Transmitted HIV Drug Resistance in Bulgaria Occurs in Clusters of Individuals from Different Transmission Groups and Various Subtypes (2012-2020).

Transmitted HIV drug resistance in Bulgaria was first reported in 2015 using data from 1988-2011. We determined the prevalence of surveillance drug resistance mutations (SDRMs) and HIV-1 genetic diversity in Bulgaria during 2012-2020 using polymerase sequences from 1053 of 2010 (52.4%) antiretroviral therapy (ART)-naive individuals. Sequences were analyzed for DRM using the WHO HIV SDRM list implemented in the calculated population resistance tool at Stanford University. Genetic diversity was inferred using automated subtyping tools and phylogenetics. Cluster detection and characterization was performed using MicrobeTrace. The overall rate of SDRMs was 5.7% (60/1053), with 2.2% having resistance to nucleoside reverse transcriptase inhibitors (NRTIs), 1.8% to non-nucleoside reverse transcriptase inhibitors (NNRTIs), 2.1% to protease inhibitors (PIs), and 0.4% with dual-class SDRMs. We found high HIV-1 diversity, with the majority being subtype B (60.4%), followed by F1 (6.9%), CRF02_AG (5.2%), A1 (3.7%), CRF12_BF (0.8%), and other subtypes and recombinant forms (23%). Most (34/60, 56.7%) of the SDRMs were present in transmission clusters of different subtypes composed mostly of male-to-male sexual contact (MMSC), including a 14-member cluster of subtype B sequences from 12 MMSC and two males reporting heterosexual contact; 13 had the L90M PI mutation and one had the T215S NRTI SDRM. We found a low SDRM prevalence amid high HIV-1 diversity among ART-naive patients in Bulgaria during 2012-2020. The majority of SDRMs were found in transmission clusters containing MMSC, indicative of onward spread of SDRM in drug-naive individuals. Our study provides valuable information on the transmission dynamics of HIV drug resistance in the context of high genetic diversity in Bulgaria, for the development of enhanced prevention strategies to end the epidemic.

Virus-Specific Stem Cell Memory CD8+ T Cells May Indicate a Long-Term Protection against Evolving SARS-CoV-2.

Immune memory to SARS-CoV-2 is key for establishing herd immunity and limiting the spread of the virus. The duration and qualities of T-cell-mediated protection in the settings of constantly evolving pathogens remain an open question. We conducted a cross-sectional study of SARS-CoV-2-specific CD4+ and CD8+ T-cell responses at several time points over 18 months (30-750 days) post mild/moderate infection with the aim to identify suitable methods and biomarkers for evaluation of long-term T-cell memory in peripheral blood. Included were 107 samples from 95 donors infected during the periods 03/2020-07/2021 and 09/2021-03/2022, coinciding with the prevalence of B.1.1.7 (alpha) and B.1.617.2 (delta) variants in Bulgaria. SARS-CoV-2-specific IFNγ+ T cells were measured in ELISpot in parallel with flow cytometry detection of AIM+ total and stem cell-like memory (TSCM) CD4+ and CD8+ T cells after in vitro stimulation with peptide pools corresponding to the original and delta variants. We show that, unlike IFNγ+ T cells, AIM+ virus-specific CD4+ and CD8+ TSCM are more adequate markers of T cell memory, even beyond 18 months post-infection. In the settings of circulating and evolving viruses, CD8+ TSCM is remarkably stable, back-differentiated into effectors, and delivers immediate protection, regardless of the initial priming strain.

Patterns of cytokine and chemokine expression in peripheral blood of patients with COVID-19 associated with disease severity.

OBJECTIVES: Cytokine dysregulation has been proposed as one of the main culprits for severe COVID-19 and poor prognosis. We examined the parallel presence of lymphopoietic, proinflammatory, Th1, Th2, regulatory cytokines, and chemokines in the serum of 47 patients with mild, moderate, and severe COVID-19 and evaluated the association between cytokine concentrations and disease severity. METHODS: A multiplex quantitative cytokine analysis ProcartaPlex™ immunoassay was applied, using the LuminexTM 200X detection system (Invitrogen). RESULTS: The concentrations of twelve cytokines: IL-18, IFN-gamma, TNF-alpha; IL-21; IL-1alpha, IL-1beta, IL-6, IL-22; IL-10, IL-1RA; IL-7 and IFN-alpha were consistently elevated in the studied serum samples. All examined chemokines-Eotaxin, GRO-alpha, IL-8, IP-10, MCP-1, MIP-1alpha, MIP-1beta, SDF-1alpha, and RANTES, were detectable in all studied groups, confirming their importance in mediating the adaptive immune response regardless of disease severity. The serum concentrations of six mediators: IL-1beta, IL-6, IL-18, IL-10, IL-8, and IP-10, showed statistically significant differences among the groups with different disease severity. IL-6, IL-1beta, and IL-10 were more significantly elevated in severe cases while milder symptoms were associated with lower levels of IL-8 and IP-10. CONCLUSION: Overall, the studied chemokines demonstrated an associated production in acute COVID-19 infection. A strong correlation was observed between the Th1 mediators IL-18 and IL-10 and the proinflammatory IL-6 in the severe COVID-19 group. Our results indicated that severe COVID-19 was characterized by a dysregulated cytokine pattern whereby the Th1 immune response is outweighed by the immunoregulatory response, while inhibitory signals cannot balance the hyperinflammatory response.

