SIV Nef Research Articles

Phylogenetic Reconstruction and Functional Characterization of the Ancestral Nef Protein of Primate Lentiviruses.

Nef is an accessory protein unique to the primate HIV-1, HIV-2, and SIV lentiviruses. During infection, Nef functions by interacting with multiple host proteins within infected cells to evade the immune response and enhance virion infectivity. Notably, Nef can counter immune regulators such as CD4 and MHC-I, as well as the SERINC5 restriction factor in infected cells. In this study, we generated a posterior sample of time-scaled phylogenies relating SIV and HIV Nef sequences, followed by reconstruction of ancestral sequences at the root and internal nodes of the sampled trees up to the HIV-1 Group M ancestor. Upon expression of the ancestral primate lentivirus Nef protein within CD4+ HeLa cells, flow cytometry analysis revealed that the primate lentivirus Nef ancestor robustly downregulated cell-surface SERINC5, yet only partially downregulated CD4 from the cell surface. Further analysis revealed that the Nef-mediated CD4 downregulation ability evolved gradually, while Nef-mediated SERINC5 downregulation was recovered abruptly in the HIV-1/M ancestor. Overall, this study provides a framework to reconstruct ancestral viral proteins and enable the functional characterization of these proteins to delineate how functions could have changed throughout evolutionary history.

The HIV-1 protein Nef activates the Tec family kinase Btk by stabilizing an intermolecular SH3-SH2 domain interaction.

The Nef protein produced by the viruses HIV-1 and SIV drives efficient viral replication partially by inducing constitutive activation of host cell tyrosine kinases, including members of the Src and Tec families. Here, we uncovered the mechanism by which both HIV-1 and SIV Nef enhanced the activity of the Tec family kinase Btk in vitro and in cells. A Nef mutant that could not bind to the SH3 domain of Src family kinases activated Btk to the same extent as did wild-type Nef, demonstrating that Nef activated Src and Tec family kinases by distinct mechanisms. The Btk SH3-SH2 region formed a homodimer requiring the CD loop in the SH2 domain, which was stabilized by the binding of Nef homodimers. Alanine substitution of Pro327 in the CD loop of the Btk SH2 domain destabilized SH3-SH2 dimers, abolished the interaction with Nef, and prevented activation by Nef in vitro. In cells, Nef stabilized and activated wild-type but not P327A Btk homodimers at the plasma membrane. These data reveal that the interaction with Nef stabilizes Btk dimers through the SH3-SH2 interface to promote kinase activity and show that the HIV-1 Nef protein evolved distinct mechanisms to activate Src and Tec family tyrosine kinases to enhance viral replication.

Substitutions in Nef That Uncouple Tetherin and SERINC5 Antagonism Impair Simian Immunodeficiency Virus Replication in Primary Rhesus Macaque Lymphocytes.

Most simian immunodeficiency viruses (SIVs) use Nef to counteract restriction by the tetherin proteins of their nonhuman primate hosts. In addition to counteracting tetherin, SIV Nef has a number of other functions, including the downmodulation of CD3, CD4, and major histocompatibility complex class I (MHC I) molecules from the surface of SIV-infected cells and the enhancement of viral infectivity by preventing the incorporation of SERINC5 into virions. Although these activities require different surfaces of Nef, they can be difficult to separate because of their dependence on similar interactions with AP-1 or AP-2 for clathrin-mediated endocytosis. We previously observed extensive overlap of the SIV Nef residues required for counteracting tetherin and SERINC5. Here, we define substitutions in Nef that separate anti-tetherin activity from SERINC5 antagonism and other activities of Nef. This information was used to engineer an infectious molecular clone of SIV (SIVmac239nefSA) that is sensitive to tetherin but retains CD3, CD4, MHC I, and SERINC5 downmodulation. In primary rhesus macaque CD4+ T cells, SIVmac239nefSA exhibits impaired replication compared to wild-type SIVmac239 under conditions of interferon-induced upregulation of tetherin. These results demonstrate that tetherin antagonism can be separated from other Nef functions and that resistance to tetherin is essential for optimal replication in primary CD4+ T cells. IMPORTANCE Tetherin is an interferon-inducible transmembrane protein that prevents the detachment of enveloped viruses from infected cells by physically tethering nascent virions to cellular membranes. SIV Nef downmodulates simian tetherin to overcome this restriction in nonhuman primate hosts. Nef also enhances virus infectivity by preventing the incorporation of SERINC5 into virions and contributes to immune evasion by downmodulating other proteins from the cell surface. To assess the contribution of tetherin antagonism to virus replication, we engineered an infectious molecular clone of SIV with substitutions in Nef that uncouple tetherin antagonism from other Nef functions. These substitutions impaired virus replication in interferon-treated macaque CD4+ T cells, revealing the impact of tetherin on SIV replication under physiological conditions in primary CD4+ lymphocytes.

