Severe West Nile virus and Sars-CoV-2 infections in a patient with thymoma and anti-type I IFN antibodies.

The current study aimed to investigate determinants of severity in a previously healthy patient who experienced two life-threatening infections, from West Nile Virus and SARS-CoV2. During COVID19 hospitalization he was diagnosed with a thymoma, retrospectively identified as already present at the time of WNV infection. Heterozygosity for p.Pro554Ser in the TLR3 gene, which increases susceptibility to severe COVID-19, and homozygosity for CCR5 c.554_585del, associated to severe WNV infection, were found. Neutralizing anti-IFN-α and anti-IFN-ω auto-antibodies were detected, likely induced by the underlying thymoma and increasing susceptibility to both severe COVID-19 pneumonia and West Nile encephalitis.

Is preexisting inflamed jaw marrow a "hidden" co-morbidity affecting outcomes of COVID-19 infections? - Clinical comparative study.

Introduction: Exceedingly high levels of the chemokine CCL5/RANTES have been found in fatty degenerated osteonecrotic alveolar bone cavities (FDOJ) and aseptic ischemic osteolysis of the jaw (AIOJ) from toothless regions. Because CCL5/RANTES seems to have a prominent role in creating the COVID-19 "cytokine storm", some researchers have used the monoclonal antibody Leronlimab to block the CCR5 on inflammatory cells.Objective: Is preexisting FDOJ/AIOJ jaw marrow pathology a "hidden" co-morbidity affecting some COVID-19 infections? To what extent does the chronic CCL5/RANTES expression from preexisting FDOJ/AIOJ areas contribute to the progression of the acute cytokine storm in COVID-19 patients?Methods: Authors report on reducing the COVID-19 "cytokine storm" by treating infected patients through targeting the chemokine receptor 5 (CCR5) with Leronlimab and interrupting the activation of CCR5 by high CCL5/RANTES signaling, thus dysregulating the inflammatory phase of the viremia. Surgical removal of FDOJ/AIOJ lesions with high CCL5/RANTES from patients with inflammatory diseases may be classified as a co-morbid disease.Results: Both multiplex analysis of 249 FDOJ/AIOJ bone tissue samples as well as serum levels of CCL5/RANTES displayed exceedingly high levels in both specimens.Discussion: By the results the authors hypothesize that chronic CCL5/RANTES induction from FDOJ/AIOJ areas may sensitize CCR5 throughout the immune system, thus, enabling it to amplify its response when confronted with the virus. As conventional intraoral radiography does little to assess the quality of the alveolar bone, ultrasonography units are available to help dentists locate the FDOJ/AIOJ lesions in an office setting.Conclusion: The authors propose a new approach to containment of the COVID-19 cytokine storm by a prophylactic focus for future viral-related pandemics, which may be early surgical clean-up of CCL5/RANTES expression sources in the FDOJ/AIOJ areas, thus diminishing a possible pre-sensitization of CCR5. A more complete dental examination includes trans-alveolar ultrasono-graphy (TAU) for hidden FDOJ/AIOJ lesions.

Development of Sensitive Anti-Mouse CCR5 Monoclonal Antibodies Using the N-Terminal Peptide Immunization.

One of the G protein-coupled receptors, C-C chemokine receptor 5 (CCR5), is an important regulator for the activation of T and B lymphocytes, dendritic cells, natural killer cells, and macrophages. Upon binding to its ligands, CCR5 activates downstream signaling, which is an important regulator in the innate and adaptive immune response through the promotion of lymphocyte migration and the secretion of proinflammatory cytokines. Anti-CCR5 monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials for tumors and inflammatory diseases. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the N-terminal peptide immunization. Among the established anti-mCCR5 mAbs, C5Mab-4 (rat IgG2a, kappa) and C5Mab-8 (rat IgG1, kappa), recognized mCCR5-overexpressing Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. The dissociation constant (KD) values of C5Mab-4 and C5Mab-8 for CHO/mCCR5 were determined as 3.5 × 10-8 M and 7.3 × 10-9 M, respectively. Furthermore, both C5Mab-4 and C5Mab-8 could detect mCCR5 by western blotting. These results indicated that C5Mab-4 and C5Mab-8 are useful for detecting mCCR5 by flow cytometry and western blotting and provide a possibility to obtain the proof of concept in preclinical studies.

