Characterization of the Plasmacytoid Dendritic Cell Response to Transmitted/Founder and Nontransmitted Variants of HIV-1.

Human immunodeficiency virus type 1 (HIV-1) infection often arises from a single transmitted/founder (TF) viral variant among a large pool of viruses in the quasispecies in the transmitting partner. TF variants are typically nondominant in blood and genital secretions, indicating that they have unique traits. The plasmacytoid dendritic cell (pDC) is the primary alpha interferon (IFN-α)-producing cell in response to viral infections and is rapidly recruited to the female genital tract upon exposure to HIV-1. The impact of pDCs on transmission is unknown. We investigated whether evasion of pDC responses is a trait of TF viruses. pDCs from healthy donors were stimulated in vitro with a panel of 20 HIV-1 variants, consisting of one TF variant and three nontransmitted (NT) variants each from five transmission-linked donor pairs, and secretion of IFN-α and tumor necrosis factor alpha (TNF-α) was measured by enzyme-linked immunosorbent assay (ELISA). No significant differences in cytokine secretion in response to TF and NT viruses were observed, despite a trend toward enhanced IFN-α and TNF-α production in response to TF viruses. NT viruses demonstrated polarization toward production of either IFN-α or TNF-α, indicating possible dysregulation. Also, for NT viruses, IFN-α secretion was associated with increased resistance of the virus to inactivation by IFN-α in vitro, suggesting in vivo evolution. Thus, TF viruses do not appear to preferentially subvert pDC activation compared to that with nontransmitted HIV-1 variants. pDCs may, however, contribute to the in vivo evolution of HIV-1.IMPORTANCE The plasmacytoid dendritic cell (pDC) is the first cell type recruited to the site of HIV-1 exposure; however, its contribution to the viral bottleneck in HIV-1 transmission has not been explored previously. We hypothesized that transmitted/founder viruses are able to avoid the pDC response. In this study, we used previously established donor pair-linked transmitted/founder and nontransmitted (or chronic) variants of HIV-1 to stimulate pDCs. Transmitted/founder HIV-1, instead of suppressing pDC responses, induced IFN-α and TNF-α secretion to levels comparable to those induced by viruses from the transmitting partner. We noted several unique traits of chronic viruses, including polarization between IFN-α and TNF-α production as well as a strong relationship between IFN-α secretion and the resistance of the virus to neutralization. These data rule out the possibility that TF viruses preferentially suppress pDCs in comparison to the pDC response to nontransmitted HIV variants. pDCs may, however, be important drivers of viral evolution in vivo.

Tim-3 is a Marker of Plasmacytoid Dendritic Cell Dysfunction during HIV Infection and Is Associated with the Recruitment of IRF7 and p85 into Lysosomes and with the Submembrane Displacement of TLR9.

In chronic diseases, such as HIV infection, plasmacytoid dendritic cells (pDCs) are rendered dysfunctional, as measured by their decreased capacity to produce IFN-α. In this study, we identified elevated levels of T cell Ig and mucin-domain containing molecule-3 (Tim-3)-expressing pDCs in the blood of HIV-infected donors. The frequency of Tim-3-expressing pDCs correlated inversely with CD4 T cell counts and positively with HIV viral loads. A lower frequency of pDCs expressing Tim-3 produced IFN-α or TNF-α in response to the TLR7 agonists imiquimod and Sendai virus and to the TLR9 agonist CpG. Thus, Tim-3 may serve as a biomarker of pDC dysfunction in HIV infection. The source and function of Tim-3 was investigated on enriched pDC populations from donors not infected with HIV. Tim-3 induction was achieved in response to viral and artificial stimuli, as well as exogenous IFN-α, and was PI3K dependent. Potent pDC-activating stimuli, such as CpG, imiquimod, and Sendai virus, induced the most Tim-3 expression and subsequent dysfunction. Small interfering RNA knockdown of Tim-3 increased IFN-α secretion in response to activation. Intracellular Tim-3, as measured by confocal microscopy, was dispersed throughout the cytoplasm prior to activation. Postactivation, Tim-3 accumulated at the plasma membrane and associated with disrupted TLR9 at the submembrane. Tim-3-expressing pDCs had reduced IRF7 levels. Furthermore, intracellular Tim-3 colocalized with p85 and IRF7 within LAMP1+ lysosomes, suggestive of a role in degradation. We conclude that Tim-3 is a biomarker of dysfunctional pDCs and may negatively regulate IFN-α, possibly through interference with TLR signaling and recruitment of IRF7 and p85 into lysosomes, enhancing their degradation.