Crystal structures of lysophospholipid-bound MHC class I molecules

Newly synthesized major histocompatibility complex (MHC) class I proteins are stabilized in the endoplasmic reticulum (ER) by binding 8-10-mer-long self-peptide antigens that are provided by transporter associated with antigen processing (TAP). These MHC class I:peptide complexes then exit the ER and reach the plasma membrane, serving to sustain the steady-state MHC class I expression on the cell surface. A novel subset of MHC class I molecules that preferentially bind lipid-containing ligands rather than conventional peptides was recently identified. The primate classical MHC class I allomorphs, Mamu-B*098 and Mamu-B*05104, are capable of binding the N-myristoylated 5-mer (C14-Gly-Gly-Ala-Ile-Ser) or 4-mer (C14-Gly-Gly-Ala-Ile) lipopeptides derived from the N-myristoylated SIV Nef protein, respectively, and of activating lipopeptide antigen-specific cytotoxic T lymphocytes. We herein demonstrate that Mamu-B*098 samples lysophosphatidylethanolamine and lysophosphatidylcholine containing up to a C20 fatty acid in the ER. The X-ray crystal structures of Mamu-B*098 and Mamu-B*05104 complexed with lysophospholipids at high resolution revealed that the B and D pockets in the antigen-binding grooves of these MHC class I molecules accommodate these lipids through a monoacylglycerol moiety. Consistent with the capacity to bind cellular lipid ligands, these two MHC class I molecules did not require TAP function for cell-surface expression. Collectively, these results indicate that peptide- and lipopeptide-presenting MHC class I subsets use distinct sources of endogenous ligands.

Loss of tetherin antagonism by Nef impairs SIV replication during acute infection of rhesus macaques.

Most simian immunodeficiency viruses use Nef to counteract the tetherin proteins of their nonhuman primate hosts. Nef also downmodulates cell-surface CD4 and MHC class I (MHC I) molecules and enhances viral infectivity by counteracting SERINC5. We previously demonstrated that tetherin antagonism by SIV Nef is genetically separable from CD4- and MHC I-downmodulation. Here we show that disruption of tetherin antagonism by Nef impairs virus replication during acute SIV infection of rhesus macaques. A combination of mutations was introduced into the SIVmac239 genome resulting in three amino acid substitutions in Nef that impair tetherin antagonism, but not CD3-, CD4- or MHC I-downmodulation. Further characterization of this mutant (SIVmac239AAA) revealed that these changes also result in partial sensitivity to SERINC5. Separate groups of four rhesus macaques were infected with either wild-type SIVmac239 or SIVmac239AAA, and viral RNA loads in plasma and sequence changes in the viral genome were monitored. Viral loads were significantly lower during acute infection in animals infected with SIVmac239AAA than in animals infected with wild-type SIVmac239. Sequence analysis of the virus population in plasma confirmed that the substitutions in Nef were retained during acute infection; however, changes were observed by week 24 post-infection that fully restored anti-tetherin activity and partially restored anti-SERINC5 activity. These observations reveal overlap in the residues of SIV Nef required for counteracting tetherin and SERINC5 and selective pressure to overcome these restriction factors in vivo.

In-vivo use of SIVmac239 with a reversing stop codon in the SIV nef gene.

In-vivo use of SIVmac239 with a reversing stop codon in the SIV nef gene.

