anti-Nef Antibodies Research Articles

SARS-CoV-2 mRNA Vaccines Induce Greater Complement Activation and Decreased Viremia and Nef Antibodies in Men With HIV-1.

Immune dysregulation in people with human immunodeficiency virus-1 (PWH) persists despite potent antiretroviral therapy and, consequently, PWH tend to have lower immune responses to licensed vaccines. However, limited information is available about the impact of mRNA vaccines in PWH. This study details the immunologic responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines in PWH and their impact on HIV-1. We quantified anti-S immunoglobulin G (IgG) binding and neutralization of 3 SARS-CoV-2 variants of concern and complement activation in blood from virally suppressed men with HIV-1 (MWH) and men without HIV-1 (MWOH), and the characteristics that may impact the vaccine immune responses. We also studied antibody levels against HIV-1 proteins and HIV-1 plasma RNA. MWH had lower anti-S IgG binding and neutralizing antibodies against the 3 variants compared to MWOH. MWH also produced anti-S1 antibodies with a 10-fold greater ability to activate complement and exhibited higher C3a blood levels than MWOH. MWH had decreased residual HIV-1 plasma viremia and anti-Nef IgG approximately 100 days after immunization. MWH respond to SARS-CoV-2 mRNA vaccines with lower antibody titers and with greater activation of complement, while exhibiting a decrease in HIV-1 viremia and anti-Nef antibodies. These results suggest an important role of complement activation mediating protection in MWH.

Co-delivery of LIGHT expression plasmid enhances humoral and cellular immune responses to HIV-1 Nef in mice

The immunogenicity and efficacy of a DNA vaccine can be greatly enhanced when a gene adjuvant is used. LIGHT, a member of TNF superfamily, can function as a costimulatory molecule for human naïve T cells to proliferate and can be a potential gene adjuvant. In the current study, the eukaryotic expression plasmid pcDNA-nef was constructed by inserting a full-length nef gene into pcDNA3.1(+), and an in vitro transfection experiment suggested that the nef gene could be expressed successfully in mammalian cells. BALB/c mice were immunized with HIV-1 nef DNA vaccine plasmids alone or in combination with LIGHT expression plasmids, and the specific humoral and cellular immune responses were measured. The data showed that HIV-1 nef DNA vaccine plasmids could induce anti-Nef antibodies, Nef-specific lymphocyte proliferation and CTL activity, whereas stronger specific immune responses were induced in mice when co-immunizing with HIV-1 nef DNA vaccine plasmids and LIGHT expression plasmids, suggesting that the eukaryotic expression vector encoding HIV-1 nef is capable of inducing specific immune responses towards HIV-1 Nef and that LIGHT could be considered as a gene adjuvant for HIV-1 DNA vaccination.

High levels of anti‐Nef antibodies may prevent AIDS disease progression in vertically HIV‐1‐infected infants

IntroductionHIV-1-associated CD4+ T-cell depletion is a consequence of uninfected cell death. Nef is one of the viral factors that trigger apoptosis on bystander cells, though the plasma Nef levels do not correlate with Th lymphocytes counts. The aim of our study was to evaluate whether anti-Nef antibodies were involved in paediatric AIDS development and whether they can prevent the CD4+ T-cell depletion in vertically infected children.MethodsTwo hundred and seventy three HIV-1 vertically infected children seen at Garrahan Paediatric Hospital were randomly included in the study, adding 13 selected cases: seven LTNP (long-term non-progressors) and six RP (rapid progressors) children (n total=286). Specific anti-HIV-1-Nef antibodies were titrated by indirect ELISA and compared between groups. The plasma blocking effect on Nef-dependent cytotoxicity was evaluated in Jurkat cells using recombinant Nef as apoptotic stimulus and patient plasmas as blockers, measuring the apoptotic levels using Annexin-V stain and flow cytometry.ResultsOnly 63.4% of the patients had specific anti-Nef antibodies, and the levels of anti-Nef antibodies found in the selected LTNPs plasmas were always significantly higher (p=1.55×10−4) than those in RPs or general HIV-1+ paediatric populations. The LTNPs’ plasma had a strong inhibitory effect on Nef-dependent cytotoxicity even at high dilutions, while RP plasmas had little or no effect on Nef-induced apoptosis.Discussion and conclusionsHigh anti-Nef antibody levels are associated and predict slow or non-progression to AIDS in vertically HIV-1-infected children. They could be an efficient tool in preventing Nef-associated bystander effect, preserving CD4+ T-cells and the immune function in the context of paediatric HIV-1 infection.