Cenicriviroc Suppresses and Reverses Steatohepatitis by Regulating Macrophage Infiltration and M2 Polarization in Mice.

The inhibition of hepatic macrophage and Kupfer cell recruitment and activation is a potential strategy for treating insulin resistance and nonalcoholic steatohepatitis (NASH). Cenicriviroc (CVC), a dual C-C chemokine receptor 2 (CCR2) and CCR5 antagonist, has shown antifibrotic activity in murine models of NASH and has been evaluated in clinical trials on patients with NASH. This study investigated the effects of CVC on macrophage infiltration and polarization in a lipotoxic model of NASH. C57BL/6 mice were fed a high-cholesterol, high-fat (CL) diet or a CL diet containing 0.015% CVC (CL + CVC) for 12 weeks. Macrophage recruitment and activation were assayed by immunohistochemistry and flow cytometry. CVC supplementation attenuated excessive hepatic lipid accumulation and peroxidation and alleviated glucose intolerance and hyperinsulinemia in the mice that were fed the CL diet. Flow cytometry analysis revealed that compared with the CL group, mice fed the CL + CVC diet had fewer M1-like macrophages, more M2-like macrophages, and fewer T cell counts, indicating that CVC caused an M2-dominant shift of macrophages in the liver. Similarly, CVC decreased lipopolysaccharide-stimulated M1-like macrophage activation, whereas it increased interleukin-4-induced M2-type macrophage polarization in vitro. In addition, CVC attenuated hepatic fibrosis by repressing hepatic stellate cell activation. Lastly, CVC reversed insulin resistance as well as steatosis, inflammation, and fibrosis of the liver in mice with pre-existing NASH. In conclusion, CVC prevented and reversed hepatic steatosis, insulin resistance, inflammation, and fibrogenesis in the liver of NASH mice via M2 macrophage polarization.

CCR5 Decorated Rilpivirine Lipid Nanoparticles Build Myeloid Drug Depots Which Sustains Antiretroviral Activities.

Antiretroviral therapy (ART) improves the quality of life for those living with the human immunodeficiency virus type one (HIV-1). However, poor compliance reduces ART effectiveness and leads to immune compromise, viral mutations, and disease co-morbidities. A novel drug formulation is made whereby a lipid nanoparticle (LNP) carrying rilpivirine (RPV) is decorated with the C-C chemokine receptor type 5 (CCR5). This facilitates myeloid drug depot deposition. Particle delivery to viral reservoirs is tracked by positron emission tomography. The CCR5-mediated RPV LNP cell uptake and retention reduce HIV-1 replication in human monocyte-derived macrophages and infected humanized mice. Focused ultrasound allows the decorated LNP to penetrate the blood-brain barrier and reach brain myeloid cells. These findings offer a role for CCR5-targeted therapeutics in antiretroviral delivery to optimize HIV suppression.

Retinoic acid enhances HIV-1 reverse transcription and transcription in macrophages via mTOR-modulated mechanisms.

The intestinal environment facilitates HIV-1 infection via mechanisms involving the gut-homing vitamin A-derived retinoic acid (RA), which transcriptionally reprograms CD4+ T cells for increased HIV-1 replication/outgrowth. Consistently, colon-infiltrating CD4+ T cells carry replication-competent viral reservoirs in people with HIV-1 (PWH) receiving antiretroviral therapy (ART). Intriguingly, integrative infection in colon macrophages, a pool replenished by monocytes, represents a rare event in ART-treated PWH, thus questioning the effect of RA on macrophages. Here, we demonstrate that RA enhances R5 but not X4 HIV-1 replication in monocyte-derived macrophages (MDMs). RNA sequencing, gene set variation analysis, and HIV interactor NCBI database interrogation reveal RA-mediated transcriptional reprogramming associated with metabolic/inflammatory processes and HIV-1 resistance/dependency factors. Functional validations uncover post-entry mechanisms of RA action including SAMHD1-modulated reverse transcription and CDK9/RNA polymerase II (RNAPII)-dependent transcription under the control of mammalian target of rapamycin (mTOR). These results support a model in which macrophages residing in the intestine of ART-untreated PWH contribute to viral replication/dissemination in an mTOR-sensitive manner.

