Five-year single-center analysis of cytomegalovirus viremia in kidney transplant recipients and possible implication for novel prophylactic therapy approaches.

BACKGROUND: Cytomegalovirus (CMV) infections are a common complication after kidney transplantation (KTx) and negatively affecting patient outcome. Valganciclovir (VGC) prophylaxis is often limited by drug-induced side effects and dose reduction due to decline in kidney function. METHOD: In the present study, episodes of CMV viremia in the first year after KTx in a cohort of 316 recipients were analyzed retrospectively to identify risk factors linked to persistent infections. RESULTS: In the studied cohort, 18.7% of patients showed a high-risk (HR) constellation (D+/R-) for CMV infections. CMV viremia affected 22% of our cohort, with HR patients being the most affected cohort (44.1%). Within this group, most viremic events (65.3%) occurred while patients were still on prophylactic therapy, showing significantly higher viral loads and a longer duration compared to seropositive recipients. CONCLUSION: The analysis at hand revealed that detection of viremia under ongoing antiviral prophylaxis bears an increased risk for sustained viral replication and antiviral drug resistance in HR patients. We identified low estimated glomerular filtration rate (eGFR) and lower dose VGC prophylaxis post-KTx as a risk factor for breakthrough infections in HR patients in our single center cohort. These patients might benefit from a closer CMV monitoring or novel prophylactic agents as letermovir.

Dynamics and durability of HIV-1 neutralization are determined by viral replication.

Human immunodeficiency virus type 1 (HIV-1)-neutralizing antibodies (nAbs) that prevent infection are the main goal of HIV vaccine discovery. But as no nAb-eliciting vaccines are yet available, only data from HIV-1 neutralizers-persons with HIV-1 who naturally develop broad and potent nAbs-can inform about the dynamics and durability of nAb responses in humans, knowledge which is crucial for the design of future HIV-1 vaccine regimens. To address this, we assessed HIV-1-neutralizing immunoglobulin G (IgG) from 2,354 persons with HIV-1 on or off antiretroviral therapy (ART). Infection with non-clade B viruses, CD4+ T cell counts <200 µl-1, being off ART and a longer time off ART were independent predictors of a more potent and broad neutralization. In longitudinal analyses, we found nAb half-lives of 9.3 and 16.9 years in individuals with no- or low-level viremia, respectively, and 4.0 years in persons who newly initiated ART. Finally, in a potent HIV-1 neutralizer, we identified lower fractions of serum nAbs and of nAb-encoding memory B cells after ART initiation, suggesting that a decreasing neutralizing serum activity after antigen withdrawal is due to lower levels of nAbs. These results collectively show that HIV-1-neutralizing responses can persist for several years, even at low antigen levels, suggesting that an HIV-1 vaccine may elicit a durable nAb response.

Torque teno virus-DNA load as individual cytomegalovirus risk assessment parameter upon allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Immune recovery following allogeneic hematopoietic stem cell transplantation (allo-HSCT) decisively influences the occurrence of opportunistic infections, one of the leading causes of death among this group of patients. Yet, today, there are no laboratory parameters mirroring immune function sufficiently. Torque teno virus (TTV) has already proven itself as a functional immune marker in other settings. AIMS: In this analysis, we investigated whether monitoring of TTV-DNA load in whole blood is able to provide additional information on the capacity of the immune system to control cytomegalovirus (CMV) replication in allo-HSCT recipients. METHODS: Whole blood samples from 59 patients were collected upon allo-HSCT (between Day -7 and +10), on Day +14, +21, +28, +56, +90, and +365 post-transplant. TTV-DNA loads and other relevant clinical information were correlated with the risk of CMV infections or reactivations, defined by evidence of viral replication in blood. RESULTS: CMV serostatus of the recipient and a TTV load below 1000 copies/mL upon allo-HSCT were significantly associated with an increased incidence of CMV infection or reactivation. CONCLUSIONS: Quantification of TTV load in the early phase of allo-HSCT procedure could provide additional information in order to identify patients at risk for CMV infection or reactivation.

Optimizing Antiretroviral Therapy in Heavily ART-Experienced Patients with Multi-Class Resistant HIV-1 Using Proviral DNA Genotypic Resistance Testing.

