DENDRITIC CELLS TRANSFECTED WITH AUTOLOGOUS SIV RNA: POTENTIAL AIDS VACCINE

The need for a therapeutic human immunodeficiency virus (HIV) vaccine is urgent for the control of the acquired immunodeficiency syndrome (AIDS) epidemic. The variability of the virus as well as its ability to undergo escape mutations in T cell epitopes are important obstacles facing the development of an AIDS vaccine. Consequently, a powerful strategy would be the induction of robust antigen specific T cell responses targeting patient-specific virus sequences expressed during the course of infection. An attractive vaccine approach to achieve this is the use of dendritic cells (DC) transfected with in vitro transcribed mRNA encoding autologous virus sequences. The rhesus macaque model provides an ideal preclinical setting to test the therapeutic potential of DC-based vaccination. We hypothesize that optimal antigen presentation and stimulation of potent T cell responses could be achieved by loading DC from SIV-infected macaques with mRNA encoding virus-derived sequences isolated during the course of infection. This represents a powerful strategy for the generation of a potential therapeutic AIDS vaccine. In support of our hypothesis, we generated the following evidence: (1) nucleofection is a superior method for efficient transfection of human and monkey monocyte-derived DC with DNA and mRNA to conventional electroporation and lipofection; (2) nucleofection of DC with mRNA led to better protein expression and DC maturation as compared to DNA transfection; (3) mRNA nucleofection of DC resulted in rapid and sustained gene expression, a critical factor in DC-based immunotherapy for durable antigen presentation; (4) nucleofection of monkey monocyte-derived DC with wild-type non codon-optimized gag mRNA was efficiently expressed and induced strong antigen-specific T cell responses whereas DNA transfection led to non-specific T cell stimulation; (5) enhanced CD4+ T cell responses were observed when Gag was redirected to the lysosomal pathway via the targeting signal of the lysosome-associated membrane protein (LAMP-1) following nucelofection of DC with mRNA; (6) rhesus DC transfected with lysosome-targeted gag mRNA encoding an escape mutation in an immunodominant CTL epitope stimulated CD4+ and CD8+ T cell responses of almost equivalent magnitude directed towards undefined epitopes outside of the mutated region; (7) gag or env mRNA transfected-DC from SIV-infected macaques stimulated significant antigen-specific T cell responses in an entirely autologous system; (8) DC cotransfected with gag mRNA as well as mRNA encoding CD70 or OX40L did not result in enhanced immunostimulatory functions. HIV/AIDS is a significant public health problem demanding action. This work demonstrates that mRNA-transfected DC expressing SIV antigen from infected monkeys stimulate broad and relevant T cell responses, thus supporting this approach for the generation of a therapeutic HIV vaccine to decrease the burden associated with the infection

Potential role of killer immunoglobulin receptor genes among individuals vaccinated against hepatitis B virus in Lebanon

AIM: To explore the role of killer immunoglobulin receptor (KIR) genes in responsiveness or non-responsiveness to vaccination against hepatitis B virus. METHODS: We recruited 101 voluntary participants between March 2010 and December 2011. Sera samples from vaccinated and non-vaccinated participants were tested for the presence of anti-HBs antibodies as a measure of protection against hepatitis B, hepatitis B surface antigen and hepatitis B core antibody as indicators of infection by enzyme-linked immunosorbent assay. KIR gene frequencies were determined by polymerase chain reaction. RESULTS: Sera samples from 99 participants were tested for the levels of anti-HBs as an indicator of protection (≥ 10 mIU/mL) following vaccination as defined by the World Health Organization international reference standard. Among the vaccinated participants, 47% (35/74) had anti-HBs titers above 100 mIU/mL, 22% (16/74) had anti-HBs ranging between 10-100 mIU/mL, and 20% (15/74) had values of less than 10 mIU/mL. We report the lack of significant association between the number of vaccine dosages and the titer of antibodies among our vaccinated participants. The inhibitory KIR2DL1, KIR2DL4, KIR3DL1, KIR3DL2, and KIR3DL were detected in more than 95%, whereas KIR2DL2, KIR2DL3, KIR2DL5 (KR2DL5A and KIR2DL5B) were expressed in 56%, 84% and 42% (25% and 29%) of participants, respectively. The observed frequency of the activating KIR genes ranged between 35% and 55% except for KIR2DS4, detected in 95% of the study participants (40.6% 2DS4*001/002; 82.2% 2DS4*003/007). KIR2DP1 pseudogene was detected in 99% of our participants, whereas KIR3DP*001/02/04 and KIR3DP1*003 had frequencies of 17% and 100%, respectively. No association between the frequency of KIR genes and anti-HBs antibodies was detected. When we compared the frequency of KIR genes between vaccinated individuals with protective antibodies titers and those who lost their protective antibody levels, we did not detect a significant difference. KIR2DL5B was significantly different among different groups of vaccinated participants (group I > 100 mIU/mL, group II 10-100 mIU/mL, group III < 10 mIU/mL and group IV with undetectable levels of protective antibodies). CONCLUSION: To our knowledge, this is the first study screening for the possible role of KIR genes among individuals vaccinated against hepatitis B virus (HBV). Our results can be used to design larger studies to better understand the role of KIR genes in protection against or susceptibility to HBV post vaccination

