Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis

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Bacteria-produced ferric exopolysaccharide nanoparticles as iron delivery system for truffles (Tuber borchii)

Applied Microbiology and Biotechnology - Iron exopolysaccharide nanoparticles were biogenerated during ferric citrate fermentation by Klebsiella oxytoca DSM 29614. Before investigating their...     

Neutralizing Antibodies in Sera from Macaques Immunized with Attenuated Simian Immunodeficiency Virus

Infection with attenuated simian immunodeficiency virus (SIV) in rhesus macaques has been shown to raise antibodies capable of neutralizing an animal challenge stock of primary SIVmac251 in CEMx174 cells that correlate with resistance to infection after experimental challenge with this virulent virus (M. S. Wyand, K. H. Manson, M. Garcia-Moll, D. C. Montefiori, and R. C. Desrosiers, J. Virol. 70:3724–3733, 1996). Here we show that these neutralizing antibodies are not detected in human and rhesus peripheral blood mononuclear cells (PBMC). In addition, neutralization of primary SIVmac251 in human and rhesus PBMC was rarely detected with plasma samples from a similar group of animals that had been infected either with SIVmac239Δnef for 1.5 years or with SIVmac239Δ3 for 3.2 years, although low-level neutralization was detected in CEMx174 cells. Potent neutralization was detected in CEMx174 cells when the latter plasma samples were assessed with laboratory-adapted SIVmac251. In contrast to primary SIVmac251, laboratory-adapted SIVmac251 did not replicate in human and rhesus PBMC despite its ability to utilize CCR5, Bonzo/STRL33, and BOB/gpr15 as coreceptors for virus entry. These results illustrate the importance of virus passage history and the choice of indicator cells for making assessments of neutralizing antibodies to lentiviruses such as SIV. They also demonstrate that primary SIVmac251 is less sensitive to neutralization in human and rhesus PBMC than it is in established cell lines. Results obtained in PBMC did not support a role for neutralizing antibodies as a mechanism of protection in animals immunized with attenuated SIV and challenged with primary SIVmac251.     

Recombinant Vaccine-Induced Protection against the Highly Pathogenic Simian Immunodeficiency Virus SIVmac251: Dependence on Route of Challenge Exposure

Vaccine protection from infection and/or disease induced by highly pathogenic simian immunodeficiency virus (SIV) strain SIV_mac251 in the rhesus macaque model is a challenging task. Thus far, the only approach that has been reported to protect a fraction of macaques from infection following intravenous challenge with SIV_mac251 was the use of a live attenuated SIV vaccine. In the present study, the gag, pol, and env genes of SIV_K6W were expressed in the NYVAC vector, a genetically engineered derivative of the vaccinia virus Copenhagen strain that displays a highly attenuated phenotype in humans. In addition, the genes for the α and β chains of interleukin-12 (IL-12), as well as the IL-2 gene, were expressed in separate NYVAC vectors and inoculated intramuscularly, in conjunction with or separate from the NYVAC-SIV vaccine, in 40 macaques. The overall cytotoxic T-lymphocyte (CTL) response was greater, at the expense of proliferative and humoral responses, in animals immunized with NYVAC-SIV and NYVAC–IL-12 than in animals immunized with the NYVAC-SIV vaccine alone. At the end of the immunization regimen, half of the animals were challenged with SIV_mac251 by the intravenous route and the other half were exposed to SIV_mac251 intrarectally. Significantly, five of the eleven vaccinees exposed mucosally to SIV_mac251 showed a transient peak of viremia 1 week after viral challenge and subsequently appeared to clear viral infection. In contrast, all 12 animals inoculated intravenously became infected, but 5 to 6 months after viral challenge, 4 animals were able to control viral expression and appeared to progress to disease more slowly than control animals. Protection did not appear to be associated with any of the measured immunological parameters. Further modulation of immune responses by coadministration of NYVAC-cytokine recombinants did not appear to influence the outcome of viral challenge. The fact that the NYVAC-SIV recombinant vaccine appears to be effective per se in the animal model that best mirrors human AIDS supports the idea that the development of a highly attenuated poxvirus-based vaccine candidate can be a valuable approach to significantly decrease the spread of human immunodeficiency virus (HIV) infection by the mucosal route.     

