In vivo CD8+ T-cell suppression of siv viremia is not mediated by CTL clearance of productively infected cells.

The CD8+ T-cell is a key mediator of antiviral immunity, potentially contributing to control of pathogenic lentiviral infection through both innate and adaptive mechanisms. We studied viral dynamics during antiretroviral treatment of simian immunodeficiency virus (SIV) infected rhesus macaques following CD8+ T-cell depletion to test the importance of adaptive cytotoxic effects in clearance of cells productively infected with SIV. As previously described, plasma viral load (VL) increased following CD8+ T-cell depletion and was proportional to the magnitude of CD8+ T-cell depletion in the GALT, confirming a direct relationship between CD8+ T-cell loss and viral replication. Surprisingly, first phase plasma virus decay following administration of antiretroviral drugs was not slower in CD8+ T-cell depleted animals compared with controls indicating that the short lifespan of the average productively infected cell is not a reflection of cytotoxic T-lymphocyte (CTL) killing. Our findings support a dominant role for non-cytotoxic effects of CD8+ T-cells on control of pathogenic lentiviral infection and suggest that cytotoxic effects, if present, are limited to early, pre-productive stages of the viral life cycle. These observations have important implications for future strategies to augment immune control of HIV.

Competition model for upregulation of the major histocompatibility complex class II-associated invariant chain by human immunodeficiency virus type 1 Nef.

The human immunodeficiency virus type 1 (HIV-1) Nef protein upregulates the expression of the invariant chain (Ii)/major histocompatibility complex class II (MHC-II) complex at the cell surface. This complex appears to reach the antigen-loading endosomal compartment at least in part via an indirect pathway in which it is internalized from the cell surface via the adaptor protein 2 (AP-2) complex. Here we provide evidence for a competition model to explain how Nef upregulates the expression of Ii at the cell surface. In this model, Nef and Ii compete for binding to AP-2. In support of this model, Nef decreased the rate of internalization of Ii from the cell surface. The AP-binding dileucine motif in Nef, ENTSLL(165), was necessary and sufficient for the upregulation of Ii. In addition, two leucine-based AP-binding motifs in the Ii cytoplasmic tail, DDQRDLI(8) and EQLPML(17), were critical for the efficient upregulation of Ii by Nef. Experiments using Nef variants in which the native dileucine-based sorting motif was replaced with similar motifs from cellular transmembrane proteins allowed modulation of AP-binding specificity. Analysis of these variants suggested that the binding of Nef to AP-2 is sufficient to upregulate Ii at the plasma membrane. Finally, interference with the expression of AP-2 caused an upregulation of Ii at the plasma membrane, and this decreased the effect of Nef. These data indicate that Nef usurps AP-2 complexes to dysregulate Ii trafficking and potentially interfere with antigen presentation in the context of MHC-II.

Nef increases infectivity of HIV via lipid rafts.

Lipid rafts, also known as detergent-resistant membranes (DRM), are microdomains in the plasma membrane enriched in sphingolipids and cholesterol (reviewed in [1, 2]). Human immunodeficiency virus 1 (HIV) buds via lipid rafts [3, 4]. However, the targeting of viral structural components to DRM and its consequences for viral replication are not understood. Moreover, the negative factor Nef from HIV increases viral infectivity (reviewed in [5, 6]). With no apparent differences in structural components and morphology between wild-type and DeltaNef virons, the latter viruses display less efficient reverse transcription in target cells. As Nef is expressed abundantly early in the viral replicative cycle [7], we hypothesized that Nef could affect viral morphogenesis and budding to render viruses more infectious. In this report, we demonstrated first that Nef increases viral budding from lipid rafts. Second, in the presence of Nef, viral envelopes contain more ganglioside (GM1), which is a major component of lipid rafts. This finding correlated directly with the increased infectivity of HIV. Finally, the depletion of exogenous and endogenous cholesterol biochemically and genetically, which disrupted lipid rafts, decreased viral infectivity only in the presence of Nef. Importantly, HIV lacking the nef gene remained unaffected by these manipulations. We conclude that lipids in virions are essential for viral infectivity. Thus, HIV becomes more infectious when it buds from lipid rafts, and Nef plays a major role in this process.

Expression, intracellular targeting and purification of HIV Nef variants in tobacco cells.