Mother-to-child anti-Nef antibody passage may protect HIV-1 vertically-infected children from AIDS disease

Event Abstract Back to Event Mother-to-child anti-Nef antibody passage may protect HIV-1 vertically-infected children from AIDS disease Guillermo Corró1, 2*, Silvia A. Marino2 and Carlos A. Rocco2 1 Universidad de Buenos Aires, Argentina 2 Hospital de Pediatría "Prof. Dr. Juan P. Garrahan", Laboratorio de Biología Celular y Retrovirus, Argentina HIV-1 Nef protein has been strongly associated with uninfected T-cell death (bystander effect). This protein exerts a cytotoxic effect upon CD4+ cells though some authors found no correlation between Nef concentration in plasma and T-cell counts. On the other hand, we have previously observed that high titers of anti-Nef antibodies were found in plasmas of long-term non-progressor children but not in typical or rapid-progressors. The aim of this preliminary study was to evaluate the protective effect that may have the pre-existent anti-Nef antibodies at acute or primary infection. Twelve children who had been clinically assisted in the Garrahan Hospital from Buenos Aires were retrospectively included in the study. Plasma samples at 1-3 months post-birth were evaluated for anti-Nef IgG. The children were then classified into 3 groups according to the anti-Nef antibody levels: IgG- (anti-Nef-IgG titer <50) n=2, IgG+ (anti-Nef-IgG titer range 200-7500) n=7 and LTNPsimil (anti-Nef-IgG >11000) n=3. We observed that the children of the last group remained AIDS-free for more than 10 years while the children that had low titers or no anti-Nef Antibodies progressed to AIDS within 2 years post-birth. Our preliminary data and results suggest that pre-existent anti-Nef antibodies may have a protective effect, preventing AIDS disease progression in HIV-1 vertically infected children. Although this is a preliminary study, our observations would be of great interest for a therapeutic vaccine design as well as alternative therapies development. Acknowledgements The authors are grateful to Ms Natalia Beltramone for her excelent technical assistance Keywords: Antibodies, Nef, passive immunization, Pediatrics, AIDS Vaccines Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Corró G, Marino SA and Rocco CA (2013). Mother-to-child anti-Nef antibody passage may protect HIV-1 vertically-infected children from AIDS disease. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00025 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 09 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Guillermo Corró, Universidad de Buenos Aires, Buenos Aires, Argentina, gcorro2@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Guillermo Corró Silvia A Marino Carlos A Rocco Google Guillermo Corró Silvia A Marino Carlos A Rocco Google Scholar Guillermo Corró Silvia A Marino Carlos A Rocco PubMed Guillermo Corró Silvia A Marino Carlos A Rocco Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Chemotactic activity of HIV-1 Nef for human monocytes

Infiltration of HIV-1 infected and uninfected monocytes/macrophages in organs and tissues is a general phenomenon observed in progression of AIDS. HIV-1 Nef protein is considered as a progression factor in AIDS, and is released from HIV-1 infected cells. Here we show that extracellular Nef increases migration of monocytes. This effect is (i) concentration dependent, (ii) reaches the order of magnitude of that induced by fMLP or CC chemokine ligand 2 MCP-1, (iii) inhibited by anti-Nef monoclonal antibodies as well as by heating and (iiii) depends on a concentration gradient of Nef. Further, Nef does not elicit monocytic THP-1 cells to express chemokines such as CCL2, CCL3 and CCL4. These data suggest that extracellular Nef may contribute to disease progression as well as HIV-1 spreading through affecting migration of monocytes.