Resistance to multiple antiretroviral drugs among people living with HIV (PLWH) can result in a high pill burden, causing toxicity and drug interactions. Thus, the goal is to simplify treatment regimens while maintaining effectiveness. However, former resistance analysis data may not be current or complete. The use of proviral DNA genotyping may assist in selecting appropriate treatment options. A retrospective study was carried out on individuals belonging to the Cologne HIV cohort with a resistance history to two or more antiretroviral (ARV) classes and on non-standard antiretroviral therapy (ART). Patients required former viral RNA and a recent proviral DNA resistance test to be available prior to the switch to ART. Potential discrepancies between resistance test results obtained through RNA and proviral DNA methods and the consequent virological and clinical outcomes following ART adjustments were analyzed. Out of 1250 patients, 35 were eligible for inclusion in this study. The median length of known HIV infection was 27 years, and the median duration of ART was 22 years. Of the 35 participants, 16 had received all five ARV classes. Based on proviral DNA genotyping results, ART was simplified in 17 patients. At the last follow-up examination after changing therapy, 15 patients had HIV RNA <50 copies/mL (median 202 days, range 21-636). The mean number of pills per day decreased from eight to three, and the median intake frequency decreased from two to one time/day (ranges 1-2). Our study supports the use of proviral DNA genotyping as a safe strategy for switching to simplified ART regimens. However, the lack of extensive research on the advantages of proviral DNA genotyping makes it challenging to fully assess its benefits in terms of treatment selection.

CMV-IgG pre-allogeneic hematopoietic stem cell transplantation and the risk for CMV reactivation and mortality.

Cytomegalovirus (CMV) represents one of the most common infectious complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Currently, a common diagnostic test used to stratify the risk for CMV infection in allo-HSCT recipients is the qualitative CMV serology of donor and recipient. A positive serostatus of the recipient is the most important risk factor for CMV reactivation and associated with reduced overall survival post-transplantation (TX). Direct and indirect effects of CMV are involved in the poorer survival outcome. The present study investigated if the quantitative interpretation of anti-CMV IgG before allo-HSCT might serve as a novel parameter for the identification of patients at risk for CMV reactivation and worse outcome post-TX. For this purpose, a cohort of 440 allo-HSCT recipients over a period of 10 years was retrospectively analyzed. Our findings indicated that patients with high CMV IgG pre-allo-HSCT had a higher risk to develop CMV reactivation, including clinically relevant infections, and a worse prognosis 36 months post-allo-HSCT as compared to recipients with low CMV IgG values. In the letermovir (LMV) era, this group of patients might benefit from a closer CMV monitoring, and hence, earlier intervention if needed, especially after discontinuation of prophylaxis.

Immune response to mRNA-based COVID-19 booster vaccination in people living with HIV.

OBJECTIVES: Our objective was to assess immune responses and their influencing factors in people living with HIV after messenger RNA (mRNA)-based COVID-19 booster vaccination (third dose). METHODS: This was a retrospective cohort study of people living with HIV who received booster vaccination with BNT-162b2 or mRNA-1273 between October 2021 and January 2022. We assessed anti-spike receptor-binding domain (RBD) immunoglobulin G (IgG), virus neutralizing activity (VNA) titres reported as 100% inhibitory dilution (ID100 ), and T-cell response (using interferon-gamma-release-assay [IGRA]) at baseline and quarterly follow-up visits. Patients with reported COVID-19 during follow-up were excluded. Predictors of serological immune response were analyzed using multivariate regression models. RESULTS: Of 84 people living with HIV who received an mRNA-based booster vaccination, 76 were eligible for analysis. Participants were on effective antiretroviral therapy (ART) and had a median of 670 CD4+ cells/µL (interquartile range [IQR] 540-850). Following booster vaccination, median anti-spike RBD IgG increased by 705.2 binding antibody units per millilitre (BAU/mL) and median VNA titres increased by 1000 ID100 at the follow-up assessment (median 13 weeks later). Multivariate regression revealed that time since second vaccination was a predictor of stronger serological responses (p < 0.0001). No association was found for other factors, including CD4+ status, choice of mRNA vaccine, or concomitant influenza vaccination. In total, 45 patients (59%) had a reactive baseline IGRA, of whom two lost reactivity during follow-up. Of 31 patients (41%) with non-reactive baseline IGRA, 17 (55%) converted to reactive and seven (23%) remained unchanged following booster vaccination. CONCLUSIONS: People living with HIV with ≥500 CD4+ cells/µL showed favourable immune responses to mRNA-based COVID-19 booster vaccination. A longer time (up to 29 weeks) since second vaccination was associated with higher serological responses, whereas choice of mRNA vaccine or concomitant influenza vaccination had no impact.

