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The availability of new classes of antiretroviral drugs is critical for treatment-experienced patients due to drug resistance to and unwanted side effects from current drugs. Our aim was therefore to evaluate the anti-HIV-1 activity of a new set of antivirals, dipeptides (WG-am or VQ-am) combined with a single-stranded oligonucleotide (ssON). The dipeptides were identified as naturally occurring and enriched in feces and systemic circulation in HIV-1-infected elite controllers and were proposed to act as entry inhibitors by binding to HIV-1 gp120. The ssON is DNA 35-mer, stabilized by phosphorothioate modifications, which acts on the endocytic step by binding to cell host receptors and inhibiting viruses through interference with binding to nucleolin.Chou-Talalay's Combination Index method for quantifying synergism was used to evaluate the drug combinations. Patient-derived chimeric viruses encoding the gp120 (env region) were produced by transient transfection and used to evaluate the antiviral profile of the combinations by drug susceptibility assays.We found that the combination WG-am:ssON or VQ-am:ssON had low combination index values, suggesting strong antiviral synergism. Of the two combinations, WG-am:ssON (1 mM:1 μM) had high efficacy against all prototype or patient-derived HIV-1 isolates tested, independent of subtype including the HIV-1-A6 sub-subtype. In addition, the antiviral effect was independent of co-receptor usage in patient-derived strains.WG-am and ssON alone significantly inhibited HIV-1 replication regardless of viral subtype and co-receptor usage, and the combination WG-am:ssON (1 mM:1 μM) was even more effective due to synergism.

The COVID-19 pandemic caused by SARS-CoV-2 presents a global health emergency. Therapeutic options against SARS-CoV-2 are still very limited but urgently required. Molecular tweezers are supramolecular agents that destabilize the envelope of viruses resulting in a loss of viral infectivity. Here, we show that first-generation tweezers, CLR01 and CLR05, disrupt the SARS-CoV-2 envelope and abrogate viral infectivity. To increase the antiviral activity, a series of 34 advanced molecular tweezers were synthesized by insertion of aliphatic or aromatic ester groups on the phosphate moieties of the parent molecule CLR01. A structure-activity relationship study enabled the identification of tweezers with a markedly enhanced ability to destroy lipid bilayers and to suppress SARS-CoV-2 infection. Selected tweezer derivatives retain activity in airway mucus and inactivate the SARS-CoV-2 wildtype and variants of concern as well as respiratory syncytial, influenza, and measles viruses. Moreover, inhibitory activity of advanced tweezers against respiratory syncytial virus and SARS-CoV-2 was confirmed in mice. Thus, potentiated tweezers are broad-spectrum antiviral agents with great prospects for clinical development to combat highly pathogenic viruses. CA Sanchez-Garcia und CA Schrader und CA extern

Artificial nanoparticles accumulate a protein corona layer in biological fluids, which significantly influences their bioactivity. As nanosized obligate intracellular parasites, viruses share many biophysical properties with artificial nanoparticles in extracellular environments and here we show that respiratory syncytial virus (RSV) and herpes simplex virus type 1 (HSV-1) accumulate a rich and distinctive protein corona in different biological fluids. Moreover, we show that corona pre-coating differentially affects viral infectivity and immune cell activation. In addition, we demonstrate that viruses bind amyloidogenic peptides in their corona and catalyze amyloid formation via surface-assisted heterogeneous nucleation. Importantly, we show that HSV-1 catalyzes the aggregation of the amyloid β-peptide (Aβ42), a major constituent of amyloid plaques in Alzheimer’s disease, in vitro and in animal models. Our results highlight the viral protein corona as an acquired structural layer that is critical for viral–host interactions and illustrate a mechanistic convergence between viral and amyloid pathologies., The protein corona around artificial nanoparticles is known to influence activity and biological fate, the formation around viruses is less well understood. Here, the authors observe the formation of protein corona on viruses and study the effects this corona has on viral infectivity and on amyloid protein assembly.

Our knowledge of the binding sites for neutralizing Abs (NAb) that recognize a broad range of HIV-1 strains (bNAb) has substantially increased in recent years. However, gaps remain in our understanding of how to focus B cell responses to vulnerable conserved sites within the HIV-1 envelope glycoprotein (Env). In this article, we report an immunization strategy composed of a trivalent HIV-1 (clade B envs) DNA prime, followed by a SIVmac239 gp140 Env protein boost that aimed to focus the immune response to structurally conserved parts of the HIV-1 and simian immunodeficiency virus (SIV) Envs. Heterologous NAb titers, primarily to tier 1 HIV-1 isolates, elicited during the trivalent HIV-1 env prime, were significantly increased by the SIVmac239 gp140 protein boost in rabbits. Epitope mapping of Ab-binding reactivity revealed preferential recognition of the C1, C2, V2, V3, and V5 regions. These results provide a proof of concept that a distally related retroviral SIV Env protein boost can increase pre-existing NAb responses against HIV-1.