First evidence for truffle production from plants inoculated with mycelial pure cultures

Truffle (Tuber spp.) cultivation is based on raising mycorrhizal trees in greenhouses that have been inoculated with suspensions of ascospores. The problem with this is that pests, pathogens, and other mycorrhizal fungi can contaminate the trees. Furthermore, because ascospores are produced sexually, each plant potentially has a different genetic mycorrhizal makeup from each other so tailoring the mycorrhizal component of plants to suit a particular set of soil and climatic conditions is out of the question. Here, we report on the production of Tuber borchii-mycorrhized plants using pure cultures, establishing a truffière with these and subsequent production of its fruiting bodies. This study opens up the possibility of producing commercial numbers of Tuber-mycorrhized trees for truffle cultivation using mycelial inoculation techniques. It also poses questions about the mechanism of fertilization between the different strains which were located in different parts of the experimental truffière.     

Lack of Viral Escape and Defective In Vivo Activation of Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Lymphocytes in Rapidly Progressive Infection

Human immunodeficiency virus type 1 (HIV-1)-specific immune responses over the course of rapidly progressive infection are not well defined. Detailed longitudinal analyses of neutralizing antibodies, lymphocyte proliferation, in vivo-activated and memory cytotoxic T-lymphocyte (CTL) responses, and viral sequence variation were performed on a patient who presented with acute HIV-1 infection, developed an AIDS-defining illness 13 months later, and died 45 months after presentation. Neutralizing-antibody responses remained weak throughout, and no HIV-1-specific lymphocyte proliferative responses were seen even early in the disease course. Strong in vivo-activated CTL directed against Env and Pol epitopes were present at the time of the initial drop in viremia but were quickly lost. Memory CTL against Env and Pol epitopes were detected throughout the course of infection; however, these CTL were not activated in vivo. Despite an initially narrow CTL response, new epitopes were not targeted as the disease progressed. Viral sequencing showed the emergence of variants within the two targeted CTL epitopes; however, viral variants within the immunodominant Env epitope were well recognized by CTL, and there was no evidence of viral escape from immune system detection within this epitope. These data demonstrate a narrowly directed, static CTL response in a patient with rapidly progressive disease. We also show that disease progression can occur in the presence of persistent memory CTL recognition of autologous epitopes and in the absence of detectable escape from CTL responses, consistent with an in vivo defect in activation of CTL.     

Neutralizing antibody responses over time in demographically and clinically diverse individuals recovered from SARS-CoV-2 infection in the United States and Peru: A cohort study

BackgroundPeople infected with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) experience a wide range of clinical manifestations, from asymptomatic and mild illness to severe illness and death, influenced by age and a variety of comorbidities. Neutralizing antibodies (nAbs) are thought to be a primary immune defense against the virus. Large, diverse, well-characterized cohorts of convalescent individuals provide standardized values to benchmark nAb responses to past SARS-CoV-2 infection and define potentially protective levels of immunity.Methods and findingsThis analysis comprises an observational cohort of 329 HIV–seronegative adults in the United States (n = 167) and Peru (n = 162) convalescing from SARS-CoV-2 infection from May through October 2020. The mean age was 48 years (range 18 to 86), 54% of the cohort overall was Hispanic, and 34% identified as White. nAb titers were measured in serum by SARS-CoV-2.D614G Spike-pseudotyped virus infection of 293T/ACE2 cells. Multiple linear regression was applied to define associations between nAb titers and demographic variables, disease severity and time from infection or disease onset, and comorbidities within and across US and Peruvian cohorts over time. nAb titers peaked 28 to 42 days post-diagnosis and were higher in participants with a history of severe Coronavirus Disease 2019 (COVID-19) (p < 0.001). Diabetes, age >55 years, male sex assigned at birth, and, in some cases, body mass index were also independently associated with higher nAb titers, whereas hypertension was independently associated with lower nAb titers. nAb titers did not differ by race, underlying pulmonary disease or smoking. Two months post-enrollment, nAb ID50 (ID80) titers declined 3.5 (2.8)-fold overall. Study limitations in this observational, convalescent cohort include survivorship bias and missing early viral loads and acute immune responses to correlate with the convalescent responses we observed.ConclusionsIn summary, in our cohort, nAb titers after SARS-CoV-2 infection peaked approximately 1 month post-diagnosis and varied by age, sex assigned at birth, disease severity, and underlying comorbidities. Our data show great heterogeneity in nAb responses among people with recent COVID-19, highlighting the challenges of interpreting natural history studies and gauging responses to vaccines and therapeutics among people with recent infection. Our observations illuminate potential correlations of demographic and clinical characteristics with nAb responses, a key element for protection from COVID-19, thus informing development and implementation of preventative and therapeutic strategies globally.Trial registrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT04403880","term_id":"NCT04403880"}}NCT04403880.