In-depth virological and immunological characterization of HIV-1 cure after CCR5Δ32/Δ32 allogeneic hematopoietic stem cell transplantation.

Despite scientific evidence originating from two patients published to date that CCR5Δ32/Δ32 hematopoietic stem cell transplantation (HSCT) can cure human immunodeficiency virus type 1 (HIV-1), the knowledge of immunological and virological correlates of cure is limited. Here we characterize a case of long-term HIV-1 remission of a 53-year-old male who was carefully monitored for more than 9 years after allogeneic CCR5Δ32/Δ32 HSCT performed for acute myeloid leukemia. Despite sporadic traces of HIV-1 DNA detected by droplet digital PCR and in situ hybridization assays in peripheral T cell subsets and tissue-derived samples, repeated ex vivo quantitative and in vivo outgrowth assays in humanized mice did not reveal replication-competent virus. Low levels of immune activation and waning HIV-1-specific humoral and cellular immune responses indicated a lack of ongoing antigen production. Four years after analytical treatment interruption, the absence of a viral rebound and the lack of immunological correlates of HIV-1 antigen persistence are strong evidence for HIV-1 cure after CCR5Δ32/Δ32 HSCT.

Implementing the Lolli-Method and pooled RT-qPCR testing for SARS-CoV-2 surveillance in schools: a pilot project.

PURPOSE: School closures have been used as part of lockdown strategies to contain the spread of SARS-CoV-2, adversely affecting children's health and education. To ensure the accessibility of educational institutions without exposing society to the risk of increased transmissions, it is essential to establish SARS-CoV-2 testing strategies that are child-friendly, scalable and implementable in a daily school routine. Self-sampling using non-invasive saliva swabs combined with pooled RT-qPCR testing (Lolli-Method) has been proven to be a sensitive method for the detection of SARS-CoV-2. METHODS: We conducted a pilot project in Cologne, Germany, designed to determine the feasibility of a large-scale rollout of the Lolli-Method for testing without any additional on-site medical staff in schools. Over a period of three weeks, students from 22 schools were sampled using the Lolli-Method. At the end of the project, teachers were asked to evaluate the overall acceptance of the project. RESULTS: We analyzed a total of 757 pooled RT-qPCRs obtained from 8,287 individual swabs and detected 7 SARS-CoV-2 infected individuals. The Lolli-Method was shown to be a feasible and accepted testing strategy whose application is only slightly disruptive to the daily school routine. CONCLUSION: Our observations suggest that the Lolli-Method in combination with pooled RT-qPCR can be implemented for SARS-CoV-2 surveillance in daily school routine, applicable on a large scale.

Effective high-throughput RT-qPCR screening for SARS-CoV-2 infections in children.

Systematic SARS-CoV-2 testing is a valuable tool for infection control and surveillance. However, broad application of high sensitive RT-qPCR testing in children is often hampered due to unpleasant sample collection, limited RT-qPCR capacities and high costs. Here, we developed a high-throughput approach ('Lolli-Method') for SARS-CoV-2 detection in children, combining non-invasive sample collection with an RT-qPCR-pool testing strategy. SARS-CoV-2 infections were diagnosed with sensitivities of 100% and 93.9% when viral loads were >106 copies/ml and >103 copies/ml in corresponding Naso-/Oropharyngeal-swabs, respectively. For effective application of the Lolli-Method in schools and daycare facilities, SEIR-modeling indicated a preferred frequency of two tests per week. The developed test strategy was implemented in 3,700 schools and 698 daycare facilities in Germany, screening over 800,000 individuals twice per week. In a period of 3 months, 6,364 pool-RT-qPCRs tested positive (0.64%), ranging from 0.05% to 2.61% per week. Notably, infections correlated with local SARS-CoV-2 incidences and with a school social deprivation index. Moreover, in comparison with the alpha variant, statistical modeling revealed a 36.8% increase for multiple (≥2 children) infections per class following infections with the delta variant. We conclude that the Lolli-Method is a powerful tool for SARS-CoV-2 surveillance and can support infection control in schools and daycare facilities.

