Surfactant protein D modulates HIV infection of both T-cells and dendritic cells

Surfactant Protein D (SP-D) is an oligomerized C-type lectin molecule with immunomodulatory properties and involvement in lung surfactant homeostasis in the respiratory tract. SP-D binds to the enveloped viruses, influenza A virus and respiratory syncytial virus and inhibits their replication in vitro and in vivo. SP-D has been shown to bind to HIV via the HIV envelope protein gp120 and inhibit infectivity in vitro. Here we show that SP-D binds to different strains of HIV (BaL and IIIB) and the binding occurs at both pH 7.4 and 5.0 resembling physiological relevant pH values found in the body and the female urogenital tract, respectively. The binding of SP-D to HIV particles and gp120 was inhibited by the presence of several hexoses with mannose found to be the strongest inhibitor. Competition studies showed that soluble CD4 and CVN did not interfere with the interaction between SP-D and gp120. However, soluble recombinant DC-SIGN was shown to inhibit the binding between SP-D and gp120. SP-D agglutinated HIV and gp120 in a calcium dependent manner. SP-D inhibited the infectivity of HIV strains at both pH values of 7.4 and 5.0 in a concentration dependent manner. The inhibition of the infectivity was abolished by the presence of mannose. SP-D enhanced the binding of HIV to immature monocyte derived dendritic cells (iMDDCs) and was also found to enhance HIV capture and transfer to the T-cell like line PM1. These results suggest that SP-D can bind to and inhibit direct infection of T-cells by HIV but also enhance the transfer of infectious HIV particles from DCs to T-cells in vivo

Inactivation of respiratory syncytial virus by zinc finger reactive compounds

<p>Abstract</p> <p>Background</p> <p>Infectivity of retroviruses such as HIV-1 and MuLV can be abrogated by compounds targeting zinc finger motif in viral nucleocapsid protein (NC), involved in controlling the processivity of reverse transcription and virus infectivity. Although a member of a different viral family (<it>Pneumoviridae</it>), respiratory syncytial virus (RSV) contains a zinc finger protein M2-1 also involved in control of viral polymerase processivity. Given the functional similarity between the two proteins, it was possible that zinc finger-reactive compounds inactivating retroviruses would have a similar effect against RSV by targeting RSV M2-1 protein. Moreover, inactivation of RSV through modification of an internal protein could yield a safer whole virus vaccine than that produced by RSV inactivation with formalin which modifies surface proteins.</p> <p>Results</p> <p>Three compounds were evaluated for their ability to reduce RSV infectivity: 2,2'-dithiodipyridine (AT-2), tetraethylthiuram disulfide and tetramethylthiuram disulfide. All three were capable of inactivating RSV, with AT-2 being the most potent. The mechanism of action of AT-2 was analyzed and it was found that AT-2 treatment indeed results in the modification of RSV M2-1. Altered intramolecular disulfide bond formation in M2-1 protein of AT-2-treated RSV virions might have been responsible for abrogation of RSV infectivity. AT-2-inactivated RSV was found to be moderately immunogenic in the cotton rats <it>S.hispidus </it>and did not cause a vaccine-enhancement seen in animals vaccinated with formalin-inactivated RSV. Increasing immunogenicity of AT-2-inactivated RSV by adjuvant (Ribi), however, led to vaccine-enhanced disease.</p> <p>Conclusions</p> <p>This work presents evidence that compounds that inactivate retroviruses by targeting the zinc finger motif in their nucleocapsid proteins are also effective against RSV. AT-2-inactivated RSV vaccine is not strongly immunogenic in the absence of adjuvants. In the adjuvanted form, however, vaccine induces immunopathologic response. The mere preservation of surface antigens of RSV, therefore may not be sufficient to produce a highly-efficacious inactivated virus vaccine that does not lead to an atypical disease.</p

Identification of HIV-1 Epitopes that Induce the Synthesis of a R5 HIV-1 Suppression Factor by Human CD4+ T Cells Isolated from HIV-1 Immunized Hu-PBL SCID Mice