Structure of HIV-2 Nef Reveals Unique Features Distinct from HIV-1 Involved in Immune Regulation

The HIV accessory protein Nef plays a major role in establishing and maintaining infection, particularly through immune evasion. Many HIV-2 infected people experience long-term viral control and survival, resembling HIV-1 elite control. HIV-2 Nef has overlapping but also distinct functions from HIV-1 Nef. Here we report the crystal structure of HIV-2 Nef core. The dileucine sorting motif forms a helix bound to neighboring molecules, and moreover, isothermal titration calorimetry demonstrated that the CD3 endocytosis motif can directly bind to HIV-2 Nef, ensuring AP-2 mediated endocytosis for CD3. The highly-conserved C-terminal region forms a α-helix, absent from HIV-1. We further determined the structure of SIV Nef harboring this region, demonstrating similar C-terminal α-helix, which may contribute to AP-1 binding for MHC-I downregulation. These results provide new insights into the distinct pathogenesis of HIV-2 infection.

Complex Problems Require Ternary Solutions: Another Lesson from SIV Nef

In this issue of Cell Host & Microbe, Buffalo etal. describe a cryo-EM structure of SIV Nef complexed with AP-2 and tetherin. The structure helps explain why human tetherin is Nef-resistant and why lentiviruses that successfully emerged in humans (HIV-1 and HIV-2) had to evolve novel anti-tetherin strategies.

Maintenance of AP-2-Dependent Functional Activities of Nef Restricts Pathways of Immune Escape from CD8 T Lymphocyte Responses.

Nef-specific CD8+ T lymphocytes (CD8TL) are linked to extraordinary control of primate lentiviral replication, but the mechanisms underlying their efficacy remain largely unknown. The immunodominant, Mamu-B*017:01+-restricted Nef195-203MW9 epitope in SIVmac239 partially overlaps a sorting motif important for interactions with host AP-2 proteins and, hence, downmodulation of several host proteins, including Tetherin (CD317/BST-2), CD28, CD4, SERINC3, and SERINC5. We reasoned that CD8TL-driven evolution in this epitope might compromise Nef's ability to modulate these important molecules. Here, we used deep sequencing of SIV from nine B*017:01+ macaques throughout infection with SIVmac239 to characterize the patterns of viral escape in this epitope and then assayed the impacts of these variants on Nef-mediated modulation of multiple host molecules. Acute variation in multiple Nef195-203MW9 residues significantly compromised Nef's ability to downregulate surface Tetherin, CD4, and CD28 and reduced its ability to prevent SERINC5-mediated reduction in viral infectivity but did not impact downregulation of CD3 or major histocompatibility complex class I, suggesting the selective disruption of immunomodulatory pathways involving Nef AP-2 interactions. Together, our data illuminate a pattern of viral escape dictated by a selective balance to maintain AP-2-mediated downregulation while evading epitope-specific CD8TL responses. These data could shed light on mechanisms of both CD8TL-driven viral control generally and on Mamu-B*017:01-mediated viral control specifically.IMPORTANCE A rare subset of humans infected with HIV-1 and macaques infected with SIV can control the virus without aid of antiviral medications. A common feature of these individuals is the ability to mount unusually effective CD8 T lymphocyte responses against the virus. One of the most formidable aspects of HIV is its ability to evolve to evade immune responses, particularly CD8 T lymphocytes. We show that macaques that target a specific peptide in the SIV Nef protein are capable of better control of the virus and that, as the virus evolves to escape this response, it does so at a cost to specific functions performed by the Nef protein. Our results help show how the virus can be controlled by an immune response, which could help in designing effective vaccines.

Vaccine Induction of Lymph Node-Resident Simian Immunodeficiency Virus Env-Specific T Follicular Helper Cells in Rhesus Macaques.

