Inositol phosphates promote HIV-1 assembly and maturation to facilitate viral spread in human CD4+ T cells.

Gag polymerization with viral RNA at the plasma membrane initiates HIV-1 assembly. Assembly processes are inefficient in vitro but are stimulated by inositol (1,3,4,5,6) pentakisphosphate (IP5) and inositol hexakisphosphate (IP6) metabolites. Previous studies have shown that depletion of these inositol phosphate species from HEK293T cells reduced HIV-1 particle production but did not alter the infectivity of the resulting progeny virions. Moreover, HIV-1 substitutions bearing Gag/CA mutations ablating IP6 binding are noninfectious with destabilized viral cores. In this study, we analyzed the effects of cellular depletion of IP5 and IP6 on HIV-1 replication in T cells in which we disrupted the genes encoding the kinases required for IP6 generation, IP5 2-kinase (IPPK) and Inositol Polyphosphate Multikinase (IPMK). Knockout (KO) of IPPK from CEM and MT-4 cells depleted cellular IP6 in both T cell lines, and IPMK disruption reduced the levels of both IP5 and IP6. In the KO lines, HIV-1 spread was delayed relative to parental wild-type (WT) cells and was rescued by complementation. Virus release was decreased in all IPPK or IPMK KO lines relative to WT cells. Infected IPMK KO cells exhibited elevated levels of intracellular Gag protein, indicative of impaired particle assembly. IPMK KO compromised virus production to a greater extent than IPPK KO suggesting that IP5 promotes HIV-1 particle assembly in IPPK KO cells. HIV-1 particles released from infected IPPK or IPMK KO cells were less infectious than those from WT cells. These viruses exhibited partially cleaved Gag proteins, decreased virion-associated p24, and higher frequencies of aberrant particles, indicative of a maturation defect. Our data demonstrate that IP6 enhances the quantity and quality of virions produced from T cells, thereby preventing defects in HIV-1 replication.

Variation in HIV-1 Nef function within and among viral subtypes reveals genetically separable antagonism of SERINC3 and SERINC5.

HIV Nef counteracts cellular host restriction factors SERINC3 and SERINC5, but our understanding of how naturally occurring global Nef sequence diversity impacts these activities is limited. Here, we quantify SERINC3 and SERINC5 internalization function for 339 Nef clones, representing the major pandemic HIV-1 group M subtypes A, B, C and D. We describe distinct subtype-associated hierarchies for Nef-mediated internalization of SERINC5, for which subtype B clones display the highest activities on average, and of SERINC3, for which subtype B clones display the lowest activities on average. We further identify Nef polymorphisms that modulate its ability to counteract SERINC proteins, including substitutions in the N-terminal domain that selectively impair SERINC3 internalization. Our findings demonstrate that the SERINC antagonism activities of HIV Nef differ markedly among major viral subtypes and between individual isolates within a subtype, suggesting that variation in these functions may contribute to global differences in viral pathogenesis.

A 10-Year-old Girl With Late Acute Lymphoblastic Leukemia Recurrence Diagnosed With COVID-19 and Treated With Remdesivir.

Patients with hemato-oncologic diseases are particularly vulnerable to severe infections. Adult patients with blood cancers infected with SARS-CoV-2 had poorer treatment outcomes and higher mortality than patients with COVID-19 without burden. However, in pediatric patients with hemato-oncologic diseases the course of COVID-19 is milder than in adults in the same group of patients. In this report, we describe the case of our patient with acute lymphoblastic leukemia infected with SARS-CoV-2 and treated with remdesivir. We also review the existing literature of pediatric patients who have been diagnosed with both hemato-oncologic diseases and COVID-19.

COVID-19-related Gastrointestinal Bleeding in a Pediatric Patient With ALL.

Coronavirus disease 2019 (COVID-19) is a contagious disease caused by severe acute respiratory syndrome coronavirus-2. Patients with hematologic malignancies have been shown to have higher risk of mortality due to COVID-19 than reported in the general adult population. Reports on acute lymphoblastic leukemia and COVID in children are scarce. We present a case of an 11-year-old male patient undergoing treatment for B-cell acute lymphoblastic leukemia with an atypical course of COVID-19. The patient received a positive result of the syndrome coronavirus-2 polymerase chain reaction test performed due to epidemiologic reasons. The chemotherapy was continued since the patient had no clinical signs of COVID-19. The disease started with intensive gastrointestinal bleeding, followed by severe respiratory tract infection over 2 weeks later.