In situ analysis of neuronal injury and neuroinflammation during HIV-1 infection.

BACKGROUND: Since the introduction of combination antiretroviral therapy (cART) the brain has become an important human immunodeficiency virus (HIV) reservoir due to the relatively low penetration of many drugs utilized in cART into the central nervous system (CNS). Given the inherent limitations of directly assessing acute HIV infection in the brains of people living with HIV (PLWH), animal models, such as humanized mouse models, offer the most effective means of studying the effects of different viral strains and their impact on HIV infection in the CNS. To evaluate CNS pathology during HIV-1 infection in the humanized bone marrow/liver/thymus (BLT) mouse model, a histological analysis was conducted on five CNS regions, including the frontal cortex, hippocampus, striatum, cerebellum, and spinal cord, to delineate the neuronal (MAP2ab, NeuN) and neuroinflammatory (GFAP, Iba-1) changes induced by two viral strains after 2 weeks and 8 weeks post-infection. RESULTS: Findings reveal HIV-infected human cells in the brain of HIV-infected BLT mice, demonstrating HIV neuroinvasion. Further, both viral strains, HIV-1JR-CSF and HIV-1CH040, induced neuronal injury and astrogliosis across all CNS regions following HIV infection at both time points, as demonstrated by decreases in MAP2ab and increases in GFAP fluorescence signal, respectively. Importantly, infection with HIV-1JR-CSF had more prominent effects on neuronal health in specific CNS regions compared to HIV-1CH040 infection, with decreasing number of NeuN+ neurons, specifically in the frontal cortex. On the other hand, infection with HIV-1CH040 demonstrated more prominent effects on neuroinflammation, assessed by an increase in GFAP signal and/or an increase in number of Iba-1+ microglia, across CNS regions. CONCLUSION: These findings demonstrate that CNS pathology is widespread during acute HIV infection. However, neuronal loss and the magnitude of neuroinflammation in the CNS is strain dependent indicating that strains of HIV cause differential CNS pathologies.

Development of a Sensitive Anti-Mouse CCR5 Monoclonal Antibody for Flow Cytometry.

C-C chemokine receptor 5 (CCR5), a member of the G protein-coupled receptor family, is the most common coreceptor for the human immunodeficiency virus type 1. CCR5 is also involved in the pathogenesis of tumors and inflammatory diseases. The CCR5 antagonists including monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the Cell-Based Immunization and Screening (CBIS) method. One of the established anti-mCCR5 mAbs, C5Mab-2 (rat IgG2b, kappa), reacted with mCCR5-overexpressed Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. Using flow cytometry, the dissociation constant (KD) of C5Mab-2 for CHO/mCCR5 was determined as 4.3 × 10-8 M. These results indicated that C5Mab-2 is useful for the detection of mCCR5 in flow cytometry and may be applicable to obtain the proof of concept in preclinical studies.

Blocking CCR5 activity by maraviroc augmentation in post-stroke depression: a proof-of-concept clinical trial.

BACKGROUND: Post-stroke depression (PSD) is a significant impediment to successful rehabilitation and recovery after a stroke. Current therapeutic options are limited, leaving an unmet demand for specific and effective therapeutic options. Our objective was to investigate the safety of Maraviroc, a CCR5 antagonist, as a possible mechanism-based add-on therapeutic option for PSD in an open-label proof-of-concept clinical trial. METHODS: We conducted a 10-week clinical trial in which ten patients with subcortical and cortical stroke, suffering from PSD. were administered a daily oral dose of 300 mg Maraviroc. Participants were then monitored for an additional eight weeks. The primary outcome measure was serious treatment-emergent adverse events (TEAEs) and TEAEs leading to discontinuation. The secondary outcome measure was a change in the Montgomery-Asberg Depression Rating Scale (MADRS). RESULTS: Maraviroc was well tolerated, with no reports of serious adverse events or discontinuations due to intolerance. The MADRS scores substantially reduced from baseline to week 10 (mean change: -16.4 ± 9.3; p < 0.001). By the conclusion of the treatment phase, a favorable response was observed in five patients, with four achieving remission. The time to response was relatively short, approximately three weeks. After the cessation of treatment, MADRS scores increased at week 18 by 6.1 ± 9.6 points (p = 0.014). CONCLUSIONS: Our proof-of-concept study suggests that a daily dosage of 300 mg of Maraviroc may represent a well-tolerated and potentially effective pharmacological approach to treating PSD. Further comprehensive placebo-controlled studies are needed to assess the impact of Maraviroc augmentation on PSD. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05932550, Retrospectively registered: 28/06/2023.