BACKGROUND: Plants may represent excellent alternatives to classical heterologous protein expression systems, especially for the production of biopharmaceuticals and vaccine components. Modern vaccines are becoming increasingly complex, with the incorporation of multiple antigens. Approaches towards developing an HIV vaccine appear to confirm this, with a combination of candidate antigens. Among these, HIV-Nef is considered a promising target for vaccine development because immune responses directed against this viral protein could help to control the initial steps of viral infection and to reduce viral loads and spreading. Two isoforms of Nef protein can be found in cells: a full-length N-terminal myristoylated form (p27, 27 kDa) and a truncated form (p25, 25 kDa). Here we report the expression and purification of HIV Nef from transgenic tobacco. RESULTS: We designed constructs to direct the expression of p25 and p27 Nef to either the cytosol or the secretory pathway. We tested these constructs by transient expression in tobacco protoplasts. Cytosolic Nef polypeptides are correctly synthesised and are stable. The same is not true for Nef polypeptides targeted to the secretory pathway by virtue of a signal peptide. We therefore generated transgenic plants expressing cytosolic, full length or truncated Nef. Expression levels were variable, but in some lines they averaged 0.7% of total soluble proteins. Hexahistidine-tagged Nef was easily purified from transgenic tissue in a one-step procedure. CONCLUSION: We have shown that transient expression can help to rapidly determine the best cellular compartment for accumulation of a recombinant protein. We have successfully expressed HIV Nef polypeptides in the cytosol of transgenic tobacco plants. The proteins can easily be purified from transgenic tissue.

High and inducible expression of human immunodeficiency virus type 1 (HIV-1) Nef by adenovirus vector does not disturb potent antigen presentation by monocyte-derived dendritic cells.

Numerous studies indicated that Nef is a pleiotropic factor. Although it has been shown that Nef impairs the antigen-presenting activity of dendritic cells, more recent studies have shown no such impairment. This issue is critical for designing a vaccine expressing Nef. To refine our knowledge regarding the effect of Nef on dendritic cells, we developed constitutive and inducible adenovirus vector systems that express high levels of Nef in monocyte-derived dendritic cells (MDDCs). We showed here that Nef expression clearly downregulated CD4 expression of MDDCs but had little or no effect on other surface molecules, including MHC class I. Nef also did not affect the functional maturation of MDDCs. Use of the inducible Nef-expression system clearly revealed that adenovirus infection per se modulates cytokine secretion and the expression of apoptosis-related molecules in MDDCs, whereas Nef had no effect on these functions. Moreover, the antigen-presenting activity of MDDCs was not disturbed by the presence of Nef. On the contrary, we found that Nef-expressing MDDCs generated from HIV-1-infected individuals efficiently activated Nef-reactive T cells. Therefore, although adenovirus vector may modulate some aspects of MDDC function, Nef-expressing adenovirus would be served as one of HIV vaccine candidates.

Codon-optimized reading frames facilitate high-level expression of the HIV-1 minor proteins.

We have constructed reading frames for the HIV-1 YU-2 minor proteins Vpr, Vpu, Vif and Nef that are codon-optimized for high-level expression in mammalian cells. We show that, in the absence of the Rev/Rev-response element system, these codon-optimized reading frames result in greatly increased levels of expression of the corresponding proteins in cell culture systems when compared with the native reading frame. Northern blot analysis shows that the increase in expression found with the codon-optimized reading frames is largely owing to increased steady-state mRNA levels.

The HIV-1 nef gene product is associated with phosphorylation of a 46 kD cellular protein.

OBJECTIVE: To study the biochemical properties of the HIV-1 nef gene product. DESIGN: Earlier reports suggested that Nef protein is phosphorylated and has kinase activity. These properties were examined using an in vitro translated product. METHODS: A DNA fragment encoding Nef of HIV-1SF2 was transcribed in vitro under the control of T7 promoter. The generated nef messenger RNA (mRNA) was translated using a rabbit reticulocyte lysate system. Immunoprecipitation was performed with a specific monoclonal antibody to Nef. Kinase activity of the translated product was evaluated using [gamma-32P]GTP and ATP. RESULTS: Translated nef mRNA was found to encode a major protein of 27 kD with two other products, of 25 and 29 kD. Using Nef immune complexes for the kinase reaction, a protein of 46 kD that reacted with the anti-Nef monoclonal antibody was found to be strongly phosphorylated in the presence of Nef. This Nef-associated kinase activity on a p46 cellular substrate was dependent on ATP. CONCLUSIONS: The in vitro-translated nef gene product was found to be associated with phosphorylation of a p46 cellular protein present in rabbit reticulocyte lysate. The Nef protein has been linked to suppression of the replication of certain HIV strains. Identification of cellular targets of Nef activity could help to elucidate the mechanisms for this antiviral effect.