Support for UNRWA's survival

The United Nations Relief and Works Agency for Palestine Refugees in the Near East (UNRWA) provides life-saving humanitarian aid for 5·4 million Palestine refugees now entering their eighth decade of statelessness and conflict. About a third of Palestine refugees still live in 58 recognised camps. UNRWA operates 702 schools and 144 health centres, some of which are affected by the ongoing humanitarian disasters in Syria and the Gaza Strip. It has dramatically reduced the prevalence of infectious diseases, mortality, and illiteracy. Its social services include rebuilding infrastructure and homes that have been destroyed by conflict and providing cash assistance and micro-finance loans for Palestinians whose rights are curtailed and who are denied the right of return to their homeland

Association between the choice of the conditioning regimen and outcomes of allogeneic hematopoietic cell transplantation for myelofibrosis

Allogeneic hematopoietic cell transplantation (allo-HCT) remains the only curative treatment for myelofibrosis. However, the optimal conditioning regimen either with reduced intensity conditioning (RIC) or myeloablative conditioning (MAC) is not well known. Using the Center for International Blood and Marrow Transplant Research database, we identified adults aged ≥18 years with myelofibrosis undergoing allo-HCT between 2008-2019 and analyzed the outcomes separately in the RIC and MAC cohorts based on the conditioning regimens used. Among 872 eligible patients, 493 underwent allo-HCT using RIC (Fludarabine/busulfan=166, Fludarabine/melphalan=327) and 379 using MAC (Fludarabine/busulfan=247, Busulfan/cyclophosphamide=132). In multivariable analysis with RIC, Fludarabine/melphalan was associated with inferior overall survival (HR 1.80, 95% CI 1.15-2.81, p=0.009), higher early non-relapse mortality (HR 1.81, 95% CI 1.12-2.91, p=0.01) and higher acute graft versus host disease (GVHD) (grade II-IV- HR 1.45, 95% CI 1.03-2.03, p=0.03; grade III-IV HR 2.21, 95%CI 1.28-3.83, p=0.004) compared to Fludarabine/busulfan. In the MAC setting, Busulfan/cyclophosphamide was associated with a higher acute GVHD (grade II-IV HR 2.33, 95% CI 1.67-3.25, p\u3c0.001; grade III-IV HR 2.31, 95% CI 1.52-3.52, p\u3c0.001) and inferior GVHD-free relapse-free survival (GRFS) (HR 1.94, 95% CI 1.49-2.53, p\u3c0.001) as compared to Fludarabine/busulfan. Hence, our study suggests that Fludarabine/busulfan is associated with better outcomes in RIC (better overall survival, lower early non-relapse mortality, lower acute GVHD) and MAC (lower acute GVHD and better GRFS) in myelofibrosis

Clinical Characteristics and Serum Cytokines Profiling in Hospitalized COVID-19 Patients in Lebanon

Since its emergence, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a public health threat worldwide. While the majority of patients recover in 3–4 weeks, complications in severely ill patients, including acute respiratory distress syndrome, cardiac injury, thrombosis, and sepsis, can lead to death. Several biomarkers, in addition to cytokine release syndrome (CRS), have been associated with severe and fatal outcomes in coronavirus disease 2019 (COVID-19) patients. The aim of this study is to assess clinical characteristics and cytokines profiles in hospitalized COVID-19 patients in Lebanon. A total of 51 hospitalized COVID-19 patients were recruited between February 2021 and May 2022. Clinical data and sera were collected at two time points: at hospital presentation (T0) and last collected results during hospitalization (T1). Our results showed that 49% of participants were >60 years with males accounting for the majority (72.5%). Hypertension, followed by diabetes and dyslipidemia, were the most frequent comorbid conditions among study participants accounting for 56.9% and 31.4%, respectively. Chronic obstructive pulmonary disease (COPD) was the only significantly different comorbid condition between intensive care unit (ICU) and non-ICU patients. Our results also showed that the median level of D-dimer was significantly elevated among patients in ICU and those who died compared to non-ICU patients and those who survived. Moreover, C-reactive protein (CRP) levels were significantly higher at T0 compared to T1 in ICU and non-ICU patients. The median level of IL-12p70 was significantly higher in patients >60 years compared to those ≤60 years (p=0.0209). Our data are in agreement with previous reports suggesting the importance of IL-6, CRP, and IL-12p70 in the assessment of risk of severe disease and mortality