CXCR3 Expression Pattern on CD4+ T Cells and IP-10 Levels with Regard to the HIV-1 Reservoir in the Gut-Associated Lymphatic Tissue.

(1) Background: The gut-associated lymphatic tissue (GALT) represents the largest lymphoid organ, and is considered to be the largest HIV reservoir. The exact size of the GALT reservoir remains unclear. Several markers, such as the chemokine receptor CXCR3 and its pro-inflammatory ligand IP-10, have been proposed to define the size of HIV reservoirs in the peripheral blood (PB). However, little is known about the role of CXCR3 and IP-10 within the GALT. (2) Methods: We compared the CXCR3 expression, IP-10 levels, and cell-associated HIV DNA of distinct memory CD4+ T cell subsets from the terminal ileum (TI), PB and rectum (RE) of 18 HIV+ patients with antiretroviral therapy (ART), 6 HIV+ treatment-naive patients and 16 healthy controls. (3) Results: While the relative distributions of CD4+ T cell subsets were similar in PB, TI and RE, HIV DNA and CXCR3 expression were markedly increased and IP-10 levels were decreased in TI when compared to PB. No significant correlation was found between the CXCR3 expression and memory CD4+ T cell subsets, IP-10 levels and the HIV DNA amounts measured in PB, TI or RE. (4) Conclusions: During a chronic HIV-1 infection, neither CXCR3 nor IP-10 are indicative of the size of the viral reservoir in the GALT (TI and RE).

Analysis of antibodies from HCV elite neutralizers identifies genetic determinants of broad neutralization.

The high genetic diversity of hepatitis C virus (HCV) complicates effective vaccine development. We screened a cohort of 435 HCV-infected individuals and found that 2%-5% demonstrated outstanding HCV-neutralizing activity. From four of these patients, we isolated 310 HCV antibodies, including neutralizing antibodies with exceptional breadth and potency. High neutralizing activity was enabled by the use of the VH1-69 heavy-chain gene segment, somatic mutations within CDRH1, and CDRH2 hydrophobicity. Structural and mutational analyses revealed an important role for mutations replacing the serines at positions 30 and 31, as well as the presence of neutral and hydrophobic residues at the tip of the CDRH3. Based on these characteristics, we computationally created a de novo antibody with a fully synthetic VH1-69 heavy chain that efficiently neutralized multiple HCV genotypes. Our findings provide a deep understanding of the generation of broadly HCV-neutralizing antibodies that can guide the design of effective vaccine candidates.

Characterization of HIV-1 drug resistance among patients with failure of second-line combined antiretroviral therapy in central Ethiopia.

BACKGROUND: As a consequence of the improved availability of combined antiretroviral therapy (cART) in resource-limited countries, an emergence of HIV drug resistance (HIVDR) has been observed. We assessed the prevalence and spectrum of HIVDR in patients with failure of second-line cART at two HIV clinics in central Ethiopia. METHODS: HIV drug resistance was analysed in HIV-1-infected patients with virological failure of second-line cART using the geno2pheno application. RESULTS: Among 714 patients receiving second-line cART, 44 (6.2%) fulfilled the criteria for treatment failure and 37 were eligible for study inclusion. Median age was 42 years [interquartile range (IQR): 20-45] and 62.2% were male. At initiation of first-line cART, 23 (62.2%) were WHO stage III, mean CD4 cell count was 170.6 (range: 16-496) cells/µL and median (IQR) HIV-1 viral load was 30 220 (7963-82 598) copies/mL. Most common second-line cART regimens at the time of failure were tenofovir disoproxil fumarate (TDF)-lamivudine (3TC)-ritonavir-boosted atazanavir (ATV/r) (19/37, 51.4%) and zidovudine (ZDV)-3TC-ATV/r (9/37, 24.3%). Genotypic HIV-1 resistance testing was successful in 35 (94.6%) participants. We found at least one resistance mutation in 80% of patients and 40% carried a protease inhibitor (PI)-associated mutation. Most common mutations were M184V (57.1%), Y188C (25.7%), M46I/L (25.7%) and V82A/M (25.7%). High-level resistance against the PI ATV (10/35, 28.6%) and lopinavir (LPV) (5/35, 14.3%) was reported. As expected, no resistance mutations conferring integrase inhibitor resistance were detected. CONCLUSIONS: We found a high prevalence of resistance mutations, also against PIs (40%), as the national standard second-line cART components. Resistance testing before switching to second- or third-line cART is warranted.