Item does not contain fulltext Cytokines and IFNs, such as TNF-alpha and IFN-alpha, upregulate costimulatory molecules in monocyte-derived dendritic cells (MDDCs), enabling effective Ag presentation to T cells. This activation of MDDCs is often accompanied by upregulation of apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) (A3) family proteins that are able to restrict HIV-1 replication in MDDCs by inducing hypermutations in the viral genome. In this study, we show that TNF-alpha upregulates costimulatory molecules and are able to restrict HIV-1BaL replication in MDDCs without significant induction of A3G, A3A, or A3F. Conversely, low quantities of IFN-alpha failed to upregulate costimulatory molecules, did not induce IL-12p40 or migration, but significantly induced A3G, A3A, and A3F mRNA expression and restricted viral replication in MDDCs. We also showed that transmission of HIV-1 from MDDCs to autologous T cells was significantly reduced in the presence of IFN-alpha. Sequence analyses detected the induction of high frequency of G-to-A hypermutations in the env genes from HIV-1BaL-infected MDDCs treated with low quantities of IFN-alpha2b. These findings show that low quantities of IFN-alpha can induce functional A3 family proteins and restrict HIV-1 replication in MDDCs while keeping an immature nonmigratory phenotype, supporting further investigations of modalities that enhance retroviral restriction factors. In addition, the findings highlight the role of IFN-alpha as a double-edged sword in HIV-1 infection, and we show that IFN-alpha can be powerful in reducing HIV-1 infection both in MDDCs and T cells.

Dendritic cells are able to present viral antigens to T-cells after uptake of apoptotic bodies derived from virus-infected cells. Immunization with virus-infected apoptotic cells was previously shown to induce HIV-specific immune responses in mice. Here we evaluate the safety and immunogenicity of immunization with activated apoptotic cells in non-human primates using autologous T-cells infected with replication defective VSV pseudotyped SIV mac239 Δ env . Animals were immunized with γ-irradiated activated T-cells carrying the VSV env SIV mac239 Δ env pseudovirus. SIV Gag-specific cellular immune responses were induced as early as two weeks after the first immunization eliciting a biased IFN-γ and IL-2 response. In addition, induction of SIV Gag-specific antibody responses and high titer neutralizing activity against the SIV pseudovirus harboring a VSV- env were detected after two immunizations. The vaccinated group and a control group of Chinese rhesus macaques were intravenously challenged with pathogenic SIV mac251. All animals became infected, but SIV-replication was effectively suppressed (below 100 copies/ml) in several animals in both groups. However the group immunized with apoptotic cells revealed better preservation of the gut CD4 + T-cell compartment. Viral control was inversely correlated with an early (4 weeks) but transient increase in the percentage of Ki67 + CD4 + peripheral blood T-cells (Spearman −0.73). We here show that immunizations with activated apoptotic lymphocytes expressing transduced SIV genes result in induction of both cellular and humoral immune responses. This study provides evidence for an immunological principle demonstrating that certain apoptotic cells can be considered as carriers of antigens directing immune responses in macaques.

Contains fulltext : 108425.pdf (Publisher’s version ) (Open Access) Dendritic cells (DCs) are professional antigen-presenting cells playing a central role in connecting innate and adaptive immunity. Maturation signals are, however, required for DCs to undergo phenotypic and functional changes to acquire a fully competent antigen-presenting capacity. We previously reported that activated apoptotic peripheral lymphocytes (ActApo) provide activation/maturation signals to human monocyte-derived DCs. In this paper, we have characterized the signaling pathways and molecules involved in ActApo-mediated DC maturation. We found that both cellular and supernatant fractions from ActApo are required for DC maturation signaling. ActApoSup-induced CD80 and CD86 expression was significantly blocked in the presence of neutralizing antibodies against tumor necrosis factor-alpha (TNF-alpha). Cell-cell contact-dependent signaling involved beta2 integrins, dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), and TLR4 because ActApo-induced up-regulation of the maturation markers CD80 and CD86 was significantly inhibited in the presence of neutralizing antibodies against CD18, CD11a, CD11b, and DC-SIGN as well as TLR4. The role of TLR4 was further confirmed by silencing of TLR4 in DCs. In addition, the endogenous adjuvant effect exerted by activated apoptotic splenocytes (ActApoSp) was reduced after immunization with human serum albumin in TLR4(-/-) mice. We detected activation of multiple signaling pathways and transcription factors in DCs upon co-culture with ActApo, including p38, JNK, PI3K-Akt, Src family kinases, NFkappaB p65, and AP1 transcription factor family members c-Jun and c-Fos, demonstrating the complex interactions occurring between ActApo and DCs. These studies provide important mechanistic insight into the responses of DCs during encounter with cells undergoing immunogenic cell death.