Leukaemia and lockdown: The delayed infection model of childhood acute lymphoblastic leukaemia and the COVID-19 pandemic.

Acute lymphoblastic leukaemia (ALL) is the most common type of leukaemia diagnosed in children. The prevailing hypothesis regarding pathogenesis of childhood ALL was developed by Greaves, and states that ALL is caused by an abnormal immune response to a common infection. The response arises either due to naivety of the immune system caused by a lack of common childhood infections, or genetic susceptibility due to specific alleles. The former explanation is known as the delayed infection hypothesis. COVID-19 is a new infection that no children in the UK were exposed to prior to 2020. Furthermore, the lockdown measures designed to prevent spread of this virus have also greatly reduced spread of other common infections. It is therefore important to examine the evidence for this hypothesis, and to consider it in the context of the pandemic to determine what effect lockdown measures may have on incidence of ALL in children.

SARS-CoV-2 viral clearance during bone marrow aplasia after allogeneic hematopoietic stem cell transplantation-A case report.

Respiratory viral infections are known causes of mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Here, we report a unique case of a child with viral pneumonia caused by coinfection with human metapneumovirus (MPV), respiratory syncytial virus (RSV), and SARS-CoV-2 after HSCT. A 9-year-old girl with acute lymphoblastic leukemia underwent allogeneic HSCT from a matched, unrelated donor. During the post-transplant period, in profound leukopenia (below 10 leukocytes/µL), she was diagnosed with SARS-CoV-2, MPV, and RSV pneumonia and was treated with ribavirin and chloroquine. Before leukocyte recovery, the girl became asymptomatic, and SARS-CoV-2 and RSV clearance was achieved. The shedding of SARS-CoV-2 stopped before immune system recovery, and one may hypothesize that the lack of an inflammatory response might have been a contributing factor to the mild clinical course. Post-transplant care in HSCT recipients with COVID-19 infection is feasible in regular transplant units, provided the patient does not present with respiratory failure. Early and repeated testing for SARS-CoV-2 in post-transplant patients with concomitant infection mitigation strategies should be considered in children after HSCT who develop fever, respiratory symptoms, and perhaps gastrointestinal symptoms to control the spread of COVID-19 both in patients and in healthcare workers in hospital environments. Training of staff and the availability of personal protective equipment are crucial for containing SARS-CoV-2 infection.

In vitro knock-out of miR-155 suppresses leukemic and HCV virus loads in pediatric HCV-4-associated acute lymphoid leukemia: A promising target therapy.

Hepatitis C virus (HCV) infection is a major public health problem, having a high prevalence in Egypt. Leukemia and lymphoma have been associated with HCV infection. MicroRNA-155 (miR-155) has been reported to play a regulatory role in cancer, inflammation, and immune response to infection. The expression level of miR-155 in HCV viremic patients is controversial; although high miR-155 levels were demonstrated in HCV genotypes 1,2, and 3, low levels of miR-155 were detected in Egyptian patients with HCV genotype 4. Several studies have investigated the correlation between the levels of miRNA-155 and the replication of HCV, others have evaluated miRNA-155 as a prognostic biomarker in different types of cancer. No studies have investigated the impact of miRNA-155 knockdown on HCV pediatric patients associated with childhood acute lymphoblastic leukemia (ALL). We knocked-out the miR_155a in cultured polymorphonuclear cells (PBMCs) obtained from 60 children with ALL; 30 were associated with HCV-4 infection and 30 were HCV negative. The miR_155a, HCV viral load, and cell proliferation werre assessed in treated and untreated cells using TaqMan assay quantitative polymerase chain reaction. We found that miRNA-155 was significantly upregulated by seven folds in the HCV-4 associated ALL group; while being linked to high HCV viral load and leukemic burden, miR_155a knock-out can improve the disease outcome. We conclude that miR-155 is a critical miRNA that is considered a therapeutic target in pediatric HCV leukemic patients.