Shelly Karuna and co-workers study variations in neutralizing antibody responses after SARS-CoV-2 infection.     

P120-Ras GTPase activating protein (RasGAP): A multi-interacting protein in downstream signaling

p120-RasGAP (Ras GTPase activating protein) plays a key role in the regulation of Ras-GTP bound by promoting GTP hydrolysis via its C-terminal catalytic domain. The p120-RasGAP N-terminal part contains two SH2, SH3, PH (pleckstrin homology) and CaLB/C2 (calcium-dependent phospholipid-binding domain) domains. These protein domains allow various functions, such as anti-/pro-apoptosis, proliferation and also cell migration depending of their distinct partners. The p120-RasGAP domain participates in protein–protein interactions with Akt, Aurora or RhoGAP to regulate functions described bellow. Here, we summarize, in angiogenesis and cancer, the various functional roles played by p120-RasGAP domains and their effector partners in downstream signaling.     

Evidence that Ecotropic Murine Leukemia Virus Contamination in TZM-bl Cells Does Not Affect the Outcome of Neutralizing Antibody Assays with Human Immunodeficiency Virus Type 1

The TZM-bl cell line that is commonly used to assess neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) was recently reported to be contaminated with an ecotropic murine leukemia virus (MLV) (Y. Takeuchi, M. O. McClure, and M. Pizzato, J. Virol. 82:12585-12588, 2008), raising questions about the validity of results obtained with this cell line. Here we confirm this observation and show that HIV-1 neutralization assays performed with a variety of serologic reagents in a similar cell line that does not harbor MLV yield results that are equivalent to those obtained in TZM-bl cells. We conclude that MLV contamination has no measurable effect on HIV-1 neutralization when TZM-bl cells are used as targets for infection.It was recently reported that TZM-bl cells, which are commonly used to assess neutralizing antibodies (Abs) against human immunodeficiency virus type 1 (HIV-1), are contaminated with an ecotropic murine leukemia virus (MLV) (22). TZM-bl (also called JC.53bl-13) is a HeLa cell derivative that was engineered by amphotropic retroviral transduction to express CD4 and CCR5 (17) and was further engineered with an HIV-1-based vector to contain Tat-responsive reporter genes for firefly luciferase (Luc) and Escherichia coli β-galactosidase (24). These engineered features made TZM-bl cells highly susceptible to HIV-1 infection in a readily quantifiable assay for neutralizing Abs. Many published studies used this cell line for assessments of HIV-1 neutralization; these include several recent reports describing the magnitude, breadth, and epitope specificity of the neutralizing Ab response in infected individuals (14, 18-20), neutralization escape (25), and the neutralization phenotype of transmitted/founder viruses (10). TZM-bl cells are also gaining popularity for assessments of vaccine-elicited neutralizing Ab responses (13). The validity of these and other published results, together with a rationale for the continued use of TZM-bl cells in assessing neutralizing Abs against HIV-1, are very dependent on establishing to what extent, if any, MLV contamination affects the outcome of the assay.It was suggested that ecotropic MLV entered TZM-bl cells via the progenitor JC.53 cell line as an amphotropic MLV pseudotype (22). In this regard, JC.53 cells were constructed from HeLa cells in two stages by using ping-pong technology to amplify the pSFF vector derived from the replication-defective and highly truncated Friend spleen focus-forming virus (3). When used with this vector, this procedure has previously resulted in stable vector expression (17) without formation of replication-competent MLV recombinants (8, 11). A panel of HeLa-CD4 clones was made that express different amounts of CD4 and where the high-expression HI-J clone was used to make a derivative panel of clones (termed JC), including JC.53, that expressed diverse levels of CCR5 (9, 16, 17). In addition, the HeLa-CD4 clone HI-R that expressed low levels of CD4 was used to make another panel of CCR5-expressing clones (termed RC). To investigate this newly reported issue, cell extracts from these clonal panels and from TZM-bl cells were analyzed for MLV Gag antigens by Western immunoblotting. Representative data, as shown in Fig. ​Fig.1A,1A, confirm that JC.53 and TZM-bl cells express MLV Gag antigens, whereas the progenitor HI-J clone of HeLa-CD4 cells and many but not all of the other HeLa-CD4/CCR5 clones in the JC panel lack MLV antigens.Open in a separate windowFIG. 