Dendritic cell derived IL-12p70 stimulates IFN-γ production in naïve T cells, thereby promoting Th1 responses, which counteracts induction of tolerance. Uptake of apoptotic cells by dendritic cells is generally considered to induce tolerance rather than immune activation and has been shown to specifically inhibit IL-12 production. However, we previously demonstrated that the activation state of apoptotic PBMC influence their immunogenic potential. Here we investigated whether dendritic cells that have engulfed apoptotic PBMC are able to produce IL-12p70 after a secondary signal. We show that dendritic cell ability to produce IL-12p70 after uptake of allogeneic apoptotic cells is dependent on the activation state of the apoptotic cells and subsequent CD40 ligation. CD40 ligation by a CD40L-transfected cell-line induced IL-12p70 in DC regardless of previous apoptotic cell uptake. Moreover, dendritic cells that were exposed to allogeneic activated apoptotic PBMC, but not to resting apoptotic PBMC, were able to produce IL-12p70 after co-culture with autologous T cells. These findings show that dendritic cells are able to produce IL-12p70 upon engulfment of apoptotic cells provided that a secondary activating signal such as CD40-ligand is delivered. In addition, resting apoptotic cell but not activated apoptotic cells reduced ongoing IL-12p70 production suggesting that the balance of activated and resting apoptotic lymphocytes influence the amount of IL-12p70 being produced.

The New Zealand white rabbit model (Oryctolagus cuniculus) is widely used to test whether HIV vaccine candidates elicit systemic antibody responses; however, its use in mucosal immunology has not been fully exploited due to the difficulty in collecting mucosal specimens longitudinally and reproducibly. Here we describe feasible and non-feasible methods to collect vaginal and nasal specimens from nulliparous rabbits. Non-feasible methods were those resulting in poor reproducibility and considerable animal twitching during sampling, whereas feasible methods resulted in no animal twitching and potential for sampling reproducibility. Standard operating procedures (SOPs) were implemented to collect vaginal swabs yielding total IgA titres ranging from 12,500 to 312,500. Intranasal immunisation with a naked DNA vaccine encoding HIV gp140 elicited HIV envelope-specific IgA detectable in nasal but not in vaginal secretions. Our methods provide an alternative to reliably assess pre- and post-vaccination mucosal antibody titres longitudinally in rabbits as part of mucosal HIV vaccine immunogenicity studies.

7C8 is a mouse monoclonal antibody that is specific for the third hypervariable loop (V3 loop) of the human immunodeficiency virus type 2 (HIV-2) associated protein gp125. Fab fragments of 7C8 effectively neutralize HIV-2. 7C8 was expressed and purified from a hybridoma cell line in order to establish the molecular basis underlying the specificity of the 7C8 antibody for the V3 loop as well as the specific role of the elongated third complementarity-determining region of the heavy chain (CDRH3). The antibody was digested with papain and Fab fragments were purified using size-exclusion chromatography. Hanging-drop vapour-diffusion crystallization techniques were employed and the protein was crystallized in 50 mM ammonium sulfate, 100 mM Tris-HCl pH 8.5, 25%(w/v) PEG 8000 and 2.5%(w/v) PEG 400 at 275 K. The analysed crystals belonged to the rhombohedral space group P3(2)21, with unit-cell parameters a = b = 100.1, c = 196.8 A, and diffracted to 2.7 A resolution.

Dendritic cells present microbial antigens to T cells after uptake of apoptotic vesicles from infected cells. We previously reported that immunizations with apoptotic HIV-1/murine leukemia virus (MuLV) infected cells lead to induction of both cellular and humoral immune responses as well as resistance to mucosal challenge with live HIV-1/MuLV infected cells. Here we extended those studies and investigated whether apoptotic cells from HIV-1/MuLV infected cells stimulate the production of HIV-1 neutralizing activity. We compared different routes of administration and were able to induce p24- and Nef-specific cellular proliferation after intraperitoneal (i.p.), intranasal (i.n.), subcutaneous (s.c.) and intramuscular (i.m.) immunizations. Serum IgG and IgA antibodies directed against gp160, p24, or Nef were also produced regardless of immunization route used. However, the induction of mucosa-associated IgAs from faeces or vaginal secretions were detected only after either i.p. or i.n. immunizations. We were able to measure neutralizing activity in sera of mice after i.p. and i.n. immunization. Neutralizing reactivity was also detected after s.c. and i.m. immunizations in the presence of the cytokine adjuvant granulocyte macrophage-colony stimulating factor (GM-CSF). Conclusively we show induction of cellular and humoral immune responses including neutralizing activity after immunization with apoptotic HIV-1/MuLV infected cells in mice. The results from this study support further evaluations using apoptotic cells as antigen delivery system for vaccination against HIV-1 in other animal models. Original Publication:Jorma Hinkula, Lilian Walther-Jallow, Anna Lauren, Barbro Makitalo, Monica Oberg, Britta Wahren, Eva-Maria Fenyo and Anna-Lena Spetz, Neutralizing activity and cellular immune responses induced in mice after immunization with apoptotic HIV-1/murine leukemia virus infected cells, 2009, VACCINE, (27), 46, 6424-6431.http://dx.doi.org/10.1016/j.vaccine.2009.06.016Copyright: Elsevier Science B.V., Amsterdam.http://www.elsevier.com/