Elevated plasma phage load as a marker for intestinal permeability in leukemic patients.

Microbial translocation (MT) and altered gut microbiota have been described in acute leukemic patients and contribute to immune activation and inflammation. However, phage translocation has not been investigated in leukemia patients yet. We recruited 44 leukemic patients and 52 healthy adults and quantified the levels of 3 phages in peripheral blood, which were the most positive phages screened from fecal samples. The content of 16S rRNA in plasma was detected by qPCR to assess the intestinal mucosa of these patients. Spearman's rank correlation was used to analyze the relationship between phage load and the relevant clinical data. We found the most prevalent phages in fecal samples were λ phage, Wphi phage, and P22 phage, and λ phage had the highest detection rate in plasma (68%). Phage content was affected by chemotherapy and course of disease and correlated with the levels of CRP (r = 0.43, p = 0.003), sCD14 (r = 0.37, p = 0.014), and sCD163 (r = 0.44, p = 0.003). Our data indicate that plasma phage load is a promising marker for gut barrier damage and that gut phage translocation correlates with monocyte/macrophage activation and systemic inflammatory response in leukemic patients.

A New Approach to Developing Long-Acting Injectable Formulations of Anti-HIV Drugs: Poly(Ethylene Phosphoric Acid) Block Copolymers Increase the Efficiency of Tenofovir against HIV-1 in MT-4 Cells.

Despite the world's combined efforts, human immunodeficiency virus (HIV), the causative agent of AIDS, remains one of the world's most serious public health challenges. High genetic variability of HIV complicates the development of anti-HIV vaccine, and there is an actual clinical need for increasing the efficiency of anti-HIV drugs in terms of targeted delivery and controlled release. Tenofovir (TFV), a nucleotide-analog reverse transcriptase inhibitor, has gained wide acceptance as a drug for pre-exposure prophylaxis or treatment of HIV infection. In our study, we explored the potential of tenofovir disoproxil (TFD) adducts with block copolymers of poly(ethylene glycol) monomethyl ether and poly(ethylene phosphoric acid) (mPEG-b-PEPA) as candidates for developing a long-acting/controlled-release formulation of TFV. Two types of mPEG-b-PEPA with numbers of ethylene phosphoric acid (EPA) fragments of 13 and 49 were synthesized by catalytic ring-opening polymerization, and used for preparing four types of adducts with TFD. Antiviral activity of [mPEG-b-PEPA]TFD or tenofovir disoproxil fumarate (TDF) was evaluated using the model of experimental HIV infection in vitro (MT-4/HIV-1IIIB). Judging by the values of the selectivity index (SI), TFD exhibited an up to 14-fold higher anti-HIV activity in the form of mPEG-b-PEPA adducts, thus demonstrating significant promise for further development of long-acting/controlled-release injectable TFV formulations.

Clearance of Treatment Refractory Adenoviremia via Adenovirus-specific Donor T-Cell Transfer During Aplasia After αßTCR-CD19-Depleted Stem Cell Transplantation.

Here, we report the case of severe adenoviremia in a 7-year-old boy with highly-resistant, acute leukemia. A combined approach of αßTCR-CD19-depleted stem cell transplantation, enabling immunosuppression-free post-transplant care, and early transfer of adenovirus-specific donor T cells during aplasia resulted in rapid and complete clearance of the treatment-refractory adenoviremia.

PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type.