1.Characterization of HeLa clones for MLV Gag expression, HIV-1 susceptibility, and cell surface expression of HIV-1 fusion receptors. (A) MLV Gag antigen expression in HeLa cells and derivative clones expressing CD4 or CD4 and CCR5. Cell lysates were prepared from the cell clones and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with Abs to MLV Gag antigens (upper blot). The lysates were also probed with anti-tubulin antibodies (lower blot). Lane 1, HeLa cells; lanes 2 and 3, HeLa CD4 clones HI-R and HI-J, respectively; lanes 4, 5, and 6, HeLa-CD4/CCR5 clones JC.10, JC.48, and JC.53, respectively; lane 7, TZM-bl cells; lane 8, psi-2 packaging cells positive for MLV Gag. (B) HIV-1 infectivity on the HeLa-CD4/CCR5 JC panel. Target cells were infected with HIV-1 isolate JRCSF that had been produced from clone JC.53 cells (black) or with JRCSF produced from transfected HEK293T cells (red). The target cells were also infected with the JR-FL isolate produced from peripheral blood mononuclear cells (PBMC; green). The HeLa-CD4/CCR5 target cells had a CCR5 expression range of 2 × 10³ (clone JC.10) to 1.3 × 10⁵ (clones JC.53 and TZM-bl) CCR5 molecules/cell. Each set of three data points at a given CCR5 expression level represents a single HeLa-CD4/CCR5 JC clone. None of the HIV-1 isolates was able to infect HeLa-CD4 cells lacking CCR5. The blue asterisks indicate clones that are negative for MLV Gag proteins. Clones JC.48 (used for subsequent infection and neutralization assays) and JC.53 (progenitor of TZM-bl cells) are specifically labeled. (C) Surface expression of CD4, CCR5, and CXCR4 on TZM-bl and JC.48 cells was assessed by flow cytometry using the same stocks of cells that were used in infection and neutralization assays in Fig. ​Fig.2.2. Surface staining was performed with phycoerythrin-conjugated mouse monoclonal Abs to CD4, CCR5 (CD195), and CXCR4 (CD184). Background staining was performed with isotype-matched control Abs. All Abs for flow cytometry were purchased from BD Biosciences Pharmingen (San Diego, CA). Results are shown as the mean fluorescence intensity (MFI) of positive cells. Most cells (>90%) stained positive in each case.Initial studies of HI-R cells and other clonal panels that were made using these methods also suggested a lack of MLV antigens (data not shown). We then determined the titers of replication-competent HIV-1_JRCSF preparations using JC.53 and TZM-bl cells as well as other representative HeLa-CD4/CCR5 clones in the JC panel. The results are plotted in Fig. ​Fig.1B1B as a function of cellular CCR5 content. Clones having more than a low threshold level of ∼8,000 CCR5/cell were equally susceptible to infection regardless of whether they contained MLV antigens, clearly demonstrating that HIV-1_JRCSF titers were not significantly affected by MLV. As expected, titers obtained with JC.53 and TZM-bl cells were also equivalent. In addition, these results demonstrate that HIV-1_JRCSF preparations made in JC.53 cells and in cells lacking MLV antigens (i.e., HEK293T cells and human peripheral blood mononuclear cells) were unable to infect HeLa cells lacking CCR5. The results in Fig. ​Fig.1B1B were expected because previous studies demonstrated that ecotropic MLVs cannot infect human cells or even bind to the human CAT-1 receptor paralog (1, 6, 21, 23). Moreover, it has been shown that ecotropic host range MLVs do not interfere with superinfection by any retrovirus capable of infecting human cells, including gibbon ape leukemia virus, amphotropic MLV, baboon endogenous virus, and feline leukemia virus subgroup C (21). In view of the report by Takeuchi et al. (22), we were surprised to find that JC.53 and TZM-bl cells express very small amounts of ecotropic MLV Env glycoproteins, as indicated by immunofluorescence microscopy and by their resistance to complement-dependent killing by a cytotoxic antiserum specific for MLV envelope glycoproteins (6). Nevertheless, the cell clones that contained MLV Gag all released ecotropic host range virions that replicated in murine NIH 3T3 cells but not in human cells (data not shown).To determine whether MLV affects the measurement of neutralizing Abs in TZM-bl cells, parallel assays were performed in TZM-bl and JC.48 cells; these latter cells were determined to be MLV free by Western blot analysis (Fig. ​(Fig.1)1) and by an inability to transfer MLV infection to NIH 3T3 cells (data not shown). Because JC.48 cells express CCR5 at somewhat lower levels than JC.53 