Dendritic cells (DCs) are antigen-presenting cells with the capacity to prime naive T cells for efficient cellular responses against pathogens such as HIV-1. DCs are also susceptible to HIV-1 infection, which may impair their ability to induce immunity. Here, we examined the ability of HIV-1-infected, in vitro-derived DCs to respond to CD40 ligand (CD40L) stimulation with the aim to study events during early HIV-1 infection. HIV-1BaL-infected p24+ DCs were detected after only 3 days of exposure to highly concentrated virus. We show that HIV-1-infected DCs up-regulated costimulatory molecules, but were skewed in their production of effector cytokines in response to CD40L stimulation. CD40L stimulation induced significant secretion of tumor necrosis factor α (TNFα) and interleukin 12 (IL-12) p70 from both HIV-1-exposed and unexposed DCs. Intracellular stainings of HIV-1-exposed DCs revealed that TNFα could be detected in both the p24- and p24+ DCs, but IL-12 p70 could be found only in the p24- DCs. Thus, although p24+ DCs showed a mature phenotype similar to p24- DCs after CD40L stimulation, they appeared to have an impaired cytokine profile. These observations suggest that HIV-1 infection disables DC function, a phenomenon that may be relevant for optimal induction of HIV-1-specific immune responses. (Blood. 2004;104:2810-2817)

Human immunodeficiency virus-1 (HIV-1) impairs tumor necrosis factor-alpha (TNF-alpha)-mediated macrophage apoptosis induced by Mycobacterium tuberculosis (Mtb). HIV Nef protein plays an important role in the pathogenesis of AIDS. We have tested the hypothesis that exogenous Nef is a factor that inhibits TNF-alpha production/apoptosis in macrophages infected with Mtb. We demonstrate that Mtb and Nef individually trigger TNF-alpha production in macrophages. However, TNF-alpha production is dampened when the two are present simultaneously, probably through cross-regulation of the individual signaling pathways leading to activation of the TNF-alpha promoter. Mtb-induced TNF-alpha production is abrogated upon mutation of the Ets, Egr, Sp1, CRE, or AP1 binding sites on the TNF-alpha promoter, whereas Nef-mediated promoter activation depends only on the CRE and AP1 binding sites, pointing to differences in the mechanisms of activation of the promoter. Mtb-dependent promoter activation depends on the mitogen-activated kinase (MAPK) kinase kinase ASK1 and on MEK/ERK signaling. Nef inhibits ASK1/p38 MAPK-dependent Mtb-induced TNF-alpha production probably by inhibiting binding of ATF2 to the TNF-alpha promoter. It also inhibits MEK/ERK-dependent Mtb-induced binding of FosB to the promoter. Nef-driven TNF-alpha production occurs in an ASK1-independent, Rac1/PAK1/p38 MAPK-dependent, and MEK/ERK-independent manner. The signaling pathways used by Mtb and Nef to trigger TNF-alpha production are therefore distinctly different. In addition to attenuating Mtb-dependent TNF-alpha promoter activation, Nef also reduces Mtb-dependent TNF-alpha mRNA stability probably through its ability to inhibit ASK1/p38 MAPK signaling. These results provide new insight into how HIV Nef probably exacerbates tuberculosis infection by virtue of its ability to dampen Mtb-induced TNF-alpha production.

Ag-presenting dendritic cells present viral Ags to T cells after uptake of apoptotic bodies derived from virus-infected cells in vitro. However, it is unclear whether apoptotic virus-infected cells are capable of generating immunity in vivo. In this study, we show that inoculation of mice with apoptotic HIV-1/murine leukemia virus (MuLV)-infected cells induces HIV-1-specific immunity. Immunization with apoptotic HIV-1/MuLV-infected syngeneic splenocytes resulted in strong Nef-specific CD8+ T cell proliferation and p24-induced CD4+ and CD8+ T cell proliferation as well as IFN-γ production. In addition, systemic IgG and IgA as well as mucosa-associated IgA responses were generated. Moreover, mice vaccinated with apoptotic HIV-1/MuLV cells were protected against challenge with live HIV-1/MuLV-infected cells, whereas mice vaccinated with apoptotic noninfected or MuLV-infected splenocytes remained susceptible to HIV-1/MuLV. These data show that i.p. immunization with apoptotic HIV-1-infected cells induces high levels of HIV-1-specific systemic immunity, primes for mucosal immunity, and induces protection against challenge with live HIV-1-infected cells in mice. These findings may have implications for the development of therapeutic and prophylactic HIV-1 vaccines.