Extracellular HIV-1 Nef increases migration of monocytes

Infiltration of human immunodeficiency virus type 1 (HIV-1)-infected and uninfected monocytes/macrophages in organs and tissues is a general phenomenon observed in progression of acquired immunodeficiency syndrome (AIDS). HIV-1 protein Nef is considered as a progression factor in AIDS, and is released from HIV-1-infected cells. Here, we show that extracellular Nef increases migration of monocytes. This effect is (i) concentration-dependent, (ii) reaches the order of magnitude of that induced by formyl-methyonyl-leucyl-proline (fMLP) or CC chemokine ligand 2 (CCL2)/monocyte chemotactic protein (MCP)-1, (iii) inhibited by anti-Nef monoclonal antibodies as well as by heating, and (iv) depends on a concentration gradient of Nef. Further, Nef does not elicit monocytic THP-1 cells to express chemokines such as CCL2, macrophage inhibitory protein-1alpha (CCL3) and macrophage inhibitory protein-1beta (CCL4). These data suggest that extracellular Nef may contribute to disease progression as well as HIV-1 spreading through affecting migration of monocytes.

Nef-induced Alteration of the Early/Recycling Endosomal Compartment Correlates with Enhancement of HIV-1 Infectivity

human immunodeficiency virus type 1 (HIV-1) Nef interacts with the clathrin-associated AP-1 and AP-3 adaptor complexes, stabilizing their association with endosomal membranes. These findings led us to hypothesize a general impact of this viral protein on the endosomal system. Here, we have shown that Nef specifically disturbs the morphology of the early/recycling compartment, inducing a redistribution of early endosomal markers and a shortening of the tubular recycling endosomal structures. Furthermore, Nef modulates the trafficking of the transferrin receptor (TfR), the prototypical recycling surface protein, indicating that it also disturbs the function of this compartment. Nef reduces the rate of recycling of TfR to the plasma membrane, causing TfR to accumulate in early endosomes and reducing its expression at the cell surface. These effects depend on the leucine-based motif of Nef, which is required for the membrane stabilization of AP-1 and AP-3 complexes. Since we show that this motif is also required for the full infectivity of HIV-1 virions, these results indicate that the positive influence of Nef on viral infectivity may be related to its general effects on early/recycling endosomal compartments.

Comparative study of immune responses induced after immunization with plasmids encoding the HIV-1 Nef protein under the control of the CMV-IE or the muscle-specific desmin promoter

The objective of this study was to compare immune responses induced against HIV-1 Nef after DNA immunization with Nef encoding plasmids under the control of an ubiquitous or a muscle-specific promoter. To this end, plasmids containing HIV-1 nef under the control of the Cytomegalovirus or the human desmin promoters, specifically expressed in muscle cells, were constructed. Different groups of BALB/c mice were immunized with 10 or 100 μg of both constructs, controls were the pcDNA3 vector or the Nef protein in Freund’s adjuvant (Nef-CFA). Our data showed that both plasmids stimulated anti-Nef humoral responses in a dose-dependent manner. The anti-Nef antibody response, however, was earlier and higher with the CMV-IE promoter than with the desmin promoter. We also showed that Nef expressing plasmids induced high titers of anti-Nef antibodies (10 4), comparable to those obtained in the Nef-CFA group (4×10 4). Analysis of the specificity of anti-Nef antibodies revealed no influence of the promoter, and in contrast to Nef-CFA, plasmid immunization elicited anti-Nef antibodies directed principally against conformational-dependent epitopes. Data on the lymphoproliferative response showed that the specificity of expression, or the plasmid dose, did not affect the onset-time or the intensity of the response. The predominant IgG2a isotype of anti-Nef antibodies and the cytokine profile, mostly IL-2 and IFN-γ, produced by Nef-stimulated spleen cells indicated a Th1 response in plasmid-immunized mice, in contrast to mice immunized with Nef-CFA, where a Th2 response was induced. In conclusion, these data indicate that antigen expression by muscle cells is sufficient to stimulate a Th1 immune response.

