human Immunodeficiency Virus, Herpes Virus Infections, and Pulmonary Vascular Disease

The following state-of-the-art seminar was delivered as part of the Aspen Lung Conference on Pulmonary Hypertension and Vascular Diseases held in Aspen, Colorado in June 2012. This paper will summarize the lecture and present results from a nonhuman primate model of infection with Simian (Human) Immunodeficiency Virus - nef chimeric virions as well as the idea that polymorphisms in the HIV-1 nef gene may be driving the immune response that results in exuberant inflammation and aberrant endothelial cell (EC) function. We will present data gathered from primary HIV nef isolates where we tested the biological consequences of these polymorphisms and how their presence in human populations may predict patients at risk for developing this disease. In this article, we also discuss how a dysregulated immune system, in conjunction with a viral infection, could contribute to pulmonary arterial hypertension (PAH). Both autoimmune diseases and some viruses are associated with defects in the immune system, primarily in the function of regulatory T cells. These T-cell defects may be a common pathway in the formation of plexiform lesions. Regardless of the route by which viruses may lead to PAH, it is important to recognize their role in this rare disease.

HIV-1 Nef Impairs Heterotrimeric G-protein Signaling by Targeting Gαi2 for Degradation through Ubiquitination

The HIV Nef protein is an important pathogenic factor that modulates cell surface receptor trafficking and impairs cell motility, presumably by interfering at multiple steps with chemotactic receptor signaling. Here, we report that a dominant effect of Nef is to trigger AIP4 E3 ligase-mediated Gα(i2) ubiquitination, which leads to Gα(i2) endolysosomal sequestration and destruction. The loss of the Gα(i2) subunit was demonstrable in many cell types in the context of gene transfection, HIV infection, or Nef protein transduction. Nef directly interacts with Gα(i2) and ternary complexes containing AIP4, Nef, and Gα(i2) form. A substantial reversal of Gα(i2) loss and a partial recovery of impaired chemotaxis occurred following siRNA knockdown of AIP4 or NEDD4 or by inhibiting dynamin. The N-terminal myristoyl group, (62)EEEE(65) motif, and (72)PXXP(75) motif of Nef are critical for this effect to occur. Nef expression does not affect a Gq(i5) chimera where the five C-terminal residues of Gq are replaced with those of Gα(i2). Lysine at position 296 of Gα(i2) was identified as the critical determinant of Nef-induced degradation. By specifically degrading Gα(i2), Nef directly subverts leukocyte migration and homing. Impaired trafficking and homing of HIV Nef-expressing lymphocytes probably contributes to early immune dysfunction following HIV infection.

Early changes in the CD8 T Cell immunodominance hierarchy in primary HIV infection prior to seroconversion

Background: Identification of the earliest CD8 T-cell responses against HIV may help select critical viral targets for inclusion in an HIV vaccine. We describe changes to the earliest detected CD8 T-cell responses and changes in genetic sequence encoding targeted epitopes in an individual who presented with Fiebig stage II, clade C acute HIV-infection, 27 days after sexual transmission. Methods: We examined HLA-restricted CD8 T-cell responses by IFNγ ELISpot and HIV Gag, Pol, Nef and Env sequence by 454 deep sequencing over 6 timepoints, from days 27-118 after HIV-transmission. HIV-specific IFNγ responses were detected against ten HIV epitopes in Gag, Nef and Env at day 27 post HIVtransmission when the patient’s CD4 T-cell count was at a nadir of 224 cells/_l, the plasma HIV RNA >106 copies/ml and prior to any detectable p24 antibody. Results: Immunodominant responses were detected to the HLA-B*07:02 restricted Env IIRRIRQGL (IL9) epitope and the B*07:02 Gag GPGHKARVL (GL9) epitope (>4000SFU). A detectable but weaker response was observed to HLA-B*08 Nef FLKEKGGL (FL8) epitope (1010SFU). These responses declined over subsequent timepoints to 470 and 1630SFU on day 118 coincident with a 2 log fall in the plasma viral load, a rise in the CD4 T cell count to 533 cells/μL and antibody seroconversion. The Nef FL8 became the immunodominant response after day 34. IFN_ responses broadened from 10 responses at day 27 to 23 responses by day 118. Analysis of Gag, Nef and Pol genes by 454 deep sequencing showed no evidence of escape within the targeted epitopes as a cause of their decline over time. Conclusion: Early changes in the CD8 T-cell immunodominance hierarchy are apparent in acute HIV-infection prior to seroconversion, including early immunodominant targeting of Env epitopes. Subsequent broadening of the CD8 T-cell response was not associated with CD8 T-cell escape in this case.