Two techniques based upon flow cytometry (FCM) and in situ image analysis were developed for quantification of intracellular cytokine and chemokine protein expression at the single cell level in dendritic cells (DCs). The qualitative and quantitative differences between the two methods were evaluated. In vitro differentiated DCs were stimulated with lipopolysaccaride (LPS) and thereafter stained for either IL-8, which is secreted through the Golgi-organelle, or IL-1ra, which localises diffusely in the cytoplasm. Microscopic examination, both for fluorophore and enzymatically stained cells, showed that DCs expressed IL-8 and IL-1ra with two different staining patterns. FCM analysis showed high frequencies of IL-1ra producing cells (76+/-13%), which was similar to the frequency obtained by in situ imaging. However, in contrast to IL-1ra, the incidence of IL-8 expressing DCs showed high variability between the donors. The numbers of positive cells were 19+/-19% as measured by FCM. The detection of IL-8 analysed by in situ imaging revealed higher frequencies (26+/-14%). The addition of brefeldin-A, leading to cytoplasmic accumulation of proteins secreted through the Golgi endoplasmatic route, generated a significantly increased signal intensity and incidence of producer cells, resulting in similar frequencies for both methods. FCM has the advantage of being less time consuming than image analysis and is also able to facilitate multiple colour analysis. However, FCM is less accurate in detecting and quantifying cytokines and chemokines with a preserved juxtanuclear staining pattern. The correct choice of detection technique therefore depends on the study question.

Activated macrophages at the sub-mucosal space play a major role in generating innate immune responses during H. pylori infection. Final disease outcome largely depends on how H. pylori and bacterium-derived products modulate macrophage responses. Here, we report that JHP0290, a functionally unknown protein from H. pylori, regulates macrophage functions. Recombinant purified JHP0290 (rJHP0290) had the ability to bind to several cell types including macrophages, human gastric epithelial cell lines, human monocyte-derived dendritic cells (MoDC) and human neutrophils. Exposure to rJHP0290 induced apoptosis in macrophages concurrent with release of proinflammatory cytokine tumor necrosis factor (TNF). A mutant strain of H. pylori disrupted in the jhp0290 gene was significantly impaired in its ability to induce apoptosis and TNF in macrophages confirming the role of endogenous protein in regulating macrophage responses. Intracellular signaling involving Src family of tyrosine kinases (SFKs) and ERK MAPK were required for rJHP0290-induced TNF release and apoptosis in macrophages. Furthermore, rJHP0290-induced TNF release was partly dependent on activation of nuclear transcription factor-κB (NF-κB). Neutralizing antibodies against TNF partially blocked rJHP0290-induced macrophage apoptosis indicating TNF-independent pathways were also involved. These results provide mechanistic insight into the potential role of the protein JHP0290 during H. pylori-associated disease development. By virtue of its ability to induce TNF, an acid suppressive proinflammatory cytokine and induction of macrophage apoptosis, JHP0290 possibly helps in persistent survival of the bacterium inside the stomach.

Mucosal inflammation is characterized by increased expression of proinflammatory cytokines and chemoattractant chemokines, resulting in infiltration of immunocompetent cells. This study compared the degree of mucosal inflammation in human immunodeficiency virus type 1 (HIV-1)-infected gut mucosa with that in tissue samples from subjects with inflammatory bowel disease (IBD) and from healthy seronegative control subjects. Gut mucosal biopsy specimens were immunohistochemically stained and were evaluated by in situ imaging. There was significantly increased expression of HIV-1 coreceptors CCR5 and CXCR4, beta-chemokine RANTES, and macrophage inflammatory protein (MIP)-1alpha and MIP-1beta, as well as increased numbers of T cells in lamina propria of HIV-1-infected patients. The results were similar in patients with IBD and in HIV-1-infected patients, suggesting increased inflammation in the colon of HIV-1-infected patients. To further investigate the effect of inflammation in HIV-1-infected lamina propria, treatments that reduce immune activation in lamina propria must be evaluated.

The role of placenta in vertical transmission is not yet fully understood. A protective role of the placenta during gestation is suggested by the finding that caesarian sections reduce the risk of transmission of human immunodeficiency virus (HIV)-1 from mother to child three- to fourfold. Here we investigated whether the immunological milieu of the placenta might be important in HIV-1 transmission. In situ imaging of immunohistochemically stained placenta sections and reverse transcriptase-polymerase chain reaction demonstrated a fourfold increase in CCR5:CXCR4 expression ratio in placentae from transmitting women compared to placentae from nontransmitting women. This chemokine receptor repertoire was consistent with an up-regulation of interleukin-4 and interleukin-10 expression in placentae from nontransmitting placentae compared to transmitting placentae. In situ imaging demonstrated that CCR5 and CXCR4 were expressed on placental macrophages and lymphocytes but not in trophoblasts. Simultaneous immunofluorescence/ultrasensitive in situ hybridization for HIV-1 gag-pol mRNA revealed that HIV-1 infects primarily CXCR4-expressing cells in placentae from nontransmitting women whereas predominantly CCR5-expressing cells were infected in placentae from transmitting women. These data are consistent with transmission of a homogeneous population of nonsyncytium-inducing HIV-1 isolates that use CCR5 as co-receptor.