Gene combination raises broad human immunodeficiency virus-specific cytotoxicity.

DNA plasmid immunization has the important advantage over traditional vaccines of making it possible to combine selected genes into one vaccine. The efficacy of a combination of DNA plasmids encoding the nef, rev, and tat HIV-1 regulatory genes in inducing cellular immune responses was analyzed in asymptomatic HIV-1-infected patients. Patients initially selected for having low or no detectable immune responses to Nef, Rev, or Tat antigens developed MHC class I-restricted cytolytic activities as well as enhanced bystander effects. The induction of memory cells against target cells infected with the whole HIV-1 genome was analyzed by using a pseudovirus HIV-1/murine leukemia virus (MuLV), and target cells infected with vaccinia virus carrying the respective gene. The most remarkable change observed after immunization with the gene combination was an increase in cytotoxic T lymphocyte (CTL) precursors to target cells infected with the whole HIV-1 genome. Infection by the pseudotype HIV-1/MuLV virus should result in a multitude of HIV-1 peptides presented on the target cell surface, representative of the in vivo situation. An in vitro assessment of the expression of the single and combined gene products showed that this was consistent with the induction of CTL responses in vivo. No clinical advantage or adverse effects were noted. Therapeutic effects of such immunization may become measurable by structured therapy interruption.

Clustered localization of oligomeric Nef protein of human immunodeficiency virus type 1 on the cell surface.

We studied human immunodeficiency virus type 1 (HIV-1) Nef protein biochemically and histologically. HIV-1 Nef, derived from baculosystem and from cells infected with HIV-1, formed homomeric monomers, dimers, trimers, and further polymers. These oligomers were non-covalently associated. In cells infected with HIV-1, Nef molecules were clustered at the cell surface as well as cytoplasm. Our previous results have indicated that the Nef on the surface of cells infected with HIV-1 is cytotoxic against uninfected CD4+ T cells. Thus, it is very likely that the HIV-1-mediated cytotoxic reaction is due, at least in part, to the clustered localization of oligomeric Nef on the cell surface.

Development of a novel allo-independent HIV-1 virus preparation for use in immunoassays.

The study of the immunologic response to whole human immunodeficiency virus type 1 (HIV-1) antigen is limited by the presence of highly immunogenic human leukocyte antigen (HLA) alloantigens on the envelope of wild type virus. This paper outlines the production of HIV-1 infectious virions free of HLA for use as whole viral antigens in immunoassays. An infectious molecular clone of HIV-1 was transfected into the K-562 cell line, which does not express HLA on the cell surface. After a 30-day selection period, to ensure stable transfection, cells and culture supernatants were analyzed for productive HIV-1 infection and virion infectivity. An enzyme-linked immunosorbent assay (ELISA) confirmed the presence of p24 in the culture supernatants. Molecular confirmation of HIV-1 transfection was achieved by gene amplification. Flow cytometric analysis was used to identify gp120 on the surface of the infected cells. Viral supernatants were tested for HIV infectivity in peripheral blood mononuclear cells (PBMCs). The usefulness of this viral preparation as whole virus antigens was validated using PBMCs from HIV-infected individuals. These results indicate the successful production of HIV-1 infectious virions, which do not have HLA molecules on their viral envelope, and demonstrate their utility for immunoassays.

Attenuated SIV imparts immunity to challenge with pathogenic spleen-derived SIV but cannot prevent repair of the nef deletion.