Safe and effective pool testing for SARS-CoV-2 detection.

OBJECTIVES: The global spread of SARS-CoV-2 is a serious public health issue. Large-scale surveillance screenings are crucial but can exceed test capacities. We (A) optimized test conditions and (B) implemented pool testing of respiratory swabs into SARS-CoV-2 diagnostics. STUDY DESIGN: (A) We determined the optimal pooling strategy and pool size. In addition, we measured the impact of vortexing prior to sample processing, compared a pipette-pooling method (by combining transport medium of several specimens) and a swab-pooling method (by combining several swabs into a test tube filled with PBS) as well as determined the sensitivities of three PCR assays. (B) Finally, we applied high-throughput pool testing for diagnostics. RESULTS: (A) In a low prevalence setting, we defined a preferable pool size of ten in a two-stage hierarchical pool testing strategy. Vortexing of swabs (n = 33) increased cellular yield by a factor of 2.34. By comparing Ct-values of 16 pools generated with two different pooling strategies, pipette-pooling was more efficient compared to swab-pooling. Measuring dilution series of 20 SARS-CoV-2 positive samples in three PCR assays simultaneously revealed detection rates of 85% (assay I), 50% (assay II), and 95% (assay III) at a 1:100 dilution. (B) We systematically pooled 55,690 samples in a period of 44 weeks resulting in a reduction of 47,369 PCR reactions. CONCLUSIONS: For implementing pooling strategies into high-throughput diagnostics, we recommend utilizing a pipette-pooling method, performing sensitivity validation of the PCR assays used, and vortexing swabs prior to analyses. Pool testing for SARS-CoV-2 detection is feasible and effective in a low prevalence setting.

Combined Therapy with Intravenous Immunoglobulins, Letermovir and (Val-)Ganciclovir in Complicated Courses of CMV-Infection in Transplant Recipients.

The treatment options for cytomegalovirus (CMV) infections in immunosuppressed patients are limited, mainly consisting of (val-)ganciclovir (VGC/GCV) as the first-line treatment. We report on three transplant recipients, one stem cell transplant (allo-HSCT) patient and two kidney transplant (KTx) recipients, with prolonged CMV viremia treated with a combined therapy based on letermovir (LMV), CMV-specific intravenous immunoglobulins (IVIg), and VGC/GCV, which led to the sustained control of CMV viremia in all patients.

RNAemia Corresponds to Disease Severity and Antibody Response in Hospitalized COVID-19 Patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a global health emergency. To improve the understanding of the systemic component of SARS-CoV-2, we investigated if viral load dynamics in plasma and respiratory samples are associated with antibody response and severity of coronavirus disease 2019 (COVID-19). SARS-CoV-2 RNA was found in plasma samples from 14 (44%) out of 32 patients. RNAemia was detected in 5 out of 6 fatal cases. Peak IgG values were significantly lower in mild/moderate than in severe (0.6 (interquartile range, IQR, 0.4-3.2) vs. 11.8 (IQR, 9.9-13.0), adjusted p = 0.003) or critical cases (11.29 (IQR, 8.3-12.0), adjusted p = 0.042). IgG titers were significantly associated with virus Ct (Cycle threshold) value in plasma and respiratory specimens ((ß = 0.4, 95% CI (confidence interval, 0.2; 0.5), p < 0.001 and ß = 0.5, 95% CI (0.2; 0.6), p = 0.002). A classification as severe or a critical case was additionally inversely associated with Ct values in plasma in comparison to mild/moderate cases (ß = -3.3, 95% CI (-5.8; 0.8), p = 0.024 and ß = -4.4, 95% CI (-7.2; 1.6), p = 0.007, respectively). Based on the present data, our hypothesis is that the early stage of SARS-CoV-2 infection is characterized by a primary RNAemia, as a potential manifestation of a systemic infection. Additionally, the viral load in plasma seems to be associated with a worse disease outcome.