Immunotherapy-activated T cells recruit and skew late-stage activated M1-like macrophages that are critical for therapeutic efficacy.

Total tumor clearance through immunotherapy is associated with a fully coordinated innate and adaptive immune response, but knowledge on the exact contribution of each immune cell subset is limited. We show that therapy-induced intratumoral CD8+ T cells recruited and skewed late-stage activated M1-like macrophages, which were critical for effective tumor control in two different murine models of cancer immunotherapy. The activated CD8+ T cells summon these macrophages into the tumor and their close vicinity via CCR5 signaling. Exposure of non-polarized macrophages to activated T cell supernatant and tumor lysate recapitulates the late-stage activated and tumoricidal phenotype in vitro. The transcriptomic signature of these macrophages is also detected in a similar macrophage population present in human tumors and coincides with clinical response to immune checkpoint inhibitors. The requirement of a functional co-operation between CD8+ T cells and effector macrophages for effective immunotherapy gives warning to combinations with broad macrophage-targeting strategies.

Involvement of CCR5 on interstitial macrophages in the development of lung fibrosis in severe asthma.

CCR5 may be involved in the pathogenesis of asthma; however, the underlying mechanisms remain unclear. In comparison with a mild asthma model, subepithelial fibrosis was more severe and CCR5 gene expression in the lungs was significantly higher in our recently developed murine model of steroid-resistant severe asthma. Treatment with the CCR5 antagonist, maraviroc, significantly suppressed the development of subepithelial fibrosis in bronchi, whereas dexamethasone did not. On the other hand, increases in leukocytes related to type 2 inflammation, eosinophils, Th2 cells, and group 2 innate lymphoid cells in the lungs were not affected by the treatment with maraviroc. Increases in neutrophils and total macrophages were also not affected by the CCR5 antagonist. However, increases in transforming growth factor (TGF)-ß-producing interstitial macrophages (IMs) were significantly reduced by maraviroc. The present results confirmed increases in CCR5-expressing IMs in the lungs of the severe asthma model. In conclusion, CCR5 on IMs plays significant roles in the development of subepithelial fibrosis in severe asthma through TGF-ß production in the lungs.

Novel artesunate and isatin hybrid CT3-1 suppresses collagen-induced arthritis through abrogating dendritic cell chemotaxis-induced by CCR5.

BACKGROUND: Chemotaxis and trafficking of dendritic cells (DCs) induced by cytokine receptors are crucial steps in rheumatoid arthritis (RA) pathogenesis. C-C chemokine receptor type 5 (CCR5) plays a key role in DC movement and has been implicated in multitudinous inflammatory and immunology diseases. Thus, targeting CCR5 to suppress DC chemotaxis is considered as a potential strategy for the management of RA. METHODS: Herein, we first synthesized a new hybrid named CT3-1 which based on artesunate and isatin. Besides, we studied the regulating effectiveness of CT3-1 on bone marrow-derived DCs (BMDCs) and on collagen-induced arthritis (CIA) through RNA-seq analysis, cell function experiments in vitro and mice model in vivo. RESULTS: The results shown that CT3-1 mainly reduced CCR5 expression of immature BMDCs and importantly inhibited immature BMDC migration induced by CCR5 in vitro, with no or minor influence on other functions of DCs, such as phagocytosis and maturation. In the mouse model, CT3-1 relieved arthritis severity and inhibited CIA development. Furthermore, CT3-1 intervention decreased the expression of CCR5 in DCs and reduced the proportion of DCs in the peripheral blood of CIA mice. CONCLUSIONS: Our findings suggest that CCR5-induced chemotaxis and trafficking of immature DCs are important in RA. Targeting CCR5 and inhibiting immature DC chemotaxis may provide a novel choice for the treatment of RA and other similar autoimmune diseases. Moreover, we synthesized a new hybrid compound CT3-1 that could inhibit immature DC trafficking and effectively relieve RA by directly reducing the CCR5 expression of immature DCs.