To date, some success has been achieved with several experimental vaccines against AIDS in the available animal models. In the simian immunodeficiency virus (SIV) macaque model protection against superinfection was obtained by preinfection with a virus attenuated by a deletion in nef. To investigate the efficacy of SIVmac32H(pC8), a nef deletion mutant of SIVmac251, as a live-attenuated vaccine, rhesus monkeys were infected intravenously (i.v.) with this virus. All monkeys became productively infected by the pC8 virus. The animals had low cell-associated viral loads but developed a strong cellular and humoral antiviral immune response. Two out of eight preinfected monkeys developed signs of immunodeficiency and were excluded from the challenge. Sequence analysis of reisolates from one of them revealed a complete repair of the nef deletion. The remaining six monkeys, two preinfected for 42 weeks and four for 22 weeks, were challenged i.v. with a pathogenic SIV derived ex vivo from the spleen of a SIV infected macaque. Four of the monkeys challenged resisted the second infection whereas in two monkeys preinfected for 22 weeks full length nef was detectable. All monkeys maintained a virus-specific CD4-cell proliferative response after challenge. Thus, even after short preinfection periods with an attenuated SIV sterilising immunity against a challenge with a pathogenic SIV can be obtained. However, such a vaccine is unsafe since the attenuated virus frequently reverts to a more virulent form.

Superinfection of a defective human immunodeficiency virus type 1 provirus-carrying T cell clone with vif or vpu mutants gives cytopathic virus particles by homologous recombination.

The partially CD4-expressing T cell clone, Vpr-1, which carries a latent vpr-defective HIV-1 genome and expresses HIV-1 Nef protein only, was permissive to superinfection by HIV-1. Superinfection of Vpr-1 with vif- or vpu-defective mutants, which were noncytopathic, reactivated the vpr-defective virus and led to homologous recombination and cytopathogenesis. The data provide an experimental model for homologous recombination being an important mechanism whereby HIV-1 acquires genetic heterogeneity, and when occurring among defective virus in vivo bestows novel biological activities and virulence.

Fusogenic activity of amino-terminal region of HIV type 1 Nef protein.

We have studied two isoforms of Nef, Nef-27 and Nef-25, which were produced in E. coli. Nef-25 lacked the first 18 N-terminal residues of Nef-27 and both were nonmyristylated. Nef-27 fuses small unilamellar dipalmitoyl phosphatidylcholine vesicles (SUVs), as indicated by enhanced light scattering of SUVs and lipid mixing using concentration-dependent fluorescence dequenching. Nef-27 also causes the appearance of a shifted isotropic peak in the 31P NMR spectra of these vesicles, suggesting that protein interactions induce nonlamellar lipid structures. Recombinant Nef-25, which lacks only the 18 N-terminal residues of Nef-27, does not fuse vesicles and has little effect on the 31P NMR spectra. On the other hand, synthetic peptides consisting of 18 or 21 of the N-terminal residues of Nef-27 are strongly membrane perturbing, causing vesicle fusion and inducing isotropic peaks in the 31P NMR spectrum. Endogenous fluorescence spectra of the N-terminal peptide (21 residues) with SUVs show that the N-terminal sequence of Nef may achieve these perturbing effects by inserting its hydrophobic side into the lipid bilayer. Theoretical calculations using hydrophobic moment plot analysis indicate that short-length stretches (i.e., six amino acid residues) of the N-terminal sequence may insert into the lipid bilayer as multimeric alpha helices or beta sheets. The above-described membrane activities of Nef-27, which principally reside in its N-terminal domain, may play critical role(s) in certain functional properties of the full-length protein. For example, the fusogenic activity of the N-terminal sequence may be involved in the extracellular release of Nef-27, much of which appears to be associated with small membrane vesicles. The fusion activity may also be relevant to the ability of Nef-27 to downregulate CD4 and IL-2 receptors when this protein is electroporated into cultured lymphocytes, an activity not possessed by Nef-25.

Expression of a novel Nef epitope on the surface of HIV type 1-infected cells.

We compared antibody responses to various structural and accessory gene products of HIV-1 between long-term nonprogressors and patients who have progressed to AIDS. On the basis of our results, we performed epitope mapping of the Nef protein and identified a novel epitope. This novel epitope of the Nef protein was found to be exposed on the surface of HIV-1-infected cells. The antibody response against it correlated with CD4+ cell counts among HIV-1-infected patients (r = 0.457, p < 0.001). Although further research is necessary, we suspect that antibody response against the epitope may be protective against disease progression.