Characterization of humoral and cellular immune responses in mice induced by immunization with HIV-1 Nef regulatory protein encapsulated in poly( dl-lactide-co-glycolide) microparticles

We have characterized the humoral and cellular immune responses of BALB/c mice immunized with HIV-1 Nef regulatory protein encapsulated in poly( dl-lactide-co-glycolide) PLG particles. Three groups of mice were immunized with Nef PLG, Nef in the presence of complete Freund’s adjuvant (CFA) or Nef alone in PBS. When titers were compared 7 months after the last injection, anti-Nef titers in mice immunized with Nef PLG were still close to the maximum, whereas a significant decrease was observed in mice immunized with Nef alone (five times lower) or with Nef in CFA (three times lower). These results indicate that Nef PLG is at least a similar or better vector/adjuvant than Nef in CFA concerning the duration of the humoral immune response. The analysis of cytokine profiles (IL-5 and IL-10) and the isotypic patterns of anti-Nef antibodies (predominantly IgG1), in the three groups of mice, indicated a predominant Th2 immune response. Using synthetic peptides covering the entire sequence of Nef, we identified at least three linear epitopes within sequences 32–64, 118–167 and 185–205 in the sera of mice immunized with Nef PLG or Nef CFA. In contrast, anti-Nef antibodies against Nef alone failed to recognize synthetic peptides, indicating that the majority of anti-Nef antibodies were primarily directed against conformational epitopes. We then examined the ability of Nef PLG to prime for the antigen-specific proliferative responses in vitro. The data obtained indicate the presence of both B-cell and T-cell epitopes in the C-terminal fragment of the protein after immunization of mice with Nef encapsulated in PLG particles.

Specific proliferative T cell responses and antibodies elicited by vaccination with simian immunodeficiency virus Nef do not confer protection against virus challenge.

The efficacy of immunizing with a combination of simian immunodeficiency virus (SIV) Nef vaccines was evaluated. Four vaccinates received three intradermal immunizations with recombinant vaccinia virus that expressed SIV Nef, followed by three intramuscular immunizations with rDNA also expressing SIV Nef. Finally, the four vaccinates received two subcutaneous boosts with recombinant SIV Nef protein. This immunization protocol elicited anti-Nef antibodies in all of the vaccinates as well as specific proliferative responses. However, specific cytotoxic T cell responses were not detected before virus challenge. All vaccinates were challenged intravenously with 10 MID(50) of SIVmacJ5 along with four controls. All eight subjects became infected after SIV challenge and there were no group-specific differences in virus load as measured by virus titration and vRNA analysis. The results of this study support indirectly the report from Gallimore and colleagues (Nat Med 1995;1:1667) suggesting that CD8(+) T lymphocyte responses are required for Nef-based vaccines to restrict SIV infection. If Nef-based vaccines are to be beneficial in controlling infection with immunodeficiency viruses, then it will be necessary to develop more effective immunization protocols that elicit potent CD8(+) cell responses reproducibly.

Low or no antibody responses to human immunodeficiency virus type 1 Nef in infected carriers with subtype E, in contrast to subtype B that showed antibodies preferentially recognizing subtype-specific Nef epitopes

The viral accessory gene product Nef has been shown to play an important role in human immunodeficiency virus type 1 (HIV-1)-induced pathogenesis. Only little information is available regarding the differences in the host immune responses against Nef protein and its function in vivo among different subtypes of HIV-1. In the present study, we showed marked differences in the immune responses to Nef protein between subtypes B and E. The amino acid sequence in subtype E Nef showed 72% homology with that in subtype B. Most murine monoclonal antibodies obtained by immunization with subtype B or E Nef protein showed cross-reactivity with both Nef proteins (80 and 67%, respectively). Next, we focused on the immune responses among infected Japanese and Thai individuals. Subtyping of the individuals into B and E was carried out by enzyme-linked immunosorbent assay (ELISA) using synthetic peptides corresponding to the V3 loop representing the principal neutralizing domain. Most of the sera from these individuals reacted strongly with Gag p24 proteins derived from subtypes B and E at similar levels. However, the immune responses among these individuals to Nef protein were markedly different. Some subtype B-infected Japanese and Thai individuals (40 and 35%, respectively) showed higher levels of anti-Nef antibodies, although these antibodies preferentially recognized epitopes specific to subtype B. On the other hand, most of the subtype E-infected Japanese and Thai individuals showed low or no antibody responses to Nef proteins. Thus, immune responses to Nef were markedly different between subtypes B- and E-infected carriers, suggesting different function(s) for Nef in AIDS pathogenesis. Further, vaccine design must take into account the different subtypes of HIV-1.