Epstein Barr virus (EBV) is a potentially oncogenic gammaherpesvirus that establishes a chronic, latent infection in memory B cells. The EBV genome persists in infected host cells as a chromatinized episome and is subject to chromatin-mediated regulation. Binding of the host insulator protein CTCF to the EBV genome has an established role in maintaining viral latency type. CTCF is posttranslationally modified by the host enzyme PARP1. PARP1, or poly(ADP-ribose) polymerase 1, catalyzes the transfer of a poly(ADP-ribose) (PAR) moiety from NAD+ onto acceptor proteins, including itself, histone proteins, and CTCF. PARylation of CTCF by PARP1 can affect CTCF's insulator activity, DNA binding capacity, and ability to form chromatin loops. Both PARP1 and CTCF have been implicated in the regulation of EBV latency and lytic reactivation. Thus, we predicted that pharmacological inhibition with PARP1 inhibitors would affect EBV latency type through a chromatin-specific mechanism. Here, we show that PARP1 and CTCF colocalize at specific sites throughout the EBV genome and provide evidence to suggest that PARP1 acts to stabilize CTCF binding and maintain the open chromatin landscape at the active Cp promoter during type III latency. Further, PARP1 activity is important in maintaining latency type-specific viral gene expression. The data presented here provide a rationale for the use of PARP inhibitors in the treatment of EBV-associated cancers exhibiting type III latency and ultimately could contribute to an EBV-specific treatment strategy for AIDS-related or posttransplant lymphomas.IMPORTANCE EBV is a human gammaherpesvirus that infects more than 95% of individuals worldwide. Upon infection, EBV circularizes as an episome and establishes a chronic, latent infection in B cells. In doing so, the virus utilizes host cell machinery to regulate and maintain the viral genome. In otherwise healthy individuals, EBV infection is typically nonpathological; however, latent infection is potentially oncogenic and is responsible for 1% of human cancers. During latent infection, EBV expresses specific sets of proteins according to the given latency type, each of which is associated with specific types of cancers. For example, type III latency, in which the virus expresses its full repertoire of latent proteins, is characteristic of AIDS-associated and posttransplant lymphomas associated with EBV infection. Understanding how viral latency type is regulated at the chromatin level may reveal potential targets for EBV-specific pharmacological intervention in EBV-associated cancers.

In utero cytomegalovirus infection and development of childhood acute lymphoblastic leukemia.

It is widely suspected, yet controversial, that infection plays an etiologic role in the development of acute lymphoblastic leukemia (ALL), the most common childhood cancer and a disease with a confirmed prenatal origin in most cases. We investigated infections at diagnosis and then assessed the timing of infection at birth in children with ALL and age, gender, and ethnicity matched controls to identify potential causal initiating infections. Comprehensive untargeted virome and bacterial analyses of pretreatment bone marrow specimens (n = 127 ALL in comparison with 38 acute myeloid leukemia cases in a comparison group) revealed prevalent cytomegalovirus (CMV) infection at diagnosis in childhood ALL, demonstrating active viral transcription in leukemia blasts as well as intact virions in serum. Screening of newborn blood samples revealed a significantly higher prevalence of in utero CMV infection in ALL cases (n = 268) than healthy controls (n = 270) (odds ratio [OR], 3.71, confidence interval [CI], 1.56-7.92, P = .0016). Risk was more pronounced in Hispanics (OR=5.90, CI=1.89-25.96) than in non-Hispanic whites (OR=2.10 CI= 0.69-7.13). This is the first study to suggest that congenital CMV infection is a risk factor for childhood ALL and is more prominent in Hispanic children. Further investigation of CMV as an etiologic agent for ALL is warranted.

Changes in hepatitis B antibody status after chemotherapy in children with acute lymphoblastic leukemia.

BACKGROUND: Children with cancer may be at an increased risk of infection with hepatitis B virus (HBV) when levels of hepatitis B antibodies are reduced owing to chemotherapy-induced immunosuppression. This study evaluated the changes in HBV antibody status and HBV infections after chemotherapy in children with acute lymphoblastic leukemia (ALL). PROCEDURE: The data of patients with ALL diagnosed between April 2007 and March 2013 were retrospectively collected. Hepatitis B surface antibody (HBsAb) titers were defined as negative at levels <10 IU/L. The HBsAb titers were individually compared before and after chemotherapy. RESULTS: A total of 88 patients were included in this study. At the time of diagnosis, 32 (36.4%) and 56 (63.6%) patients were HBsAb negative and HBsAb positive, respectively. The 56 HBsAb-positive patients were categorized into two groups, namely, group A with 44 patients (78.6%, 44/56) who became HBsAb negative after chemotherapy, and group B with 12 patients (21.4%) who remained HBsAb positive. On multivariate analysis, lower initial levels of HBsAb titers were associated with HBsAb negativity after chemotherapy (relative risk: 1.003, 95% confidence interval: 1.001-1.006; P = .009). CONCLUSION: This study demonstrated that patients with a low level of prechemotherapy HBsAb titers were likely to become HBsAb negative after chemotherapy. Therefore, evaluation of HBsAb status may be necessary after the completion of chemotherapy in children with ALL.