Measurement of Ag-specific T follicular helper (TFH) cell activity in rhesus macaques has not previously been reported. Given that rhesus macaques are the animal model of choice for evaluating protective efficacy of HIV/SIV vaccine candidates and that TFH cells play a pivotal role in aiding B cell maturation, quantifying vaccine induction of HIV/SIV-specific TFH cells would greatly benefit vaccine development. In this study, we quantified SIV Env-specific IL-21-producing TFH cells for the first time, to our knowledge, in a nonhuman primate vaccine study. Macaques were primed twice mucosally with adenovirus 5 host range mutant recombinants encoding SIV Env, Rev, Gag, and Nef followed by two i.m. boosts with monomeric SIV gp120 or oligomeric SIV gp140 proteins. At 2 wk after the second protein boost, we obtained lymph node biopsy specimens and quantified the frequency of total and SIV Env-specific IL-21(+) TFH cells and total germinal center B cells, the size and number of germinal centers, and the frequency of SIV-specific Ab-secreting cells in B cell zones. Multiple correlation analyses established the importance of TFH for development of B cell responses in systemic and mucosally localized compartments, including blood, bone marrow, and rectum. Our results suggest that the SIV-specific TFH cells, initially induced by replicating adenovirus-recombinant priming, are long lived. The multiple correlations of SIV Env-specific TFH cells with systemic and mucosal SIV-specific B cell responses indicate that this cell population should be further investigated in HIV vaccine development as a novel correlate of immunity.

Lentiviral Nef suppresses iron uptake in a strain specific manner through inhibition of Transferrin endocytosis

BackgroundIncreased cellular iron levels are associated with high mortality in HIV-1 infection. Moreover iron is an important cofactor for viral replication, raising the question whether highly divergent lentiviruses actively modulate iron homeostasis. Here, we evaluated the effect on cellular iron uptake upon expression of the accessory protein Nef from different lentiviral strains.ResultsSurface Transferrin receptor (TfR) levels are unaffected by Nef proteins of HIV-1 and its simian precursors but elevated in cells expressing Nefs from most other primate lentiviruses due to reduced TfR internalization. The SIV Nef-mediated reduction of TfR endocytosis is dependent on an N-terminal AP2 binding motif that is not required for downmodulation of CD4, CD28, CD3 or MHCI. Importantly, SIV Nef-induced inhibition of TfR endocytosis leads to the reduction of Transferrin uptake and intracellular iron concentration and is accompanied by attenuated lentiviral replication in macrophages.ConclusionInhibition of Transferrin and thereby iron uptake by SIV Nef might limit viral replication in myeloid cells. Furthermore, this new SIV Nef function could represent a virus-host adaptation that evolved in natural SIV-infected monkeys.

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.

Tetherin/BST-2 Antagonism by Nef Depends on a Direct Physical Interaction between Nef and Tetherin, and on Clathrin-mediated Endocytosis

Nef is the viral gene product employed by the majority of primate lentiviruses to overcome restriction by tetherin (BST-2 or CD317), an interferon-inducible transmembrane protein that inhibits the detachment of enveloped viruses from infected cells. Although the mechanisms of tetherin antagonism by HIV-1 Vpu and HIV-2 Env have been investigated in detail, comparatively little is known about tetherin antagonism by SIV Nef. Here we demonstrate a direct physical interaction between SIV Nef and rhesus macaque tetherin, define the residues in Nef required for tetherin antagonism, and show that the anti-tetherin activity of Nef is dependent on clathrin-mediated endocytosis. SIV Nef co-immunoprecipitated with rhesus macaque tetherin and the Nef core domain bound directly to a peptide corresponding to the cytoplasmic domain of rhesus tetherin by surface plasmon resonance. An analysis of alanine-scanning substitutions identified residues throughout the N-terminal, globular core and flexible loop regions of Nef that were required for tetherin antagonism. Although there was significant overlap with sequences required for CD4 downregulation, tetherin antagonism was genetically separable from this activity, as well as from other Nef functions, including MHC class I-downregulation and infectivity enhancement. Consistent with a role for clathrin and dynamin 2 in the endocytosis of tetherin, dominant-negative mutants of AP180 and dynamin 2 impaired the ability of Nef to downmodulate tetherin and to counteract restriction. Taken together, these results reveal that the mechanism of tetherin antagonism by Nef depends on a physical interaction between Nef and tetherin, requires sequences throughout Nef, but is genetically separable from other Nef functions, and leads to the removal of tetherin from sites of virus release at the plasma membrane by clathrin-mediated endocytosis.