Humoral and cellular immune responses to HIV-1 Nef in mice DNA-immunised with non-replicating or self-replicating expression vectors

Objective: HIV accessory protein Nef is expressed early in the infectious cycle of the virus and has been shown to be an effective immunogen in humoral and cellular immune responses. We have used two different self-replicating pBN vectors and one non-replicating pCGal2 derived (pCG) vector expressing HIV-1 Nef in DNA immunisation of mice in order to determine their efficiency in raising humoral and cellular immune responses. Design and methods: The expression of Nef by the three plasmids was tested by transfections into COS-1 cells. Balb/c mice were immunised with the pBN-NEF and pCGE2-NEF constructs using gold particle bombardment. Immunoblotting and immunocytochemistry were used to detect in vitro expression of Nef. 51 Cr release assay, ELISA and immunoblotting were used to detect cellular and humoral immune responses in immunised mice. Results: Efficient in vitro expression of Nef was detected in pBN and pCGE2-NEF transfected cells, in pBN-NEF transfected cells the expression lasting up to three weeks. Anti-Nef antibodies in sera of 13 of 16 pBN-NEF immunised mice were detected within four weeks after the last immunisation, whereas only 2 of 12 pCGE2-NEF immunised mice had very weak anti-Nef antibodies. Twelve of the pBN-NEF immunised mice (75%) and 6 the pCGE2-NEF immunised mice (50%) showed Nef-specific cytotoxic T lymphocyte (CTL) responses within four weeks. Conclusions: We conclude that the three eukaryotic expression vectors tested are capable of inducing a cell mediated immune response towards HIV-1 Nef and should be considered as part of a genetic HIV vaccine.

Specificity of anti-Nef antibodies produced in mice immunized with DNA encoding the HIV-1 nef gene product

Mice immunized with plasmid DNA encoding Nef regulatory protein of human immunodeficiency virus type 1 developed high levels of anti-Nef antibodies. After 4 intramuscular injections of 100 μg plasmid DNA, anti-Nef antibodies reached titers up to 2×10 4. A significant specific antibody response was maintained for at least 16 months. Using a set of seven 31–66 mer synthetic peptides covering the entire sequence of Nef, we analysed the specificity of ant-Nef antibodies. Interestingly, specific antibodies produced in response to Nef expressing plasmid DNA did not recognize the linear peptides except the long C-terminal peptide (aa 141–205) for 3 of the 10 sera. With anti-Nef antibodies produced in mice immunized with the protein Nef without any adjuvant, the same restraint epitope binding was found. Only 3 of the 5 Nef positive sera reacted with the C-terminal peptide. This suggests that specific antibodies induced by plasmid DNA as well as by the non-denatured protein recognize conformation-dependent epitopes. On the contrary, anti-Nef antibodies from mice immunized with the protein in Freund's adjuvant showed a broader epitope reactivity pattern. Interestingly, the analysis of immunoglobulin isotype profiles of antibodies generated by the different protocols of immunization showed that plasmid DNA immunization induced predominantly IgG2a, whereas immunization with Nef protein, with or without adjuvant, yielded a preponderance of IgG1 antibodies.

Characterization of humoral immune responses induced by immunization with plasmid DNA expressing HIV-1 Nef accessory protein

Mice immunized with plasmid DNA encoding Nef accessory protein of human immunodeficiency virus type 1 developed high levels of anti-Nef antibodies which were maintained for at least 16 months. These antibodies produced in response to Nef-expressing plasmid DNA did not recognize the linear peptides except the long C-terminal peptide for three of the ten sera. With anti-Nef antibodies produced in mice immunized with the protein Nef without any adjuvant, the same restraint epitope binding was found. On the contrary, anti-Nef antibodies from mice immunized with the protein in Freund's adjuvant showed a broader epitope reactivity pattern. Interestingly, the analysis of immunoglobulin isotype profiles of antibodies generated by the different protocols of immunization showed that plasmid DNA immunization induced predominantly IgG2a, whereas immunization with Nef protein, with or without adjuvant, yielded a preponderance of IgG1 antibodies.