We have developed a direct immunocytochemical technique to identify cytokine and chemokine production in epidermal Langerhans cells (LC) and in vitro derived CD14-, CD1a+, CD83+, CD40+ dendritic cells (DC) at the single cell level. Formaldehyde fixation combined with saponin permeabilization preserved cellular morphology and generated a characteristic juxtanuclear staining signal due to the accumulation of cytokine to the Golgi organelle. This approach was used for the assessment of TNF-alpha, IL-6, IL-8, IL-10, IL-12, GM-CSF, MIP-1alpha, MIP-1beta and RANTES producing cells. In contrast, a diffuse cytoplasmic staining was evident for IL-1ra, IL-1alpha and IL-1beta production. IL-1ra and IL-1alpha were expressed in 10-25% of unstimulated cultured cells, while all the other cytokines were undetectable. IL-1ra, IL-1alpha and IL-1beta were also the dominating cytokines, expressed in up to 85% of the DC, after 3 h of LPS stimulation. A significantly lower number of cells (0-5%) synthesized TNF-alpha, IL-6, IL-10, IL-12 and GM-CSF. The incidence of chemokine producing cells (IL-8, RANTES, MIP-1alpha, MIP-1beta) peaked 10 h after LPS stimulation in up to 60% of the DC. Both immature CD83- and mature CD83+ DC as well as LC had a similar cytokine production pattern. Thus, in comparison to monocytes, LPS stimulation of DC generated a lower incidence of TNF-alpha, IL-6, IL-10 and IL-12 producing cells while IL-1 was expressed in a comparable number of cells.

In order to study whether positive selection of T cells plays any role in the MHC-dependent protection from diabetes in the non-obese-diabetic (NOD) mouse, the T cell V beta repertoire has been studied in NOD mice and in NOD mice either transgenic for the wildtype MHC class II E alpha gene, or for delta Y, a promotor-mutagenized E alpha gene with a restricted tissue expression. The E alpha transgenic line is protected from both insulitis and diabetes. The delta Y transgenic line is neither protected from insulitis nor from diabetes, although it can perform both positive and negative E-mediated selection in the thymus. The V beta repertoire was studied in the pancreatic lymph nodes as these drain the area which is the target for the autoimmune attack. We see no evidence for E alpha TCR V beta repertoire differing from both nontransgenic NOD mice and delta Y mice despite its striking difference in susceptibility to autoimmunity. We conclude that none of the differences in the TCR V beta repertoire of E alpha-transgenic NOD mice hitherto observed are likely to explain the protective effect of E molecule expression in NOD mice.

7C8 is a mouse monoclonal antibody specific for the third hypervariable region (V3) of the human immunodeficiency virus type 2 (HIV-2)-associated protein gp125. The three-dimensional crystal structure of the Fab fragment of 7C8, determined to 2.7 A resolution, reveals a deep and narrow antigen-binding cleft with architecture appropriate for an elongated epitope. The highly hydrophobic cleft is bordered on one side by the negatively charged second complementarity determining region (CDR2) and the unusually long positively charged CDR3 of the heavy chain and, on the other side, by the CDR1 of the light chain. Analysis of 7C8 in complex with molecular models of monomeric and trimeric gp125 highlights the importance of a conserved stretch of residues FHSQ that is localized centrally on the V3 region of gp125. Furthermore, modeling also indicates that the Fab fragment neutralizes the virus by sterically impairing subsequent engagement of the gp125 trimer with the co-receptor on the target cell.

Dendritic cells (DCs) are activated by signaling via pathogen-specific receptors or exposure to inflammatory mediators. Here we show that co-culturing DCs with apoptotic HIV-infected activated CD4(+) T cells (ApoInf) or apoptotic uninfected activated CD4(+) T cells (ApoAct) induced expression of co-stimulatory molecules and cytokine release. In addition, we measured a reduced HIV infection rate in DCs after co-culture with ApoAct. A prerequisite for reduced HIV infection in DCs was activation of CD4(+) T cells before apoptosis induction. DCs exposed to ApoAct or ApoInf secreted MIP-1α, MIP-1β, MCP-1, and TNF-α; this effect was retained in the presence of exogenous HIV. The ApoAct-mediated induction of co-stimulatory CD86 molecules and reduction of HIV infection in DCs were partially abrogated after blocking TNF-α using monoclonal antibodies. APOBEC3G expression in DCs was increased in co-cultures of DCs and ApoAct but not by apoptotic resting CD4(+) T cells (ApoRest). Silencing of APOBEC3G in DC abrogated the HIV inhibitory effect mediated by ApoAct. Sequence analyses of an env region revealed significant induction of G-to-A hypermutations in the context of GG or GA dinucleotides in DNA isolated from DCs exposed to HIV and ApoAct. Thus, ApoAct-mediated DC maturation resulted in induction of APOBEC3G that was important for inhibition of HIV-infection in DCs. These findings underscore the complexity of differential DC responses evoked upon interaction with resting as compared with activated dying cells during HIV infection.