We have previously reported that immunization of the severe combined immunodeficiency (SCID) mice reconstituted with human peripheral blood mononuclear cells (PBMC) (hu-PBL-SCID mice) with inactivated human immunodeficiency virus type-1 (HIV-1)-pulsed-autologous dendritic cells (HIV-DC) elicits HIV-1-reactive CD4+ T cells that produce an as yet to be defined novel soluble factor in vitro with anti-viral properties against CCR5 tropic (R5) HIV-1 infection. These findings led us to perform studies designed to identify the lineage of the cell that synthesizes such a factor in vitro and define the epitopes of HIV-1 protein that have specificity for the induction of such anti-viral factor. Results of our studies show that this property is a function of CD4+ but not CD8+ T cells. Human CD4+ T cells were thus recovered from the HIV-DC-immunized hu-PBL-SCID mice and were re-stimulated in vitro by co-culture for 2 days with autologous adherent PBMC as antigen presenting cells, APC previously pulsed with inactivated HIV in IL-2-containing medium to expand HIV-1-reactive CD4+ T cells. Aliquots of these re-stimulated CD4+ T cells were then co-cultured with similar APC's that were previously pulsed with 10 μg/ml of a panel of HIV peptides for an additional 2 days, and their culture supernatants were examined for the production of both the R5 HIV-1 suppression factor and IFN-Υ. The data presented herein show that the HIV-1 primed CD4+ T cells produced the R5 suppression factor in response to a wide variety of HIV-1 gag, env, pol, nef or vif peptides, depending on the donor of the CD4+ T cells. Simultaneous production of human interferon (IFN)-Υ was observed in some cases. These results indicate that human CD4+ T cells in PBMC of HIV-1 naive donors have a wide variety of HIV-1 epitope-specific CD4+ T cell precursors that are capable of producing the R5 HIV-1 suppression factor upon DC-based vaccination with whole inactivated HIV-1

Molecular Architectures of Trimeric SIV and HIV-1 Envelope Glycoproteins on Intact Viruses: Strain-Dependent Variation in Quaternary Structure

The initial step in target cell infection by human, and the closely related simian immunodeficiency viruses (HIV and SIV, respectively) occurs with the binding of trimeric envelope glycoproteins (Env), composed of heterodimers of the viral transmembrane glycoprotein (gp41) and surface glycoprotein (gp120) to target T-cells. Knowledge of the molecular structure of trimeric Env on intact viruses is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogen-based vaccines to combat HIV/AIDS. Previous analyses of intact HIV-1 BaL virions have already resulted in structures of trimeric Env in unliganded and CD4-liganded states at ∼20 Å resolution. Here, we show that the molecular architectures of trimeric Env from SIVmneE11S, SIVmac239 and HIV-1 R3A strains are closely comparable to that previously determined for HIV-1 BaL, with the V1 and V2 variable loops located at the apex of the spike, close to the contact zone between virus and cell. The location of the V1/V2 loops in trimeric Env was definitively confirmed by structural analysis of HIV-1 R3A virions engineered to express Env with deletion of these loops. Strikingly, in SIV CP-MAC, a CD4-independent strain, trimeric Env is in a constitutively “open” conformation with gp120 trimers splayed out in a conformation similar to that seen for HIV-1 BaL Env when it is complexed with sCD4 and the CD4i antibody 17b. Our findings suggest a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses

Cryoelectron Tomography of HIV-1 Envelope Spikes: Further Evidence for Tripod-Like Legs

A detailed understanding of the morphology of the HIV-1 envelope (Env) spike is key to understanding viral pathogenesis and for informed vaccine design. We have previously presented a cryoelectron microscopic tomogram (cryoET) of the Env spikes on SIV virions. Several structural features were noted in the gp120 head and gp41 stalk regions. Perhaps most notable was the presence of three splayed legs projecting obliquely from the base of the spike head toward the viral membrane. Subsequently, a second 3D image of SIV spikes, also obtained by cryoET, was published by another group which featured a compact vertical stalk. We now report the cryoET analysis of HIV-1 virion-associated Env spikes using enhanced analytical cryoET procedures. More than 2,000 Env spike volumes were initially selected, aligned, and sorted into structural classes using algorithms that compensate for the “missing wedge” and do not impose any symmetry. The results show varying morphologies between structural classes: some classes showed trimers in the head domains; nearly all showed two or three legs, though unambiguous three-fold symmetry was not observed either in the heads or the legs. Subsequently, clearer evidence of trimeric head domains and three splayed legs emerged when head and leg volumes were independently aligned and classified. These data show that HIV-1, like SIV, also displays the tripod-like leg configuration, and, unexpectedly, shows considerable gp41 leg flexibility/heteromorphology. The tripod-like model for gp41 is consistent with, and helps explain, many of the unique biophysical and immunological features of this region

Human Immunodeficiency Virus-1 Uses the Mannose-6-Phosphate Receptor to Cross the Blood-Brain Barrier