Nef protein of HIV-1 induces apoptotic cytolysis of murine lymphoid cells independently of CD95 (Fas) and its suppression by serine/threonine protein kinase inhibitors

The Nef protein of HIV-1 is suggested to play a role in depletion of uninfected CD4 + T cells leading to the development of AIDS. The recombinant soluble Nef protein was shown to bind to cell surfaces of various murine lymphoid cell lines, including T and B lymphocytes, mastocytoma cells and macrophages. Cross-linking of the cell-bound Nef protein with anti-Nef antibodies induced apoptotic cytolysis of the cells. Although primary lymphocytes from young mice resisted Nef binding and Nef-induced cytolysis, treatment of the cells with concanavalin A or phytohemagglutinin made them susceptible to these activities, indicating that cellular activation is required for the apoptosis. The Nef-induced apoptosis also occurred with murine cells not expressing CD95 (Fas). These findings were quite similar to those obtained for human blood cells, suggesting that the mouse is applicable for analysis of Nef activities. The Nef-induced apoptosis was efficiently suppressed by serine/threonine protein kinase inhibitors, H7, fasudil hydrochloride and M3, which did not inhibit CD95 (Fas)-mediated apoptosis. On the other hand, bisindolylmaleimide, a protein kinase C inhibitor which inhibits CD95 (Fas)-mediated apoptosis, did not affect Nef-induced apoptosis. These results suggest that the Nef-induced apoptosis of murine cells involved a serine/threonine protein kinase-dependent signal transduction pathway distinct from the CD95 (Fas)-mediated system.

Binding of HIV-1 Nef to a Novel Thioesterase Enzyme Correlates with Nef-mediated CD4 Down-regulation

Nef is a 27-kDa myristoylated protein conserved in primate lentiviruses. In vivo, simian immunodeficiency virus Nef is required in macaques to produce a high viral load and full pathological effects. Nef has at least three major effects in vitro, induction of CD4 down-regulation, alteration of T cell activation pathways, and enhancement of viral infectivity. We have used the yeast two-hybrid system to identify cellular proteins that interact with HIV-1Lai Nef and could mediate Nef function. A human cDNA was isolated that encodes a new type of thioesterase, an enzyme that cleaves thioester bonds. This novel thioesterase is unlike the animal types I and II thioesterases previously cloned but is homologous to the Escherichia coli thioesterase II. Nef and this thioesterase interact in vitro and are co-immunoprecipitated by anti-Nef antibodies in CEM cells expressing Nef. Nef alleles from human immunodeficiency virus-1 (HIV-1) isolates unable to down-regulate CD4 do not react or react poorly with thioesterase. An HIV-1 NefLai mutant selected for its lack of interaction with thioesterase was also unable to down-regulate CD4 cell-surface expression. These observations suggest that this human thioesterase is a cellular mediator of Nef-induced CD4 down-regulation.

Soluble Nef antigen of HIV-1 is cytotoxic for human CD4+ T cells

We have previously shown that Nef-gene 10 fusion protein induces marked growth arrest of human primary CD4+ T cells. Here, in vitro cytostatic and cytotoxic activities of human immunodeficiency virus type 1 (HIV-1) Nef against CD4+ T cells were extensively investigated. Growth of human CD4+ cells was inhibited significantly just by the addition of purified full-length Nef to cultures. When Nef was cross-linked by anti-Nef antibodies, it became very cytocidal for CD4+ T cells. A high percentage of sera from HIV-1-infected individuals contained soluble Nef. Thus, soluble Nef in vivo may play an important role in immunodysfunction of CD4+ T lymphocytes in HIV-1 infection.

Involvement of HIV nef protein in abnormal hematopoiesis in AIDS: in vitro study on bone marrow progenitor cells.