Oligomerization of the HIV type 2 Nef protein: mutational analysis of the heptad leucine repeat motif and cysteine residues.

The human immunodeficiency virus type 2 (HIV-2) Nef protein expressed in Escherichia coli forms highly stable homooligomeric complexes in vitro. Similarly, the native protein synthesized in the persistently infected H9 T cell line also forms stable homooligomers in vivo. To determine whether homooligomer formation is mediated by the leucine zipper-type sequence located in the middle region of the protein, site-directed mutagenesis was used to introduce double and triple point mutations at heptad leucine positions L1, L2, and L4 within the HIV-2NIHZ Nef protein sequence. Here, we show that substitution of a serine residue for the L1 (residue 108) and L2 (residue 115) heptad leucines, and a glutamine residue for the L4 (residue 129) heptad leucine, did not prevent Nef homooligomer formation in vitro. However, a more drastic substitution of alpha-helix-breaking proline residue for the L2 and L4 heptad leucines significantly abrogated ability of the protein to form stable homooligomers. In addition, because significantly higher levels of the Nef oligomers were consistently observed under the nonreducing SDS-PAGE condition, site-specific mutagenesis was also used to examine the role of cysteine residues in generating disulfide-linked Nef dimers in vitro. Here, we also show that single cysteine-to-glycine substitutions at positions 28, 32, or 55 drastically reduced covalent Nef dimer formation and thermal stability of the Nef protein in vitro. Therefore, these results demonstrate that the leucine zipper-type motif in the HIV-2 Nef protein mediates stable homooligomer formation in vitro, and also establish a role for covalent disulfide bonds in the formation of linked Nef dimers and thermal stability of the monomer Nef in vitro.

Two-step purification of His-tagged Nef protein in native condition using heparin and immobilized metal ion affinity chromatographies.

The Nef protein encoded by human immunodeficiency virus type 1 (HIV-1) has been shown to be an important factor of progression of viral growth and pathogenesis in both in vitro and in vivo. The lack of a simple procedure to purify Nef in its native conformation has limited molecular studies on Nef function. A two-step procedure that includes heparin and immobilized metal ion affinity chromatographies (IMACs) was developed to purify His-tagged Nef (His(6)-Nef) expressed in bacteria in native condition. During the elaboration of this purification procedure, we identified two closely SDS-PAGE-migrating contaminating bacterial proteins, SlyD and GCHI, that co-eluted with His(6)-Nef in IMAC in denaturing condition and developed purification steps to eliminate these contaminants in native condition. Overall, this study describes a protocol that allows rapid purification of His(6)-Nef protein expressed in bacteria in native condition and that removes metal affinity resin-binding bacterial proteins that can contaminate recombinant His-tagged protein preparation.

HIV-1 mRNA electroporation of PBMC: a simple and efficient method to monitor T-cell responses against autologous HIV-1 in HIV-1-infected patients.

Efficient monitoring of HIV-1-specific T-cells is crucial for the development of HIV-1 vaccines and immunotherapies. Currently, mainly peptides and vaccinia vectors are used for detection of HIV-1-specific cytotoxic T-lymphocytes (CTL), however, as HIV-1 is a variable virus, it is unknown to what extent the T-cell response against the autologous virus is under- or overestimated by using antigens from heterologous viral strains. Therefore, we established a new method for immunomonitoring of CTL using electroporation of peripheral blood mononuclear cells (PBMC) with mRNA derived from autologous viral strains. From six HIV-1-infected patients virus derived mRNA was produced after PCR-based cloning of autologous gag (n=5) and/or nef genes (n=3) from plasma and electroporated into PBMC from patients and healthy donors. Electroporation of PBMC with mRNA resulted in efficient protein expression with good induction of γ-interferon (γ-IFN) release by specific T-cells comparable to peptide pools and better than recombinant vaccinia viruses. Three mRNA encoded autologous Gag proteins and one autologous mRNA encoded Nef protein were better recognized by autologous PBMC in comparison to heterologous mRNA encoded Gag or Nef proteins (SF2 or HXB2). However, in one case each, mRNA encoded autologous Gag or Nef, respectively, was recognized less efficiently due to the presence of CTL escape mutations. In summary, electroporation of PBMC with mRNA is a very efficient, easy and rapid method for immunomonitoring of HIV-1-specific T-cell responses against autologous viral strains. Our data demonstrate that patients' CTL responses against autologous viral strains may be under- or overestimated by using antigens from heterologous viral strains.