Eicosanoid and cytokine levels differentiate between stages of MTB infection.

Abstract.

Assessment of hepatitis E seropositivity among HIV-infected patients in Bulgaria.

It is debatable whether HIV-infected patients are at greater risk for hepatitis E virus (HEV) infection compared with healthy subjects. The reported anti-HEV seroprevalence among different groups in Bulgaria varied from 9.04% to 25.9%, but the information regarding the HIV population is still missing. The aim of the present study was to evaluate hepatitis E seroprevalence among HIV-infected patients in Bulgaria and to analyze demographic and immunological factors associated with HEV infection. Serum samples of 312 HIV-infected patients were analyzed retrospectively. Age, sex, residence and laboratory markers for HEV, HBV, HCV and HIV infection, and lymphocytes subpopulations were collected for all patients. None of the tested samples were positive for HEV RNA. HEV seroprevalence among HIV-infected patients was 10.9%. Males were more affected with the highest prevalence of positivity in the age group > 30 to ≤ 40 years. The documented HIV transmission routes in HIV/HEV co-infected group were heterosexual, homosexual, intravenous drug use (IDU), and vertical with predominace of the heterosexual route (z = 0.2; p = 0.804). There was a statistically significant trend of HIV mixed infection with routes of HIV transmission other than homosexual - heterosexual in HIV/HEV group and injection drug use in HIV/HBV/HCV co-infected group. The route of HIV transmission, in contexts of patients' behavior, was associated with HEV prevalence among HIV-infected patients.

Induction of humoral and cellular immune responses to COVID-19 mRNA and vector vaccines: A prospective cohort study in Bulgarian healthcare workers.

Installing efficient protective immunity by anti-SARS-CoV-2 vaccines is the only current means to overcome coronavirus disease 2019 pandemics. The cellular and humoral immune responses induced with an messenger RNA (mRNA) (BNT162b2) or with a vector (ChAdOx1nCoV-19) vaccine among Bulgarian healthcare workers (n = 123, aged 23-71 years) were studied in the course of 16 weeks after priming. Receptor-binding domain (RBD)-blocking Abs and SARS-CoV-2 RBD immunoglobulin A  (IgA) were evaluated in parallel with interferon gamma (IFNγ)-producing virus-specific T cells. Both vaccines induced RBD-blocking Abs in 100% of the participants after complete immunization while the levels of protection after a single dose largely varied (22%-98%). Advanced age had a negative impact on the level and longevity of virus-neutralizing activity induced by one dose mRNA, but not by the vector vaccine. RBD-binding IgA was detected in 100% of tested donors from the mRNA vaccine cohort, and in 67% of tested from the vector vaccine cohort, at least 1 month after completed immunization. One month after completing mRNA immunization, the number of IFNγ-producing T cells correlated significantly with the levels of RBD-specific IgA and virus-neutralizing activity induced after priming. Enumeration of circulating virus-specific IFNγ+ T cells is not recommended for evaluation of protective immunity as their detection may require longer stimulation beyond the firstmonth postimmunization. In conclusion, BNT162B2 and ChAdOx1nCoV-19 induced potent and comparable humoral and cellular anti-SARS-CoV-2 immune responses, peaking between 10 and 30 days after complete immunization. A single dose of any vaccine did not induce adequate protection in a great part of donors, making the shorter interval between mRNA vaccine doses preferable in the settings of increased risk of infection.

Assessment of hepatitis E seropositivity among HIV-infected patients in Bulgaria

Abstract It is debatable whether HIV-infected patients are at greater risk for hepatitis E virus (HEV) infection compared with healthy subjects. The reported anti-HEV seroprevalence among different groups in Bulgaria varied from 9.04% to 25.9%, but the information regarding the HIV population is still missing. The aim of the present study was to evaluate hepatitis E seroprevalence among HIV-infected patients in Bulgaria and to analyze demographic and immunological factors associated with HEV infection. Serum samples of 312 HIV-infected patients were analyzed retrospectively. Age, sex, residence and laboratory markers for HEV, HBV, HCV and HIV infection, and lymphocytes subpopulations were collected for all patients. None of the tested samples were positive for HEV RNA. HEV seroprevalence among HIV-infected patients was 10.9%. Males were more affected with the highest prevalence of positivity in the age group > 30 to ≤ 40 years. The documented HIV transmission routes in HIV/HEV co-infected group were heterosexual, homosexual, intravenous drug use (IDU), and vertical with predominace of the heterosexual route (z = 0.2; p = 0.804). There was a statistically significant trend of HIV mixed infection with routes of HIV transmission other than homosexual - heterosexual in HIV/HEV group and injection drug use in HIV/HBV/HCV co-infected group. The route of HIV transmission, in contexts of patients' behavior, was associated with HEV prevalence among HIV-infected patients.