Comprehensive Cross-Clade Characterization of Antibody-Mediated Recognition, Complement-Mediated Lysis, and Cell-Mediated Cytotoxicity of HIV-1 Envelope-Specific Antibodies toward Eradication of the HIV-1 Reservoir.

Immunotherapy with passive administration of broadly neutralizing HIV-1 envelope-specific antibodies (bnAbs) in the setting of established infection in vivo has yielded mixed results. The contribution of different antibodies toward the direct elimination of infected cells is poorly understood. In this study, we determined the ability of 12 well-characterized anti-HIV-1 neutralizing antibodies to recognize and eliminate primary CD4 T cells infected with HIV-1 belonging to clades A, B, C, and D, via antibody-dependent complement-mediated lysis (ADCML) and antibody-dependent cell-mediated cytotoxicity (ADCC), in vitro We further tested unique combinations of these antibodies to determine the optimal antibody cocktails to be tested in future clinical trials. We report that antibody binding to infected CD4 T cells is highly variable and correlates with ADCML and ADCC processes. Particularly, antibodies targeting the envelope glycan shield (2G12) and V1/V2 site (PG9, PG16, and PGT145) are best at recognizing HIV-1-infected CD4 T cells. However, only PG9 and PG16 and their combinations with other bnAbs sufficiently induced the elimination of HIV-1-infected CD4 T cells by ADCML, ADCC, or both. Notably, CD4 binding site antibodies VRC01, 3BNC117, and NIH45-46 G54W did not exhibit recognition of infected cells and were unable to induce their killing. Future trials geared toward the development of a cure for HIV/AIDS should incorporate V1/V2 antibodies for maximal clearance of infected cells. With the use of only primary immune cells, we conducted a comprehensive cross-clade physiological analysis to aid the direction of antibodies as therapeutics toward the development of a cure for HIV/AIDS.IMPORTANCE Several antibodies capable of neutralizing the majority of circulating HIV-1 strains have been identified to date and have been shown to prevent infection in animal models. However, the use of combinations of such broadly neutralizing antibodies (bnAbs) for the treatment and eradication of HIV-1 in infected humans remains uncertain. In this study, we tested the ability of bnAbs to directly recognize and eliminate primary human CD4 T cells infected with diverse HIV-1 strains representative of the global epidemic by antibody-dependent pathways. We also tested several combinations of bnAbs in our assays in order to maximize the clearance of infected cells. We show that the ability of bnAbs to identify and kill infected cells is highly variable and that only a few of them are able to exert this function. Our data will help guide the formulation of bnAbs to test in future human trials aimed at the development of a cure.

A sphingosine-1-phosphate receptor 1-directed agonist reduces central nervous system inflammation in a plasmacytoid dendritic cell-dependent manner.

Gradients of the sphingolipid sphingosine-1-phosphate (S1P) are responsible for the egress of lymphocytes from lymph nodes by activating the S1P1 receptor expressed on the surface of lymphocytes. Small molecule drugs that downregulate S1P receptors induce the sequestration of lymphocytes within lymph nodes, thus preventing lymphocytes from accessing sites of inflammation. In particular, FTY720, a pan-S1P receptor agonist, has been efficacious in the treatment of multiple sclerosis as well as its animal model, experimental autoimmune encephalomyelitis (EAE), by virtue of its ability to restrain lymphocytes within the lymph nodes, thus precluding their migration into the CNS. However, multiple leukocyte subsets express S1P receptors of varying types, and although it is beneficial to prevent transmigration of proinflammatory lymphocytes into the CNS, allowing access of regulatory leukocyte subsets to the CNS is desirable. In this study, we show that an S1P1-specific agonist (AUY954) is clinically efficacious in ameliorating pre-established EAE in SJL/J mice. Efficacy of AUY954 correlated with a reduction of lymphocytes in the CNS, but access of plasmacytoid dendritic cells (pDCs) to the CNS was unimpaired, and the presence of pDCs was found to be an important cofactor in mediating the clinical efficacy of AUY954. These results indicate that pDCs are important in quieting autoimmune responses during EAE, and that trafficking inhibitors that are permissive for pDC accumulation in the CNS may be of therapeutic value for the treatment of multiple sclerosis.