High-Level Antigen Expression and Sustained Antigen Presentation in Dendritic Cells Nucleofected with Wild-Type Viral mRNA but Not DNA▿

Dendritic cells (DC) are potent antigen-presenting cells that hold promise as cell-based therapeutic vaccines for infectious diseases and cancer. Ideally, DC would be engineered to express autologous viral or tumor antigens to ensure the presentation of relevant antigens to host T cells in vivo; however, expression of wild-type viral genes in primary cell lines can be problematic. Nucleofection is an effective means of delivering transgenes to primary cell lines, but its use in transfecting DNA or mRNA into DC has not been widely investigated. We show that nucleofection is a superior means of transfecting human and monkey monocyte-derived DC with DNA and mRNA compared to lipofection and conventional electroporation. However, the delivery of DNA and mRNA had significantly different outcomes in transfected DC. DC nucleofected with DNA encoding green fluorescent protein (GFP) had poor antigen expression and viability and were refractory to maturation with CD40 ligand. In contrast, >90% of DC expressed uniform and high levels of GFP from 3 h to 96 h postnucleofection with mRNA while maintaining a normal maturation response to CD40 ligation. Monkey DC nucleofected with wild-type, non-codon-optimized mRNA encoding simian immunodeficiency virus Gag stimulated robust antigen-specific effector T-cell responses at 24 h and 48 h postnucleofection, reflecting sustained antigen presentation in transfected DC, whereas no detectable T-cell response was noted when DC were nucleofected with DNA encoding the same Gag sequence. These data indicate that mRNA nucleofection may be an optimal means of transfecting DC with autologous tumor or viral antigen for DC-based immunotherapy

The changing pattern of hepatitis A in Lebanese adults

Objective: A shift in the age of hepatitis A virus (HAV) infection from early childhood to adulthood has been observed in many developing countries. This epidemiological shift has been attributed to improved socioeconomic status and sanitary conditions resulting in growing cohorts of susceptible young people and hence an increased risk of HAV outbreaks. The aim of this study was to investigate the evolutionary trend of anti-HAV seroprevalence in Lebanon in a cohort of Lebanese adults. Methods: This was a cross-sectional study employing a convenience sample (voluntary blood donors) along with secondary data analysis. Sera from 283 healthy blood donors were tested for anti-HAV IgG antibodies. Moreover, we analyzed the national reports of HAV cases published by the Lebanese Ministry of Public Health since 2001. Results: Anti-HAV seropositivity increased steadily from 60% in the younger age group (19–29 years) to 91% in the older age group (50–59 years), leaving the younger group at higher risk of acquiring HAV. The national data show that the number of acute hepatitis A infections is higher in the age groups 5–9 and 10–19 years. Conclusion: Our seroprevalence data reveal that young adults are becoming more at risk of acquiring HAV infection. Thus the introduction of hepatitis A vaccine is highly recommended

Primary Human Immunodeficiency Virus Type 1-Specific CD8+ T-Cell Responses Induced by Myeloid Dendritic Cells▿

Induction of an antigenically broad and vigorous primary T-cell immune response by myeloid dendritic cells (DC) in blood and tissues could be important for an effective prophylactic or therapeutic vaccine to human immunodeficiency virus type 1 (HIV-1). Here we show that a primary CD8+ T-cell response can be induced by HIV-1 peptide-loaded DC derived from blood monocytes of HIV-1-negative adults and neonates (moDC) and by Langerhans cells (LC) and interstitial, dermal-intestinal DC (idDC) derived from CD34+ stem cells of neonatal cord blood. Optimal priming of single-cell gamma interferon (IFN-γ) production by CD8+ T cells required CD4+ T cells and was broadly directed to multiple regions of Gag, Env, and Nef that corresponded to known and predicted major histocompatibility complex class I epitopes. Polyfunctional CD8+ T-cell responses, defined as single-cell production of more than one cytokine (IFN-γ, interleukin 2, or tumor necrosis factor alpha), chemokine (macrophage inhibitory factor 1β), or cytotoxic degranulation marker CD107a, were primed by moDC, LC, and idDC to HIV-1 Gag and reverse transcriptase epitopes, as well as to Epstein-Barr virus and influenza A virus epitopes. Thus, three major types of blood and tissue myeloid DC targeted by HIV-1, i.e., moDC, LC, and idDC, can prime multispecific, polyfunctional CD8+ T-cell responses to HIV-1 and other viral antigens