The Size and Conservation of a Coiled-coil Structure in the Ectodomain of Human BST-2/Tetherin Is Dispensable for Inhibition of HIV-1 Virion Release

BST-2/CD317/tetherin is a host factor that inhibits HIV-1 release and is counteracted by HIV-1 Vpu. Structural studies indicate that the BST-2 ectodomain assumes a coiled-coil conformation. Here we studied the role of the BST-2 ectodomain for tethering function. First, we addressed the importance of the length and structure of the ectodomain by adding or substituting heterologous coiled-coil or non-coiled-coil sequences. We found that extending or replacing the BST-2 ectodomain using non-coiled-coil sequences resulted in loss of BST-2 function. Doubling the size of the BST-2 ectodomain by insertion of a heterologous coiled-coil motif or substituting the BST-2 coiled-coil domain with a heterologous coiled-coil motif maintained tethering function. Reductions in the size of the BST-2 coiled-coil domain were tolerated as well. In fact, deletion of the C-terminal half of the BST-2 ectodomain, including a series of seven consecutive heptad motifs did not abolish tethering function. However, slight changes in the positioning of deletions affecting the relative placing of charged or hydrophobic residues on the helix severely impacted the functional properties of BST-2. Overall, we conclude that the size of the BST-2 ectodomain is highly flexible and can be reduced or extended as long as the positioning of residues important for the stability of the dimer interface is maintained.

A trivalent recombinant Ad5 gag/pol/nef vaccine fails to protect rhesus macaques from infection or control virus replication after a limiting-dose heterologous SIV challenge

It has been suggested that poor immunogenicity may explain the lack of vaccine efficacy in preventing or controlling HIV infection in the Step trial. To investigate this issue we vaccinated eight Indian rhesus macaques with a trivalent replication-incompetent adenovirus serotype 5 vaccine expressing SIV Gag, Pol, and Nef using a regimen similar to that employed in the Step trial. We detected broad vaccine-induced CD8+ (2–7 pool-specific responses) and CD4+ (5–19 pool-specific responses) T-cell responses in IFN-γ ELISPOT assays at one week post-boost using fresh PBMC. However, using cryopreserved cells at one and four weeks post-boost we observed a reduction in both the number and magnitude of most vaccine-induced responses. This demonstrates that the time points and conditions chosen to perform immune assays may influence the observed breadth and frequency of vaccine-induced T-cell responses. To evaluate protective efficacy, we challenged the immunized macaques, along with naïve controls, with repeated, limiting doses of the heterologous swarm isolate SIVsmE660. Vaccination did not significantly affect acquisition or control of virus replication in vaccinees compared to naïve controls. Post-infection we observed an average of only two anamnestic CD8+ T-cell responses per animal, which may not have been sufficiently broad to control heterologous virus replication. While the trivalent vaccine regimen induced relatively broad T-cell responses in rhesus macaques, it failed to protect against infection or control viral replication. Our results are consistent with those observed in the Step trial and indicate that SIV immunization and challenge studies in macaque models of HIV infection can be informative in assessing pre-clinical HIV vaccines.

Therapeutic DNA vaccine induces broad T cell responses in the gut and sustained protection from viral rebound and AIDS in SIV-infected rhesus macaques.

Immunotherapies that induce durable immune control of chronic HIV infection may eliminate the need for life-long dependence on drugs. We investigated a DNA vaccine formulated with a novel genetic adjuvant that stimulates immune responses in the blood and gut for the ability to improve therapy in rhesus macaques chronically infected with SIV. Using the SIV-macaque model for AIDS, we show that epidermal co-delivery of plasmids expressing SIV Gag, RT, Nef and Env, and the mucosal adjuvant, heat-labile E. coli enterotoxin (LT), during antiretroviral therapy (ART) induced a substantial 2–4-log fold reduction in mean virus burden in both the gut and blood when compared to unvaccinated controls and provided durable protection from viral rebound and disease progression after the drug was discontinued. This effect was associated with significant increases in IFN-γ T cell responses in both the blood and gut and SIV-specific CD8+ T cells with dual TNF-α and cytolytic effector functions in the blood. Importantly, a broader specificity in the T cell response seen in the gut, but not the blood, significantly correlated with a reduction in virus production in mucosal tissues and a lower virus burden in plasma. We conclude that immunizing with vaccines that induce immune responses in mucosal gut tissue could reduce residual viral reservoirs during drug therapy and improve long-term treatment of HIV infection in humans.