HIV-1 circulates both as free virus and within immune cells, with the level of free virus being predictive of clinical course. Both forms of HIV-1 cross the blood-brain barrier (BBB) and much progress has been made in understanding the mechanisms by which infected immune cells cross the blood-brain barrier BBB. How HIV-1 as free virus crosses the BBB is less clear as brain endothelial cells are CD4 and galactosylceramide negative. Here, we found that HIV-1 can use the mannose-6 phosphate receptor (M6PR) to cross the BBB. Brain perfusion studies showed that HIV-1 crossed the BBB of all brain regions consistent with the uniform distribution of M6PR. Ultrastructural studies showed HIV-1 crossed by a transcytotic pathway consistent with transport by M6PR. An in vitro model of the BBB was used to show that transport of HIV-1 was inhibited by mannose, mannan, and mannose-6 phosphate and that enzymatic removal of high mannose oligosaccharide residues from HIV-1 reduced transport. Wheatgerm agglutinin and protamine sulfate, substances known to greatly increase transcytosis of HIV-1 across the BBB in vivo, were shown to be active in the in vitro model and to act through a mannose-dependent mechanism. Transport was also cAMP and calcium-dependent, the latter suggesting that the cation-dependent member of the M6PR family mediates HIV-1 transport across the BBB. We conclude that M6PR is an important receptor used by HIV-1 to cross the BBB

α1Proteinase Inhibitor Regulates CD4+ Lymphocyte Levels and Is Rate Limiting in HIV-1 Disease

Background: The regulation of adult stem cell migration through human hematopoietic tissue involves the chemokine CXCL12 (SDF-1) and its receptor CXCR4 (CD184). In addition, human leukocyte elastase (HLE) plays a key role. When HLE is located on the cell surface (HLE CS), it acts not as a proteinase, but as a receptor for a 1proteinase inhibitor (a 1PI, a 1antitrypsin, SerpinA1). Binding of a1PI to HLECS forms a motogenic complex. We previously demonstrated that a1PI deficiency attends HIV-1 disease and that a1PI augmentation produces increased numbers of immunocompetent circulating CD4 + lymphocytes. Herein we investigated the mechanism underlying the a 1PI deficiency that attends HIV-1 infection. Methods and Findings: Active a 1PI in HIV-1 subjects (median 17 mM, n = 35) was significantly below normal (median 36 mM, p,0.001, n = 30). In HIV-1 uninfected subjects, CD4 + lymphocytes were correlated with the combined factors a1PI, HLECS + lymphocytes, and CXCR4 + lymphocytes (r 2 = 0.91, p,0.001, n = 30), but not CXCL12. In contrast, in HIV-1 subjects with.220 CD4 cells/ml, CD4 + lymphocytes were correlated solely with active a 1PI (r 2 =0.93,p,0.0001, n = 26). The monoclonal anti-HIV-1 gp120 antibody 3F5 present in HIV-1 patient blood is shown to bind and inactivate human a 1PI. Chimpanzee a 1PI differs from human a1PI by a single amino acid within the 3F5-binding epitope. Unlike human a1PI, chimpanzee a1PI did not bind 3F5 or become depleted following HIV-1 challenge, consistent with the normal CD4 + lymphocyte levels and benign syndrome of HIV-1 infected chimpanzees. The presence of IgG-a 1PI immune complexes correlated with decreased CD4 + lymphocytes in HIV-1 subjects

Topology of the C-Terminal Tail of HIV-1 gp41: Differential Exposure of the Kennedy Epitope on Cell and Viral Membranes

The C-terminal tail (CTT) of the HIV-1 gp41 envelope (Env) protein is increasingly recognized as an important determinant of Env structure and functional properties, including fusogenicity and antigenicity. While the CTT has been commonly referred to as the “intracytoplasmic domain” based on the assumption of an exclusive localization inside the membrane lipid bilayer, early antigenicity studies and recent biochemical analyses have produced a credible case for surface exposure of specific CTT sequences, including the classical “Kennedy epitope” (KE) of gp41, leading to an alternative model of gp41 topology with multiple membrane-spanning domains. The current study was designed to test these conflicting models of CTT topology by characterizing the exposure of native CTT sequences and substituted VSV-G epitope tags in cell- and virion-associated Env to reference monoclonal antibodies (MAbs). Surface staining and FACS analysis of intact, Env-expressing cells demonstrated that the KE is accessible to binding by MAbs directed to both an inserted VSV-G epitope tag and the native KE sequence. Importantly, the VSV-G tag was only reactive when inserted into the KE; no reactivity was observed in cells expressing Env with the VSV-G tag inserted into the LLP2 domain. In contrast to cell-surface expressed Env, no binding of KE-directed MAbs was observed to Env on the surface of intact virions using either immune precipitation or surface plasmon resonance spectroscopy. These data indicate apparently distinct CTT topologies for virion- and cell-associated Env species and add to the case for a reconsideration of CTT topology that is more complex than currently envisioned