In previous studies (1-4), we described disrupted hematopoiesis in long-term human bone marrow cultures infected with HIV. Conditioned medium (CM) from HIV-1 non-productively infected liquid cultures inhibits the proliferation of granulo-monocytic progenitor cells (CFU-GM) in clonogenic assays. A growth-inhibitory soluble factor was therefore suspected and we report here that this factor has been identified as the nef gene product. These nef-containing supernatants are specifically neutralized by anti-nef antibodies and recombinant nef has the same growth inhibitory properties. We used a nef-deficient virus to confirm that nef protein is responsible for the in vitro growth inhibition of CFU-GM.

Evidence for the role of human immunodeficiency virus type 1 Nef protein as a growth inhibitor to CD4+ T lymphocytes and for the blocking of the Nef function by anti-Nef antibodies

Human immunodeficiency virus type 1 (HIV-1) can lead to a profound CD4+ T-cell deficiency. To examine the functional role of HIV-1 Nef protein on the marked loss of CD4+ cells, Nef protein was expressed in and purified from Escherichia coli as a fusion protein with T7 phage gene10 product (Nef-gene10). When peripheral blood mononuclear cells (PBMC) from healthy donors were cultivated in the presence of Nef-gene10 or the gene10 product as well as interleukin-2 (IL-2), it was found that the Nef-gene10, but not the gene10 product, induced a remarkable decline in the CD4/CD8 ratio and in the response to phytohaemagglutinin of PBMC as well as of nylon wool-passed purified T cells. Nef-gene10 inhibited the proliferation of CD4+ cells, but did not kill the cells. This suppression of the IL-2-dependent proliferation of CD4+ cells by Nef-gene10 seemed to be due to enhanced production of several lymphokines, especially of interferon-γ. Thus, Nef protein might be partly responsible for the selective depletion of CD4+ cells in HIV-1 infection. Furthermore, the Nef-induced decline in the CD4 CD8 ratio was interrupted by anti-Nef antibodies, suggesting the possibility that a vaccine which resulted in the production of such functional Nef antibodies would be useful in the treatment of HIV-1-induced immunodysfunction.

Epitopes of Human Immunodeficiency Virus Regulatory Proteins &lt;i&gt;Tat&lt;/i&gt;&lt;i&gt;,&lt;/i&gt;&lt;i&gt;Ne&lt;/i&gt;&lt;i&gt;f&lt;/i&gt; and &lt;i&gt;Rev&lt;/i&gt; are Expressed in Skin in Atopic Dermatitis

We extend our previous documentation that epitopes of HIV regulatory proteins tat, rev, and nef are expressed in tissue from uninfected individuals by the immunohistochemical analysis of normal skin (n = 10) and skin in some selected inflammatory dermatoses including urticaria (n = 6), systemic lupus erythematosus (n = 6), and atopic dermatitis (affected skin, n = 10, and after epicutaneous patch test for allergens, n = 8). A rabbit antibody to HIV-2 tat did not show immunolabeling of skin. Blood vessel endothelium was immunolabeled by one of two antibodies applied to HIV-1 tat, by an antibody to HIV-1 rev, and by two antibodies to HIV-1 nef. In addition one of the anti-nef antibodies labeled Langerhans cells. The anti-rev antibody labeled Langerhans cells and melanocytes in the epidermis, and dendritic cells in the dermis. The labeling of these skin components did not differ in prevalence between controls and groups of dermatosis. For other components, diseased skin conditions especially atopic dermatitis showed additional labeling. In affected skin, keratinocytes were labeled by antibodies to rev and one of two antibodies to nef. Skin after epicutaneous allergen patch testing also showed a statistically significantly increased prevalence of immunolabeling of dendritic cells and Langerhans cells by one of the anti-tat antibodies. We conclude that skin components show expression of epitopes recognized by antibodies to HIV-1 tat, rev, and nef; this expression is more extensive in atopic dermatitis than in normal skin, and can be further increased after epicutaneous allergen patch testing.(ABSTRACT TRUNCATED AT 250 WORDS)