Iatrogenic immunodeficiency-associated lymphoproliferative disorder in a child with B-cell acute lymphoblastic leukemia.

Iatrogenic immunodeficiency-associated lymphoproliferative disorders (LPDs) are a group of lymphoid proliferations or lymphomas that are well known to be associated with an immunosuppressed state. These disorders most commonly occur following hematopoietic or solid organ transplantation (called post-transplant lymphoproliferative disorders or PTLD), but cases have also been described during the treatment of autoimmune and rheumatologic disorders by immunosuppressive and immunomodulatory medications. These disorders are strongly associated with infection by the Epstein-Barr virus (EBV) as a result of impaired immune function in the immunosuppressed state. While this phenomenon has been well documented in autoimmune conditions, cases affecting pediatric patients while on anti-leukemia chemotherapy are lacking. In this report, we describe a case of a pediatric immunosuppressed patient with recurrent sinusitis found to have a nasopharyngeal mass consistent with EBV-positive B-cell lymphoproliferative disorder resembling a polymorphic PTLD during the maintenance phase of B-cell Acute Lymphoblastic Leukemia (ALL) therapy. The patient was successfully treated with rituximab without any cytotoxic chemotherapy, highlighting the importance of recognizing this clinical entity in non-transplant patients with hematologic malignancies.

Vaccine-associated paralytic poliomyelitis in a patient with acute lymphocytic leukemia.

We report a case of vaccine-associated paralytic poliomyelitis (VAPP) in an immunocompromised patient with acute lymphocytic leukemia who was initially diagnosed with aseptic meningitis. Isolation of Sabin-like type 1 poliovirus from the patient's cerebrospinal fluid made this a case of vaccine-related poliovirus (VRPV) infection. The patient developed paralysis and respiratory distress and deceased a few months after onset of paralysis with respiratory failure. This tragic case report highlights the emergence of VAPP and indicates the importance of timely diagnosis of VRPV infections to improve clinical management of VRPV-infected patients and to prevent the devastating consequences of silent introduction of VRPVs in treatment wards and eventually in the society.

Virome characterisation from Guthrie cards in children who later developed acute lymphoblastic leukaemia.

BACKGROUND: Some childhood acute lymphoblastic leukaemias (ALL) can be traced back to a prenatal origin, where a virus infection could be involved in the first pre-leukaemic clone development. The DNA virome of 95 children who later developed ALL was characterised from neonatal blood spots (NBS) using unbiased next-generation sequencing (NGS) and compared with the virome of 95 non-ALL controls. METHODS: DNA was individually extracted from the ALL-patients and controls, pooled, randomly amplified and sequenced using the Illumina MiSeq Sequencing System. RESULTS: Virus-like sequences identified in both groups mapped to human endogenous retroviruses and propionibacterium phage, considered a part of the normal microbial flora. Potential pathogens human herpesvirus type 6 (HHV-6) and parvovirus B19 were also identified, but only few samples in both ALL and controls tested positive by PCR follow-up. CONCLUSIONS: Unbiased NGS was employed to search for DNA from potential infectious agents in neonatal samples of children who later developed ALL. Although several viral candidates were identified in the NBS samples, further investigation by PCR suggested that these viruses did not have a major role in ALL development.

Association Between Combined Presence of Hepatitis C Virus and Polymorphisms in Different Genes With Toxicities of Methotrexate and 6-Mercaptopurine in Children With Acute Lymphoblastic Leukemia.