Head-to-head comparison on the immunogenicity of two HIV/AIDS vaccine candidates based on the attenuated poxvirus strains MVA and NYVAC co-expressing in a single locus the HIV-1BX08 gp120 and HIV-1(IIIB) Gag-Pol-Nef proteins of clade B.

In this investigation we have generated and defined the immunogenicity of two novel HIV/AIDS vaccine candidates based on the highly attenuated vaccinia virus strains, MVA and NYVAC, efficiently expressing in the same locus (TK) and under the same viral promoter the codon optimized HIV-1 genes encoding gp120 and Gag-Pol-Nef antigens of clade B (referred as MVA-B and NYVAC-B). In infected human HeLa cells, gp120 is released from cells and GPN is produced as a polyprotein; NYVAC-B induces severe apoptosis but not MVA-B. The two poxvirus vectors showed genetic stability of the inserts. In BALB/c and in transgenic HHD mice for human HLA-A2 class I, both vectors are efficient immunogens and induced broad cellular immune responses against peptides represented in the four HIV-1 antigens. Some differences were observed in the magnitude and breadth of the immune response in the mouse models. In DNA prime/poxvirus boost protocols, the strongest immune response, as measured by fresh IFN-gamma and IL-2 ELISPOT, was obtained in BALB/c mice boosted with NYVAC-B, while in HHD mice there were no differences between the poxvirus vectors. When the prime/boost was performed with homologous or with combination of poxvirus vectors, the protocols MVA-B/MVA-B and NYVAC-B/NYVAC-B, or the combination NYVAC-B/MVA-B gave the most consistent broader immune response in both mouse models, although the magnitude of the overall response was higher for the DNA-B/poxvirus-B regime. All of the immunization protocols induced some humoral response against the gp160 protein from HIV-1 clone LAV. Our findings indicate that MVA-B and NYVAC-B meet the criteria to be potentially useful vaccine candidates against HIV/AIDS.

Production of recombinant HIV-1 Nef (negative factor) protein using Pichia pastoris and a low-temperature fed-batch strategy.

In the present paper we describe the cloning and extracellular expression of the HIV-1 Nef (negative factor) protein utilizing the yeast Pichia pastoris, as well as the successful use of a low-temperature fed-batch strategy for decreasing end-product degradation by proteases. The nef gene in a pPICZalphaA vector was integrated into the genome of three different P. pastoris strains, namely X-33, GS115 and KM71H. On the basis of its efficient growth and production characteristics the wild-type strain (X-33) was found to be the best choice. The decreased end-product degradation at low temperatures was not due to lower amounts of proteases but due to their diminished activity. The yield of biomass from methanol was improved 1.44-fold utilizing the low-temperature strategy compared with the standard fermentation. Purification of histidine-tagged Nef was performed in one step using a Ni(2+)-nitrilotriacetate-Sepharose column. The purified product was characterized by SDS/PAGE, Western blotting, matrix-assisted laser-desorption ionization-time-of-flight MS, reversed-phase HPLC and N-terminal-sequence analysis.

Evasion of cytotoxic T lymphocytes is a functional constraint maintaining HIV-1 Nef expression.

Nef expression is not required for HIV-1 replication and is highly targeted by CD8+ CTL, raising the question of why Nef expression is not lost in order to evade immunity in vivo. We explore whether MHC class I (MHC-I) down-regulation to evade CTL in general is a selective pressure maintaining Nef. HIV-1 with functional Nef (wild type, WT) is compared to virus containing a Nef point mutation (M20A) that selectively ablates MHC-I down-regulation. WT-infected cells are relatively resistant to cytolysis and less suppressed for viral replication by Gag- and RT-specific CTL compared to M20A. These viruses grow similarly in vitro in the absence of CTL, but the presence of Gag- or RT-specific CTL strongly favors WT overgrowth of M20A. Finally, while in vitro selection by Nef-specific CTL readily drives disruption of the nef reading frame, the addition of Gag- or RT-specific CTL markedly limits such escape. These data indicate that MHC-I down-regulation is an important function favoring Nef maintenance due to a net selective advantage in the setting of the general CTL response.