Modulation of autoimmune diseases by interleukin (IL)-17 producing regulatory T helper (Th17) cells.

Following the discovery of interleukin (IL)-17 producing T helper (Th17) cells as a distinct lineage of CD4+ T helper cells it became clear that these cells play an important role in the host defense against extracellular fungal and bacterial pathogens and participate in the pathogenesis of multiple inflammatory and autoimmune disorders. Depending on the microenvironment, Th17 cells can alter their differentiation programme ultimately giving rise to either protective or pro-inflammatory pathogenic cells. We found that besides the conventional in vitro protocol for Th17 differentiation by transforming growth factor-beta (TGF-ß) plus IL-6 cytokines, a combination of IL-23 plus IL-6 can also induce Th17 cells. The Th17 cells induced by IL-23 plus IL-6 (termed as effector Th17, Teff17 cells) are pathogenic upon adoptive transfer into non-obese diabetic (NOD) mice contributing to the development of type 1 diabetes (T1D) while cells induced by TGF-ß plus IL-6 (termed as regulatory T cells, Treg17 cells) are non pathogenic and regulatory, and suppressed the pathogenic T cells in T1D. These cells differentially expressed a number of cytokines where Teff17 cells exhibited an increase in granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-22 whereas Treg17 cells demonstrated increased expression of IL-21 and immunosuppressive cytokine IL-10. Differentiation of Th17 cells is controlled by a transcription factor, RORγT although these cells also express variable levels of T-bet and FoxP3 transcription factors. This points to a dual functional role of Th17 subsets in autoimmune diseases particularly T1D. We suggest that similar to conventional regulatory T cells (Treg), induction of regulatory Treg17 cells could play an important role in modulating and preventing certain autoimmune diseases.

Th17 polarized cells from nonobese diabetic mice following mycobacterial adjuvant immunotherapy delay type 1 diabetes.

IL-17-producing T cells are regarded as potential pathogenic T cells in the induction of autoimmune diseases. Previously, we have shown that injection of adjuvants containing Mycobacterium, such as CFA or bacillus Calmette-Guérin, can prevent type 1 diabetes in NOD mice. We injected NOD mice with mycobacterial products s.c. and analyzed the IL-17-producing cells from the draining lymph nodes and spleen by restimulating whole-cell populations or CD4(+) T cells in vitro with or without IL-17-polarizing cytokines. Mice receiving CFA had a concomitant rise in the level of IL-17, IL-22, IL-10, and IFN-gamma in the draining lymph node and spleen. Adoptive transfer of splenocytes from CFA-injected NOD mice polarized with TGF-beta plus IL-6 or IL-23 delayed the development of diabetes in recipient mice. IL-17-producing cells induced by CFA maintained their IL-17-producing ability in the recipient mice. Injection of CFA also changed the cytokine profile of cells in pancreatic tissue by increasing IL-17, IL-10, and IFN-gamma cytokine gene expression. We suggest that the rise in the level of IL-17 after adjuvant therapy in NOD mice has a protective effect on type 1 diabetes development.

Preclinical evaluation of a SARS-CoV-2 mRNA vaccine PTX-COVID19-B.

Safe and effective vaccines are needed to end the COVID-19 pandemic. Here, we report the preclinical development of a lipid nanoparticle­formulated SARS-CoV-2 mRNA vaccine, PTX-COVID19-B. PTX-COVID19-B was chosen among three candidates after the initial mouse vaccination results showed that it elicited the strongest neutralizing antibody response against SARS-CoV-2. Further tests in mice and hamsters indicated that PTX-COVID19-B induced robust humoral and cellular immune responses and completely protected the vaccinated animals from SARS-CoV-2 infection in the lung. Studies in hamsters also showed that PTX-COVID19-B protected the upper respiratory tract from SARS-CoV-2 infection. Mouse immune sera elicited by PTX-COVID19-B vaccination were able to neutralize SARS-CoV-2 variants of concern, including the Alpha, Beta, Gamma, and Delta lineages. No adverse effects were induced by PTX-COVID19-B in either mice or hamsters. Based on these results, PTX-COVID19-B was authorized by Health Canada to enter clinical trials in December 2020 with a phase 2 clinical trial ongoing.