HIV-Induced Type I Interferon and Tryptophan Catabolism Drive T Cell Dysfunction Despite Phenotypic Activation

Infection by the human immunodeficiency virus (HIV) is characterized by functional impairment and chronic activation of T lymphocytes, the causes of which are largely unexplained. We cultured peripheral blood mononuclear cells (PBMC) from HIV-uninfected donors in the presence or absence of HIV. HIV exposure increased expression of the activation markers CD69 and CD38 on CD4 and CD8 T cells. IFN-α/β, produced by HIV-activated plasmacytoid dendritic cells (pDC), was necessary and sufficient for CD69 and CD38 upregulation, as the HIV-induced effect was inhibited by blockade of IFN-α/β receptor and mimicked by recombinant IFN-α/β. T cells from HIV-exposed PBMC showed reduced proliferation after T cell receptor stimulation, partially prevented by 1-methyl tryptophan, a competitive inhibitor of the immunesuppressive enzyme indoleamine (2,3)-dioxygenase (IDO), expressed by HIV-activated pDC. HIV-induced IDO inhibited CD4 T cell proliferation by cell cycle arrest in G1/S, and prevented CD8 T cell from entering the cell cycle by downmodulating the costimulatory receptor CD28. Finally, the expression of CHOP, a marker of the stress response activated by IDO, was upregulated by HIV in T cells in vitro and is increased in T cells from HIV-infected patients. Our data provide an in vitro model for HIV-induced T cell dysregulation and support the hypothesis that activation of pDC concomitantly contribute to phenotypic T cell activation and inhibition of T cell proliferative capacity during HIV infection

Generation and characterization of a defective HIV-1 Virus as an immunogen for a therapeutic vaccine

BACKGROUND: The generation of new immunogens able to elicit strong specific immune responses remains a major challenge in the attempts to obtain a prophylactic or therapeutic vaccine against HIV/AIDS. We designed and constructed a defective recombinant virus based on the HIV-1 genome generating infective but non-replicative virions able to elicit broad and strong cellular immune responses in HIV-1 seropositive individuals. RESULTS: Viral particles were generated through transient transfection in producer cells (293-T) of a full length HIV-1 DNA carrying a deletion of 892 base pairs (bp) in the pol gene encompassing the sequence that codes for the reverse transcriptase (NL4-3/ΔRT clone). The viral particles generated were able to enter target cells, but due to the absence of reverse transcriptase no replication was detected. The immunogenic capacity of these particles was assessed by ELISPOT to determine γ-interferon production in a cohort of 69 chronic asymptomatic HIV-1 seropositive individuals. Surprisingly, defective particles produced from NL4-3/ΔRT triggered stronger cellular responses than wild-type HIV-1 viruses inactivated with Aldrithiol-2 (AT-2) and in a larger proportion of individuals (55% versus 23% seropositive individuals tested). Electron microscopy showed that NL4-3/ΔRT virions display immature morphology. Interestingly, wild-type viruses treated with Amprenavir (APV) to induce defective core maturation also induced stronger responses than the same viral particles generated in the absence of protease inhibitors. CONCLUSIONS: We propose that immature HIV-1 virions generated from NL4-3/ΔRT viral clones may represent new prototypes of immunogens with a safer profile and stronger capacity to induce cellular immune responses than wild-type inactivated viral particles.This study was supported by grants FIS PI050265, FIS PI040503, FIS PI070291, FIS Intrasalud 080752, FIS PS09/01297, FIS PI10/02984, SAF2006-26667-E, FIT 09-010-205-9, FIPSE 36780/08, Fundación Mútua Madrileña, TRA-094, EC10-153, ISCIII-RETIC RD06/0006, HIVACAT–HIV Development Program in Catalonia, FIPSE 36630/07, UE Program Health 2009 CHAARM. Spanish Health Institute Carlos III (ISCIII) and the Health Department of the Catalan Government (Generalitat de Catalunya). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S