Drug Resistance Spread in 6 Metropolitan Regions, Germany, 2001-20181.

We analyzed 1,397 HIV-1 pol sequences of antiretroviral therapy-naive patients in a total of 7 university hospitals in Bonn, Cologne, Frankfurt, Hamburg, Hannover, and Munich, Germany. Phylogenetic and network analysis elucidated numerous cases of shared drug resistance mutations among genetically linked patients; K103N was the most frequently shared mutation.

Vulnerability to reservoir reseeding due to high immune activation after allogeneic hematopoietic stem cell transplantation in individuals with HIV-1.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only medical intervention that has led to an HIV cure. Whereas the HIV reservoir sharply decreases after allo-HSCT, the dynamics of the T cell reconstitution has not been comprehensively described. We analyzed the activation and differentiation of CD4+ and CD8+ T cells, and the breadth and quality of HIV- and CMV-specific CD8+ T cell responses in 16 patients with HIV who underwent allo-HSCT (including five individuals who received cells from CCR5Δ32/Δ32 donors) to treat their underlying hematological malignancy and who remained on antiretroviral therapy (ART). We found that reconstitution of the T cell compartment after allo-HSCT was slow and heterogeneous with an initial expansion of activated CD4+ T cells that preceded the expansion of CD8+ T cells. Although HIV-specific CD8+ T cells disappeared immediately after allo-HSCT, weak HIV-specific CD8+ T cell responses were detectable several weeks after transplant and could still be detected at the time of full T cell chimerism, indicating that de novo priming, and hence antigen exposure, occurred during the time of T cell expansion. These HIV-specific T cells had limited functionality compared with CMV-specific CD8+ T cells and persisted years after allo-HSCT. In conclusion, immune reconstitution was slow, heterogeneous, and incomplete and coincided with de novo detection of weak HIV-specific T cell responses. The initial short phase of high T cell activation, in which HIV antigens were present, may constitute a window of vulnerability for the reseeding of viral reservoirs, emphasizing the importance of maintaining ART directly after allo-HSCT.

HIV-1 Sub-Subtype A6: Settings for Normalised Identification and Molecular Epidemiology in the Southern Federal District, Russia.

Russia has one of the largest and fastest growing HIV epidemics. However, epidemiological data are scarce. Sub-subtype A6 is most prevalent in Russia but its identification is challenging. We analysed protease/reverse transcriptase-, integrase-sequences, and epidemiological data from 303 patients to develop a methodology for the systematisation of A6 identification and to describe the HIV epidemiology in the Russian Southern Federal District. Drug consumption (32.0%) and heterosexual contact (27.1%) were the major reported transmission risks. This study successfully established the settings for systematic identification of A6 samples. Low frequency of subtype B (3.3%) and large prevalence of sub-subtype A6 (69.6%) and subtype G (23.4%) were detected. Transmitted PI- (8.8%) and NRTI-resistance (6.4%) were detected in therapy-naive patients. In therapy-experienced patients, 17.3% of the isolates showed resistance to PIs, 50.0% to NRTI, 39.2% to NNRTIs, and 9.5% to INSTIs. Multiresistance was identified in 52 isolates, 40 corresponding to two-class resistance and seven to three-class resistance. Two resistance-associated-mutations significantly associated to sub-subtype A6 samples: A62VRT and G190SRT. This study establishes the conditions for a systematic annotation of sub-subtype A6 to normalise epidemiological studies. Accurate knowledge on South Russian epidemiology will allow for the development of efficient regional frameworks for HIV-1 infection management.