Trimeric HIV-1 gp140 fused with APRIL, BAFF, and CD40L on the mucosal gp140-specific antibody responses in mice.

HIV-1 envelope (Env)-specific antibody present at mucosal surfaces can block entry of HIV-1 into these portals and thus should be elicited by an HIV-1 preventive vaccine. Since three molecules of tumor necrosis factor superfamily (TNFSF), APRIL, BAFF, and CD40L, could promote mucosal antibody responses, we made fusion constructs of them with an HIV-1 gp140 trimer and tested the mucosal gp140-specific antibody elicited by the fusion constructs in mice using a DNA prime-protein boost vaccination regimen. The fusion constructs formed trimers and displayed both broadly neutralizing antibody epitopes and non-broadly neutralizing antibody epitopes. Compared with the control construct, trimeric gp140, trimeric gp140-APRIL and gp140-BAFF fusion proteins mildly promoted B cell proliferation in vitro, enhanced HIV-1 gp140-binding IgG responses in vaginal lavage or fecal pellets, respectively, and decreased HIV-1 gp140-binding IgA in sera. Gp140-APRIL also augmented HIV-1 gp140-binding IgG in sera. Surprisingly, gp140-CD40L did not promote B cell proliferation in vitro and inhibited mucosal and systemic HIV-1 gp140-binding IgG or IgA. These results suggest that APRIL and BAFF should be further explored as molecular adjuvants for HIV-1 vaccines to enhance mucosal antibody responses, but covalent fusion of TNFSFs to gp140 may hinder their adjuvancy due to steric interactions.

Sonographically guided percutaneous liver biopsy in infants: a retrospective review.

OBJECTIVE: The purpose of our study was to assess the technical success and complication rate of sonographically guided percutaneous liver biopsies performed in infants under 1 year old at a tertiary pediatric center. MATERIALS AND METHODS: Retrospective analysis of 65 biopsies performed in 61 infants between January 1999 and December 2003 was conducted. Data collected included patient demographics; details of the biopsy procedure including indication, needle type and size, number of passes, and samples; pathology results; and procedure-related complications. RESULTS: The 61 infants studied included 37 males and 24 females with a mean age of 119 days (age range, 7-348 days; median age, 83 days) and a mean weight of 4.5 kg (1.9-8.3 kg). A total of 65 biopsies were performed in these 61 infants. General anesthesia was used in 66.1% of procedures. An 18-gauge needle was used in 47 (72.3%) procedures. Coaxial technique was used in seven procedures, and five biopsy tracts were embolized. In 63 of 65 procedures, the mean number of passes was 1.8. In two procedures, using a coaxial technique, 11 and 12 passes were made. One biopsy was considered technically unsuccessful, and 64 of 65 (98.5%) of the biopsies provided adequate tissue for pathologic analysis. There were three (4.6%) major complications related to bleeding: one requiring a blood transfusion, one requiring surgery, and one arteriobiliary fistula requiring transarterial embolization. Three (4.6%) minor complications also occurred. There were no deaths. CONCLUSION: Sonographically guided percutaneous liver biopsy in infants is a good and effective diagnostic tool. The complication rate, however, even when performed by an experienced physician, is not insignificant in this age group of patients.

IL-10-producing B cells are induced early in HIV-1 infection and suppress HIV-1-specific T cell responses.