cells (∼2-fold lower; Fig. ​Fig.1B),1B), it may be expected that they would be less susceptible to HIV-1 infection than are TZM-bl cells. Differences in susceptibility to HIV-1 infection may require the use of adjusted virus doses to achieve equivalent assay performance when measuring neutralizing Abs. Indeed, levels of CD4 and CCR5 were approximately twofold lower on JC.48 cells than on TZM-bl cells, whereas levels of CXCR4 were approximately equal (Fig. ​(Fig.1C).1C). We therefore measured the susceptibility of both cell lines to infection by three molecularly cloned Env-pseudotyped viruses, each bearing an Env from a different CCR5-tropic HIV-1 subtype B virus (SF162.LS, Bal.26, and QH0692.42). Infection was quantified by Luc activity expressed as relative luminescence units (RLU). Because JC.48 cells do not contain a reporter gene, the Env-pseudotyped viruses were prepared by cotransfection with the NL4-3.Luc.R-E- reporter backbone plasmid (7). Identical Luc-containing, Env-pseudotyped virus stocks were used in both cell lines. As shown in Fig. ​Fig.2A,2A, the infectivity of each pseudotyped virus was somewhat diminished in JC.48 cells compared to the infectivity in TZM-bl cells. Nonetheless, the levels of infectivity in JC.48 cells remained acceptable for neutralization assays.Open in a separate windowFIG. 2.HIV-1 infectivity and neutralization in TZM-bl and JC.48.CD4.CCR5 cells. (A) TZM-bl and JC.48 cells were incubated with serial fourfold dilutions (11 dilutions total) of three HIV-1 Env-pseudotyped viruses in quadruplicate in 96-well culture plates. Luc activity was measured after 48 h of incubation and is expressed as RLU after subtraction of background luminescence from cell control wells. Squares, TZM-bl cells; triangles, JC.48 cells. (B) Neutralization assays were performed with three HIV-1 Env-pseudotyped viruses in either TZM-bl or JC.48 cells. Input virus doses were adjusted to yield equivalent infectivity in both cell lines. Black bars, TZM-bl; gray bars, JC.48. Top panel: sCD4, monoclonal Abs, and HIVIG (purified immunoglobulin G from pooled HIV-1-positive plasmas). Bottom panel: individual HIV-1-positive plasma samples. The same three stocks of virus were used in both experiments. All three Env-pseudotyped viruses were prepared with the NL4-3.Luc.R-E- reporter backbone plasmid.With this information in hand, neutralization assays were performed in JC.48 and TZM-bl cells using adjusted virus doses that yielded equivalent infectivity levels in both cell lines. These neutralization assays were performed in a 96-well format as described previously (12), where the 50% inhibitory dose (ID₅₀) was reported as either the concentration or sample dilution at which RLU were reduced by 50% compared to RLU in virus control wells (cells plus virus without test sample) after subtraction of background RLU from cell control wells (cells only). A wide variety of serologic reagents was tested, including sCD4, a monoclonal Ab to the CD4 binding site of gp120 (immunoglobulin G1b12) (15); a monoclonal Ab that recognizes a glycan-specific epitope on gp120 (2G12) (5); two monoclonal Abs that recognize adjacent epitopes in the membrane proximal external region of gp41 (2F5 and 4E10) (2, 4); and serum samples from seven antiretroviral-naive HIV-1-infected individuals. As shown in Fig. ​Fig.2B,2B, results in the two cell lines were similar for all three viruses and all serologic reagents tested. Indeed, ID₅₀ values in the two cell types agreed within twofold, which is within the normal range of variability of the assay. These results indicate that equivalent neutralization results were obtained in both cell lines.In summary, we found no evidence that ecotropic MLV contamination in TZM-bl cells has a measurable effect on HIV-1 neutralization when these cells are used as targets for infection. This outcome indicates that the presence of ecotropic MLV in TZM-bl cells does not alter the ability of Ab to neutralize HIV-1, nor does it interfere with the detection of neutralization by using HIV-1 Tat-regulated reporter gene expression in a single-cycle infection assay. However, we discourage the use of TZM-bl cells to generate HIV-1 stocks, because the latter would likely be contaminated with ecotropic MLV and contain pseudovirions with mixtures of HIV-1 and ecotropic MLV Env glycoproteins. For this reason, we have begun efforts to produce an uncontaminated, second-generation panel of HeLa-CD4/CCR5 cell clones that express diverse amounts of CCR5 and to isolate a TZM-bl variant lacking MLV antigens.     