Allergen-Specific IgE and IgG4 as Biomarkers for Immunologic Changes during Subcutaneous Allergen Immunotherapy.

(1) Background: Biomarkers of efficacy for subcutaneous immunotherapy (SCIT) on allergic rhinitis have not been evaluated in details. The present study aims to assess the relevance of measuring of sIgE, sIgG4 and IgE/IgG4 ratio during SCIT in patients with allergic rhinitis; (2) Methods: 20 patients, 13 men and 7 women aged 19 to 58 years, with clinically manifested seasonal and perennial allergic rhinitis were studied. At the initiation and in the end of the three-year course of SCIT serum allergen-specific IgE and IgG4 were measured with ImmunoCAP system. The sIgE/sIgG4 ratio was calculated as a biomarker for immunologic effectiveness; (3) Results: There was a significant increase of sIgG4 antibodies (p < 0.05), while at the end of SCIT for the sIgE levels no significant changes were seen (p > 0.05). Moreover, 90% of patients showed a decrease of the IgE/IgG4 ratio; (4) Conclusions: In most of treated patients with AR, SCIT with Bulgarian allergen products leads to clear immunological changes. After a 3-year of SCIT there is a significant increase in allergen specific IgG4 levels and both decrease of sIgE and IgE/IgG4 ratio. sIgE, sIgG4 and IgE/IgG4 ratio can be used as a substantial biomarker for predicting immunological effectiveness of SCIT.

Short Communication: Elevated Labile Iron Levels in CD4 and CD8 T Cells from HIV-Positive Individuals with Undetectable Viral Load.

Iron is a key factor at various stages of HIV life cycle and determines the progression of HIV infection. Data about cellular labile iron pool (LIP) in the settings of contemporary antiretroviral therapy (cART) are lacking. Yet LIP is directly related to the generation of reactive oxygen species, and may contribute to immune activation, dysfunction, and exhaustion. Using multiparameter flow cytometry, we evaluated LIP in CD4 and CD8 T cells from HIV+ patients with sustained viral suppression (SVS) as a result of continuous long-term cART. Based on the recovery of CD4/CD8 ratio, two patients' subgroups were defined: A (n = 26), CD4/CD8 > 0.9, and B (n = 37), CD4/CD8 < 0.9, with significantly differing CD4 absolute count (AC) (mean 752 vs. 571 cells/µL, p < .05). Although hemoglobin and serum iron had recovered in all patients, CD4 T cell LIP and CD8 T cell LIP were significantly higher than that of controls, both in the subgroup with complete (A) and with incomplete (B) immune recovery [mean CD4 mean fluorescence intensity (ΔMFI) 318.7 and 777.8 vs. 157.6; mean CD8 ΔMFI 359.5 and 628.7 vs. 179.2, analysis of variance p < .05 for both]. CD4 LIP correlated inversely with CD4 AC (R = -0.4, p < .01), and both CD4 LIP and CD8 LIP-with CD4/CD8 ratio (R = -0.4, p < .01). Thus, increased CD4 T cell LIP and CD8 T cell LIP in the settings of SVS and immune recovery are a sensitive marker of residual immune activation and may predict immune exhaustion in long-term cART-treated patients.

Cystathionine gamma-lyase of perivascular adipose tissue with reversed regulatory effect in diabetic rat artery.

The aim of this study is to reveal the regulatory role of cystathionine gamma-lyase (CSE), the main source of hydrogen sulphide (H2S) in perivascular adipose tissue (PVAT), of diabetic rats. Diabetes was induced in male rats by a single intraperitoneal injection of streptozotocin. Animals with glucose levels above 20 mmol/L were determined as diabetic. The rat gracilis arteries (a. gracilis) were dissected with or without PVAT. In all in vitro experiments endothelium-denuded preparations were used for isometric contraction measurements. Increasing concentrations of 5-hydroxytryptamine (5-HT) from 10-10 to 10-5 mol/L were applied to induce gradual increase in force of contractions of circular artery segments. The relaxing effect of CSE was inhibited by DL-propargyl glycine (PGG). The presence of PVAT decreases the contractile response to 5-HT of a. gracilis from control rats. This response is reversed in contraction studies in the same rat artery from diabetic rats. DL-PPG (1 mmol/L) induced significant increase of the force of contraction in artery preparations with PVAT from control rats in the whole range of 5-HT. In contrast, PGG had a relaxing effect in high concentrations of 5-HT (10-6 and 10-5 mol/L) in diabetic rat arteries with PVAT. It is concluded that in skeletal muscle artery from diabetic rats, a mediator related to H2S is released from PVAT. This paracrine mediator increases the maximal force of contraction of endothelium-denuded preparations at higher concentrations of 5-HT.