BACKGROUND: The aim of the present study is to determine the correlation of hepatitis C virus (HCV) infection and polymorphisms in different genes with toxicity of either methotrexate (MTX) or 6-mercaptopurine (6-MP) administered to children with acute lymphoblastic leukemia (ALL). PROCEDURE: One hundred children with low-risk ALL, who were treated according to the St. Jude Total therapy XV, were recruited. The recruited children were receiving MTX and 6-MP during maintenance phase. Patients were excluded from the study if they had other types of leukemia. Genotyping analyses for the thiopurine methyltransferase (TPMT), methylenetetrahydrofolate reductase (MTHFR), and glutathione S-transferase (GST) genes were performed using a combination of polymerase chain reaction (PCR) and PCR-RFLP (where RFLP is restriction fragment length polymorphism) protocols. Relevant clinical data on adverse drug reactions were collected objectively (blinded to genotypes) from the patient medical records. RESULTS: There was a significant correlation between the combined presence of HCV and TPMT*3B G460A gene polymorphisms and grades 2-4 hepatotoxicity as aspartate aminotransferase (AST) elevation (P < 0.04). The same observation was seen when comparing either the presence of HCV alone or the presence of the gene polymorphism alone. A significant association between the combined presence of HCV and MTHFR C677T polymorphism and grades 2-4 hepatotoxicity as alanine aminotransferase (ALT), AST, and alkaline phosphatase (ALP) elevation was observed (P values <0.001, 0.02, and 0.001, respectively). The presence of HCV infection had a significant negative effect on hepatic transaminases. CONCLUSIONS: The present data support a role for combining analysis of genetic variation in drug-metabolizing enzymes and the presence of HCV in the assessment of specific drugs toxicities in multiagent chemotherapeutic treatment regimens.

Cytokine and cellular responses to human herpesvirus-6B in patients with B-acute lymphoblastic leukemia.

Primary infection with human herpesvirus-6 (HHV-6), is followed by its lifelong persistence in the host. Most T-cell responses to HHV-6 have been characterized using peripheral blood from healthy adults; however, the role of HHV-6 infection in immune modulation has not been elucidated for some diseases. Therefore, in this study the immune response to HHV-6 infection in patients with B-acute lymphoblastic leukemia (B-ALL) was analyzed. HHV-6 load was quantified in blood samples taken at the time of diagnosis of leukemia and on remission. The same concentrations of anti- and pro-inflammatory cytokines (IL-4, IL-1, IL-6, IL-8, IL-12p70, IL-17a, TNF-α and IFN-γ) were detected in plasma samples from 20 patients with and 20 without detectable HHV-6 virus loads in blood. Characterization of T-cell responses to HHV-6 showed low specific T-cells frequencies of 2.08% and 1.46% in patients with and without detectable viral loads, respectively. IFN-γ-producing T cells were detected in 0.03%-0.23% and in 0%-0.2% of CD4+T cells, respectively. Strong production of IL-6 was detected in medium supernatants of challenged T-cells whatever the HHV-6 status of the patients (973.51 ± 210.06 versus 825.70 ± 210.81 pg/mL). However, concentrations of TNF-α and IFN-γ were low. Thus, no association between plasma concentrations of cytokines and detection of HHV-6 in blood was identified, suggesting that HHV-6 is not strongly associated with development of B-ALL. The low viral loads detected may correspond with latently infected cells. Alternatively, HHV-6B specific immune responses may be below the detection threshold of the assays used.

Cytomegalovirus disease in children with acute lymphoblastic leukemia.

Viral infections are an underrecognized problem in children on standard chemotherapy for acute lymphoblastic leukemia (ALL). In countries with high baseline seroprevalence of cytomegalovirus (CMV) such as India, it may be an important pathogen leading to fever, end-organ damage, and cytopenia. Data regarding the incidence and manifestations of CMV disease in pediatric ALL patients are scanty. The authors prospectively assessed all children on chemotherapy for ALL with prolonged febrile neutropenia (FN) for CMV disease over a 3-year period. Children with end-organ damage, including pneumonia, retinitis, and colitis, were also evaluated. Quantitative and qualitative polymerase chain reaction (PCR) from blood, body fluids, or tissue was done along with ophthalmologic evaluation. CMV disease was detected in 10% of the children with prolonged FN. In addition, other children were identified due to end-organ damage, lung and eye being the common organs of involvement. Time of CMV reactivation was essentially during nonintense phase of chemotherapy. Lymphopenia was present in most children, and prolonged lymphopenia was associated with relapse of CMV infection after therapy. The authors conclude that CMV is an important pathogen in children on standard chemotherapy for ALL. It has a good outcome with early detection and directed therapy. Parenteral ganciclovir is needed for a period of 14-21 days to prevent recurrence.