CRISPR/Cas9 genome editing of CCR5 combined with C46 HIV-1 fusion inhibitor for cellular resistant to R5 and X4 tropic HIV-1.

Hematopoietic stem-cell (HSC) transplantation using a donor with a homozygous mutation in the HIV co-receptor CCR5 (CCR5Δ32/Δ32) holds great promise as a cure for HIV-1. Previously, there were three patients that had been reported to be completely cured from HIV infection by this approach. However, finding a naturally suitable Human Leukocyte Antigen (HLA)-matched homozygous CCR5Δ32 donor is very difficult. The prevalence of this allele is only 1% in the Caucasian population. Therefore, additional sources of CCR5Δ32/Δ32 HSCs are required. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is one method to mediate CCR5 knockout in HSCs that has been successfully employed as a gene editing tool in clinical trials. Additional anti-HIV-1 strategies are still required for broad-spectrum inhibition of HIV-1 replication. Here in this study, we combined an additional anti-HIV-1 therapy, which is C46, a cell membrane-anchored HIV-1 fusion inhibitor with the CRISPR/Cas9 mediated knockout CCR5. The combined HIV-1 therapeutic genes were investigated for the potential prevention of both CCR5 (R5)- and CXCR4 (X4)-tropic HIV-1 infections in the MT4CCR5 cell line. The combinatorial CRISPR/Cas9 therapies were superior compared to single method therapy for achieving the HIV-1 cure strategy and shows potential for future applications.

A temperature-sensitive and interferon-silent Sendai virus vector for CRISPR-Cas9 delivery and gene editing in primary human cells.

The transformative potential of gene editing technologies hinges on the development of safe and effective delivery methods. In this study, we developed a temperature-sensitive and interferon-silent Sendai virus (ts SeV) as a novel delivery vector for CRISPR-Cas9 and for efficient gene editing in sensitive human cell types without inducing IFN responses. ts SeV demonstrates unprecedented transduction efficiency in human CD34+ hematopoietic stem and progenitor cells (HSPCs) including transduction of the CD34+/CD38-/CD45RA-/CD90+(Thy1+)/CD49fhigh stem cell enriched subpopulation. The frequency of CCR5 editing exceeded 90% and bi-allelic CCR5 editing exceeded 70% resulting in significant inhibition of HIV-1 infection in primary human CD14+ monocytes. These results demonstrate the potential of the ts SeV platform as a safe, efficient, and flexible addition to the current gene-editing tool delivery methods, which may help to further expand the possibilities in personalized medicine and the treatment of genetic disorders.

HIV-1 envelope facilitates the development of protease inhibitor resistance through acquiring mutations associated with viral entry and immune escape.

Introduction: There is increasing evidence supporting a role for HIV-1 envelope in the development of Protease Inhibitor drug resistance, and a recent report from our group suggested that Env mutations co-evolve with Gag-Protease mutations in the pathway to Lopinavir resistance. In this study, we investigated the effect of co-evolving Env mutations on virus function and structure. Methods: Co-receptor usage and n-linked glycosylation were investigated using Geno2Pheno as well as tools available at the Los Alamos sequence database. Molecular dynamics simulations were performed using Amber 18 and analyzed using Cpptraj, and molecular interactions were calculated using the Ring server. Results: The results showed that under Protease Inhibitor drug selection pressure, the envelope gene modulates viral entry by protecting the virus from antibody recognition through the increased length and number of N-glycosylation sites observed in V1/V2 and to some extent V5. Furthermore, gp120 mutations appear to modulate viral entry through a switch to the CXCR4 coreceptor, induced by higher charge in the V3 region and specific mutations at the coreceptor binding sites. In gp41, S534A formed a hydrogen bond with L602 found in the disulfide loop region between the Heptad Repeat 1 and Heptad Repeat 2 domains and could negatively affect the association of gp120-gp41 during viral entry. Lastly, P724Q/S formed both intermolecular and intramolecular interactions with residues within the Kennedy loop, a known epitope. Discussion: In conclusion, the results suggest that mutations in envelope during Protease Inhibitor treatment failure are related to immune escape and that S534A mutants could preferentially use the cell-to-cell route of infection.