Dynamic antibody specificities and virion concentrations in circulating immune complexes in acute to chronic HIV-1 infection

Understanding the interactions between human immunodeficiency virus type 1 (HIV-1) virions and antibodies (Ab) produced during acute HIV-1 infection (AHI) is critical for defining antibody antiviral capabilities. Antibodies that bind virions may prevent transmission by neutralization of virus or mechanically prevent HIV-1 migration through mucosal layers. In this study, we quantified circulating HIV-1 virion-immune complexes (ICs), present in approximately 90% of AHI subjects, and compared the levels and antibody specificity to those in chronic infection. Circulating HIV-1 virions coated with IgG (immune complexes) were in significantly lower levels relative to the viral load in acute infection than in chronic HIV-1 infection. The specificities of the antibodies in the immune complexes differed between acute and chronic infection (anti-gp41 Ab in acute infection and anti-gp120 in chronic infection), potentially suggesting different roles in immunopathogenesis for complexes arising at different stages of infection. We also determined the ability of circulating IgG from AHI to bind infectious versus noninfectious virions. Similar to a nonneutralizing anti-gp41 monoclonal antibody (MAb), purified plasma IgG from acute HIV-1 subjects bound both infectious and noninfectious virions. This was in contrast to the neutralizing antibody 2G12 MAb that bound predominantly infectious virions. Moreover, the initial antibody response captured acute HIV-1 virions without selection for different HIV-1 envelope sequences. In total, this study demonstrates that the composition of immune complexes are dynamic over the course of HIV-1 infection and are comprised initially of antibodies that nonselectively opsonize both infectious and noninfectious virions, likely contributing to the lack of efficacy of the antibody response during acute infection.