Neutralizing antibodies (NAb) able to react to heterologous viruses are generated during natural HIV-1 infection in some individuals. Further knowledge is required in order to understand the factors contributing to induction of cross-reactive NAb responses. Here a well-established model of experimental pathogenic infection in cynomolgus macaques, which reproduces long-lasting HIV-1 infection, was used to study the NAb response as well as the viral evolution of the highly neutralization-resistant SIVmac239. Twelve animals were infected intravenously with SIVmac239. Antiretroviral therapy (ART) was initiated ten days post-inoculation and administered daily for four months. Viral load, CD4+ T-cell counts, total IgG levels, and breadth as well as strength of NAb in plasma were compared simultaneously over 14 months. In addition, envs from plasma samples were sequenced at three time points in all animals in order to assess viral evolution. We report here that seven of the 12 animals controlled viremia to below 104 copies/ml of plasma after discontinuation of ART and that this control was associated with a low level of evolutionary divergence. Macaques that controlled viral load developed broader NAb responses early on. Furthermore, escape mutations, such as V67M and R751G, were identified in virus sequenced from all animals with uncontrolled viremia. Bayesian estimation of ancestral population genetic diversity (PGD) showed an increase in this value in non-controlling or transient-controlling animals during the first 5.5 months of infection, in contrast to virus-controlling animals. Similarly, non- or transient controllers displayed more positively-selected amino-acid substitutions. An early increase in PGD, resulting in the generation of positively-selected amino-acid substitutions, greater divergence and relative high viral load after ART withdrawal, may have contributed to the generation of potent NAb in several animals after SIVmac239 infection. However, early broad NAb responses correlated with relatively preserved CD4+ T-cell numbers, low viral load and limited viral divergence., Author Summary In a longitudinal study of clinical and evolutionary responses to transient treatment in 12 experimentally-infected macaques, subjects show clear stratification into two groups based on viral load, immunological response, and evolutionary factors. Subjects that controlled viremia following withdrawal of treatment developed broadly neutralizing antibody responses earlier than subjects with no or transient control of viremia. Moreover, this latter group of macaques with higher viral loads showed greater divergence of SIV sequences, greater numbers of positively-selected amino-acid substitutions and a stronger neutralizing antibody response. The increase in viral genetic diversity started at an early stage of infection. The authors propose that this early phase of evolution is principally responsible for the later failure to control viremia and resulted in the development of potent neutralizing capacity.

A key gap in the development and evaluation of HIV-1 vaccines is insufficient knowledge with regard to sampling techniques and assessment of mucosal immune responses required for early prevention and inhibition of viral dissemination. In an attempt to start bridging this gap, the EUROPRISE network of scientists working on HIV-1 vaccine and microbicide research organized a workshop with the aim to review the types of mucosal responses/biomarkers currently measured in mucosal immunology and to define how the mucosal responses/biomarkers are measured and/or the assays and sampling methods used. The Workshop addressed two critical questions: first whether, with current knowledge, it would be possible to define a consensus set of mucosal sampling methods to facilitate cross-species comparisons and ensure standardized implementation in clinical trials; second to determine the remaining challenges (technical and logistical) and their possible solutions for assessing mucosal responses to HIV-1 vaccines.

Human immunodeficiency virus-1 (HIV-1) impairs tumor necrosis factor-α (TNF-α)-mediated macrophage apoptosis induced by Mycobacterium tuberculosis (Mtb). HIV Nef protein plays an important role in the pathogenesis of AIDS. We have tested the hypothesis that exogenous Nef is a factor that inhibits TNF-α production/apoptosis in macrophages infected with Mtb. We demonstrate that Mtb and Nef individually trigger TNF-α production in macrophages. However, TNF-α production is dampened when the two are present simultaneously, probably through cross-regulation of the individual signaling pathways leading to activation of the TNF-α promoter. Mtb-induced TNF-α production is abrogated upon mutation of the Ets, Egr, Sp1, CRE, or AP1 binding sites on the TNF-α promoter, whereas Nef-mediated promoter activation depends only on the CRE and AP1 binding sites, pointing to differences in the mechanisms of activation of the promoter. Mtb-dependent promoter activation depends on the mitogen-activated kinase (MAPK) kinase kinase ASK1 and on MEK/ERK signaling. Nef inhibits ASK1/p38 MAPK-dependent Mtb-induced TNF-α production probably by inhibiting binding of ATF2 to the TNF-α promoter. It also inhibits MEK/ERK-dependent Mtb-induced binding of FosB to the promoter. Nef-driven TNF-α production occurs in an ASK1-independent, Rac1/PAK1/p38 MAPK-dependent, and MEK/ERK-independent manner. The signaling pathways used by Mtb and Nef to trigger TNF-α production are therefore distinctly different. In addition to attenuating Mtb-dependent TNF-α promoter activation, Nef also reduces Mtb-dependent TNF-α mRNA stability probably through its ability to inhibit ASK1/p38 MAPK signaling. These results provide new insight into how HIV Nef probably exacerbates tuberculosis infection by virtue of its ability to dampen Mtb-induced TNF-α production.