Down-Modulation of CD8αβ Is a Fundamental Activity of Primate Lentiviral Nef Proteins

It is well established that the Nef proteins of human and simian immunodeficiency viruses (HIV and SIV) modulate major histocompatibility complex class I (MHC-I) cell surface expression to protect infected cells against lysis by cytotoxic T lymphocytes (CTLs). Recent data supported the observation that Nef also manipulates CTLs directly by down-modulating CD8αβ (J. A. Leonard, T. Filzen, C. C. Carter, M. Schaefer, and K. L. Collins, J. Virol. 85:6867-6881, 2011), but it remained unknown whether this Nef activity is conserved between different lineages of HIV and SIV. In this study, we examined a total of 42 nef alleles from 16 different primate lentiviruses representing most major lineages of primate lentiviruses, as well as nonpandemic HIV-1 strains and the direct precursors of HIV-1 (SIVcpz and SIVgor). We found that the vast majority of these nef alleles strongly down-modulate CD8β in human T cells. Primate lentiviral Nefs generally interacted specifically with the cytoplasmic tail of CD8β, and down-modulation of this receptor was dependent on the conserved dileucine-based motif and two adjacent acidic residues (DD/E) in the C-terminal flexible loop of SIV Nef proteins. Both of these motifs are known to be important for the interaction of HIV-1 Nef with AP-2, and they were also shown to be critical for down-modulation of CD4 and CD28, but not MHC-I, by SIV Nefs. Our results show that down-modulation of CD4, CD8β, and CD28 involves largely overlapping (but not identical) domains and is most likely dependent on conserved interactions of primate lentiviral Nefs with cellular adaptor proteins. Furthermore, our data demonstrate that Nef-mediated down-modulation of CD8αβ is a fundamental property of primate lentiviruses and suggest that direct manipulation of CD8+ T cells plays a relevant role in viral immune evasion.

Nef interferes with development of thymic T cell precursors: differential mechanisms in HIV and SIV

Background HIV infection of the thymus results in a decreased output of naive T cells. We previously showed that expression of the Nef protein alone is sufficient to disturb human thymopoiesis. Additional structure-function studies with mutant Nef alleles suggested a role of PAK2 in this process. In this study we evaluated the effect of Nef alleles from different clinical HIV-1, HIV-2 and SIV isolates and Nef mutants which were specifically mutated in the PAK2 interaction surface for their effect on T cell development.

One Protein to Rule them All: Modulation of Cell Surface Receptors and Molecules by HIV Nef

The HIV-1, HIV-2 and SIV Nef protein are known to modulate the expression of several cell surface receptors and molecules to escape the immune system, to alter T cell activation, to enhance viral replication, infectivity and transmission and overall to ensure the optimal environment for infection outcome. Consistent and continuous efforts have been made over the years to characterize the modulation of expression of each of these molecules, in the hope that a better understanding of these processes essential for HIV infection and/or pathogenesis will eventually highlight new therapeutic targets. In this article we provide an extensive review of the knowledge gained so far on this important and evolving topic.

Cationic nanoglycolipidic particles as vector and adjuvant for the study of the immunogenicity of SIV Nef protein

The objective of this study was to test the immunogenicity of SIV Nef protein formulated in cationic nanoglycolipidic particles of 100 nm of diameter. In parallel, the adjuvant effect of these nanoglycolipidic particles was compared in similar experiments using GST-Nef in association with the commonly strongest used complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant in association with MDP or MDP alone. Our results showed that these particles do not alter the integrity of our immunogen GST-Nef, which remains stable for more than three months at 4 °C. We demonstrated that in the presence of nanoglycolipidic particles antibodies against Nef were produced since the first injection and remained stable after the third injection with high titers for long lasting periods as observed with CFA and IFA/MDP adjuvant. The analysis of immunoglobulin isotype profiles of antibodies generated by the different protocols of immunization showed the preponderance of IgG1 isotypes suggesting the predominance of Th2-type immune response.