A rare subset of IL-10-producing B cells, named regulatory B cells (Bregs), suppresses adaptive immune responses and inflammation in mice. In this study, we examined the role of IL-10-producing B cells in HIV-1 infection. Compared to uninfected controls, IL-10-producing B cell frequencies were elevated in both blood and sigmoid colon during the early and chronic phase of untreated HIV-1 infection. Ex vivo IL-10-producing B cell frequency in early HIV-1 infection directly correlated with viral load. IL-10-producing B cells from HIV-1 infected individuals were enriched in CD19(+)TIM-1(+) B cells and were enriched for specificity to trimeric HIV-1 envelope protein. Anti-retroviral therapy was associated with reduced IL-10-producing B cell frequencies. Treatment of B cells from healthy donors with microbial metabolites and Toll-like receptor (TLR) agonists could induce an IL-10 producing phenotype, suggesting that the elevated bacterial translocation characteristic of HIV-1 infection may promote IL-10-producing B cell development. Similar to regulatory B cells found in mice, IL-10-producing B cells from HIV-1-infected individuals suppressed HIV-1-specific T cell responses in vitro, and this suppression is IL-10-dependent. Also, ex vivo IL-10-producing B cell frequency inversely correlated with contemporaneous ex vivo HIV-1-specific T cell responses. Our findings show that IL-10-producing B cells are induced early in HIV-1 infection, can be HIV-1 specific, and are able to inhibit effective anti-HIV-1 T cell responses. HIV-1 may dysregulate B cells toward Bregs as an immune evasion strategy.

HIV delays IFN-α production from human plasmacytoid dendritic cells and is associated with SYK phosphorylation.

Plasmacytoid dendritic cells (pDC) are the major producers of type I interferons (IFNs) in humans and rapidly produce IFN-α in response to virus exposure. Although HIV infection is associated with pDC activation, it is unclear why the innate immune response is unable to effectively control viral replication. We systematically compared the effect of HIV, Influenza, Sendai, and HSV-2 at similar target cell multiplicity of infection (M.O.I.) on human pDC function. We found that Influenza, Sendai, HSV-2 and imiquimod are able to rapidly induce IFN-α production within 4 hours to maximal levels, whereas HIV had a delayed induction that was maximal only after 24 hours. In addition, maximal IFN-α induction by HIV was at least 10 fold less than that of the other viruses in the panel. HIV also induced less TNF-α and MIP-1ß but similar levels of IP-10 compared to other viruses, which was also mirrored by delayed upregulation of pDC activation markers CD83 and CD86. BDCA-2 has been identified as an inhibitory receptor on pDC, signaling through a pathway that involves SYK phosphorylation. We find that compared to Influenza, HIV induces the activation of the SYK pathway. Thus, HIV delays pDC IFN-α production and pDC activation via SYK phosphorylation, allowing establishment of viral populations.

Therapeutic benefits of regulating inflammation in autoimmunity.

Autoimmunity results from the dysregulation of the immune system leading to tissue damage. Th1 and Th17 cells are known to be cellular mediators of inflammation in autoimmune diseases. The specific cytokine milieu within the site of inflammation or within secondary lymphatic tissues is important during the priming and effector phases of T cell response. In this review, we will address the nature of the inflammatory response in the context of autoimmune disease, specifically we will discuss the role of dendritic cells following stimulation of their innate pathogen recognition receptors in directing the development of T cell responses. We will focus on how dendritic cell subsets change the balance between major players in autoimmunity, namely Th1, Th17 and regulatory T cells. Th17 cells, once thought to only act as pathogenic effectors through production of IL-17, have been shown to have regulatory properties as well with co-production of the anti-inflammatory cytokine IL-10 by a subset now referred to as regulatory Th17 cells. IL-17 is important in the induction of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) and inflammatory bowel disease (IBD). Study of the inflammatory process following encounter with agents that stimulate the innate immune responses such as adjuvants opens a new horizon for the discovery of therapeutic agents including those derived from microorganisms. Microbial products such as adjuvants that function as TLR ligands may stimulate the immune system by interacting with Toll-like receptors (TLR) on antigen-presenting cells. Microbial agents such as Bacille Calmette-Guérin (BCG) or Freund's adjuvant (CFA) that induce a Th17 response are protective in models of autoimmune diseases particularly EAE and type 1 diabetes (T1D). The induction of innate immunity by these microbial products alters the balance in the cytokine microenvironment and may be responsible for modulation of the inflammation and protection from autoimmunity.