Background The kinetics of cellular and humoral immune responses was evaluated in a macaque model after early introduction of antiretroviral treatment. Twelve cynomolgus macaques were infected intravenously with the patogenic SIVmac239 strain. Treatment with the antiretroviral drug Tenofovir was initiated 10 days after inoculation with SIVmac239 and was administered subcutaneously as daily injections for 4 months.

Delivery of DNA encoding foreign antigens into mammalian cells can induce adaptive immune responses. There are currently many DNA-based vaccines in clinical trials against infectious diseases and cancer but there is a lack of adjuvants for improvement of responses to DNA-based vaccines. Here, we show augmented systemic and mucosa-associated B cell responses after immunization with a cocktail of seven different plasmids (3 env, 2 gag, 1 rev, 1 RT) combined with mitogen activated apoptotic syngeneic lymphocytes in mice. In addition we show that apoptotic cells can function as adjuvant for induction of cellular immune responses in a magnitude comparable to the cytokine adjuvant GM-CSF in mice. These data suggest that activated apoptotic lymphocytes can act independent as adjuvants to improve antigen-specific DNA vaccines.

HIV-1 has proved to be notoriously difficult to tackle despite the availability of more than 20 clinically approved drugs. The majority of these drugs, however, target viral genes and their continued use will select for drug-resistant strains. Since NF-kappaB signaling is critical for viral replication, we wanted to investigate the effect of proteasome inhibitors on viral gene expression. We herein demonstrate that proteasome and NF-kappaB inhibitors effectively shut down transcription from the HIV-1 LTR-promoter. We further show that replication of HIV-1 in PBMC was severely compromised following treatment with proteasome inhibitors alone or in combination with other antiretroviral drugs. Finally, incubation of PBMC with these drugs reduced expression of IL-2 inducible T cell kinase (Itk), a Tec-family kinase, recently shown to be required for HIV-1 replication. These results suggest that proteasome inhibitors suppress LTR-promoter activity by interfering with cellular targets required for viral replication.

Thymocytes differentiate upon interactions with microenvironmental components, but the precise role of different stromal cells, or other T cells, in early differentiative events remains unclear. Here we have analyzed the in vitro differentiation of double-negative (DN) thymocytes from young adult mice. We demonstrate that a substantial proportion of DN thymocytes differentiate into CD8+ gamma/delta T cells upon stimulation with concanavalin A and recombinant interleukin 2. However, if alpha/beta T cells are excluded from the initial population of DN thymocytes, the CD8+ gamma/delta T cells do not appear in the cultures. These results suggest a role for T-T cell interactions in thymic differentiative events, and provide evidence for physiological interactions between the alpha/beta and gamma/delta T cell compartments within the thymus.

Dendritic cells (DCs) process and present bacterial and endogenous lipid antigens in complex with CD1 molecules to T cells and invariant natural killer T (NKT) cells. However, different types of DCs, such as blood myeloid DCs and skin Langerhans cells, exhibit distinct patterns of CD1a, CD1b, CD1c, and CD1d expression. The regulation of such differences is incompletely understood. Here, we initially observed that monocyte-derived DCs cultured in an immunoglobulin-rich milieu expressed CD1d but not CD1a, CD1b, and CD1c, whereas DCs cultured in the presence of low levels of immunoglobulins had an opposite CD1 profile. Based on this, we tested the possibility that immunoglobulins play a central role in determining these differences. IgG depletion and intravenous immunoglobulin (IVIg) add-in experiments strongly supported a role for IgG in directing the CD1 expression profile. Blocking experiments indicated that this effect was mediated by FcγRIIa (CD32a), and quantitative polymerase chain reaction data demonstrated that regulation of the CD1 profile occurred at the gene expression level. Finally, the ability of DCs to activate CD1-restricted NKT cells and T cells was determined by this regulatory effect of IgG. Our data demonstrate an important role for FcγRIIa in regulating the CD1 antigen presentation machinery of human DCs.

The usage of four different T cell receptor (TcR) V beta gene families within normal, non-primed T cell populations in response to various types of antigen-presenting cells (APC) in primary mixed lymphocyte reaction has been studied. We demonstrate that distinct patterns of V beta gene usage are obtained within a given T cell population in response to different types of APC with the same allo-H-2. When responder T cells are stimulated with one type of allogeneic APC, from various H-2-disparate mice, the same V beta gene preference is observed. Furthermore, when H-2- and Mls-mismatched APC gene used as stimulators, the Mls-associated V beta 6 and V beta 8.1 gene families are highly elevated in response to both B and T cell blasts from certain Mls-positive strains. The results demonstrate that different types of allogeneic APC have the capacity to generate biases in TcR V beta gene usage and imply that functional Mls-like determinants are presented by T cell blasts. The findings are discussed with respect to TcR-major histocompatibility complex interactions in allostimulation.