Corrigendum: Case Series: Maraviroc and pravastatin as a therapeutic option to treat long COVID/Post-acute sequalae of COVID (PASC).

[This corrects the article DOI: 10.3389/fmed.2023.1122529.].

CCR5-overexpressing mesenchymal stem cells protect against experimental autoimmune uveitis: insights from single-cell transcriptome analysis.

Autoimmune uveitis is a leading cause of severe vision loss, and animal models provide unique opportunities for studying its pathogenesis and therapeutic strategies. Here we employ scRNA-seq, RNA-seq and various molecular and cellular approaches to characterize mouse models of classical experimental autoimmune uveitis (EAU), revealing that EAU causes broad retinal neuron degeneration and marker downregulation, and that Müller glia may act as antigen-presenting cells. Moreover, EAU immune response is primarily driven by Th1 cells, and results in dramatic upregulation of CC chemokines, especially CCL5, in the EAU retina. Accordingly, overexpression of CCR5, a CCL5 receptor, in mesenchymal stem cells (MSCs) enhances their homing capacity and improves their immunomodulatory outcomes in preventing EAU, by reducing infiltrating T cells and activated microglia and suppressing Nlrp3 inflammasome activation. Taken together, our data not only provide valuable insights into the molecular characteristics of EAU but also open an avenue for innovative MSC-based therapy.

Multiple treatment interruptions and protecting HIV-specific CD4 T cells enable durable CD8 T cell response and viral control.

Human Immunodeficiency Virus (HIV) remains a global health challenge, and novel approaches to improve HIV control are significantly important. The cell and gene therapy product AGT103-T was previously evaluated (NCT04561258) for safety, immunogenicity, and persistence in seven patients for up to 180 days post infusion. In this study, we sought to investigate the impact of AGT103-T treatment upon analytical treatment interruptions (ATIs). Six patients previously infused with AGT103-T were enrolled into an ATI study (NCT05540964), wherein they suspended their antiretroviral therapy (ART) until their viral load reached 100,000 copies/mL in two successive visits, or their CD4 count was reduced to below 300 cells/µL. During the ATI, all patients experienced viral rebound followed by a notable expansion in HIV specific immune responses. The participants demonstrated up to a five-fold increase in total CD8 counts over baseline approximately 1-2 weeks followed by the peak viremia. This coincided with a rise in HIV-specific CD8 T cells, which was attributed to the increase in antigen availability and memory recall. Thus, the protocol was amended to include a second ATI with the first ATI serving as an "auto-vaccination." Four patients participated in a second ATI. During the second ATI, the Gag-specific CD8 T cells were either maintained or rose in response to viral rebound and the peak viremia was substantially decreased. The patients reached a viral set point ranging from 7,000 copies/mL to 25,000 copies/mL. Upon resuming ART, all participants achieved viral control more rapidly than during the first ATI, with CD4 counts remaining within 10% of baseline measurements and without any serious adverse events or evidence of drug resistance. In summary, the rise in CD8 counts and the viral suppression observed in 100% of the study participants are novel observations demonstrating that AGT103-T gene therapy when combined with multiple ATIs, is a safe and effective approach for achieving viral control, with viral setpoints consistently below 25,000 copies/mL and relatively stable CD4 T cell counts. We conclude that HIV cure-oriented cell and gene therapy trials should include ATI and may benefit from designs that include multiple ATIs when induction of CD8 T cells is required to establish viral control.