Treatment with integrase inhibitors alters SARS-CoV-2 neutralization levels measured with HIV-based pseudotypes in people living with HIV.

The presence of neutralizing antibodies (NAbs) is a major correlate of protection for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Thus, different in vitro pseudoviruses-based assays have been described to detect NAbs against SARS-CoV-2. However, the determination of NAbs against SARS-CoV-2 in people living with HIV (PLWH) through HIV-based pseudoparticles could be influenced by cross-neutralization activity or treatment, impeding accurate titration of NAbs. Two assays were compared using replication-defective HIV or VSV-based particles pseudotyped with SARS-CoV-2 spike to measure NAbs in COVID-19-recovered and COVID-19-naïve PLWH. The assay based on HIV-pseudoparticles displayed neutralization activity in all COVID-19-recovered PLWH with a median neutralizing titer 50 (NT50) of 1417.0 (interquartile range [IQR]: 450.3-3284.0), but also in 67% of COVID-19-naïve PLWH (NT50: 631.5, IQR: 16.0-1535.0). Regarding VSV-pseudoparticles system, no neutralization was observed in COVID-19-naïve PLWH as expected, whereas in comparison with HIV-pseudoparticles assay lower neutralization titers were measured in 75% COVID-19-recovered PLWH (NT50: 100.5; IQR: 20.5-1353.0). Treatment with integrase inhibitors was associated with inaccurate increase in neutralization titers when HIV-based pseudoparticles were used. IgG purification and consequent elimination of drugs from samples avoided the interference with retroviral cycle and corrected the lack of specificity observed in HIV-pseudotyped assay. This study shows methodological alternatives based on pseudoviruses systems to determine specific SARS-CoV-2 neutralization titers in PLWH.

Longer intervals between SARS-CoV-2 infection and mRNA-1273 doses improve the neutralization of different variants of concern.

The humoral immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern elicited by vaccination was evaluated in COVID-19 recovered individuals (Rec) separated 1-3 months (Rec2m) or 4-12 months (Rec9m) postinfection and compared to the response in naïve participants. Antibody-mediated immune responses were assessed in 66 participants by three commercial immunoassays and a SARS-CoV-2 lentiviral-based pseudovirus neutralization assay. Immunoglobulin (Ig) levels against SARS-CoV-2 spike were lower in naïve participants after two doses than in Rec after a single dose (p < 0.05). After two doses in Rec, levels of total Ig to receptor-binding domain were significantly increased in Rec9m compared to Rec2m (p < 0.001). The neutralizing potency observed in Rec9m was consistently higher than in Rec2m against variants of concern (VOCs) Alpha, Beta, Delta, and BA.1 sublineage of Omicron with 2.2-2.8-fold increases. Increasing the interval between SARS-CoV-2 infection and the vaccination with messenger RNA-based vaccines to more than 3 months generates a more efficient heterologous humoral immune response against VOCs by allowing enough time to mount a strong recall memory B cell response.

Deep-Sequencing Analysis of the Dynamics of HIV-1 Quasiespecies in Naive Patients during a Short Exposure to Maraviroc.

In this study, we have characterized quasispecies dynamics and the evolution of viral tropism in naive HIV-1-infected patients treated with a short course of maraviroc monotherapy (ClinicalTrials.gov registration no. NCT01060618) independently of the tropism of the infecting virus. We randomly selected 20 patients infected with viruses displaying different basal tropisms-10 carrying R5 and 10 carrying dual/mixed X4 (DM/X4) viruses-at recruitment as determined by phenotypic assay (Trofile). Evolution of viral quasiespecies at the end of treatment was determined by ultradeep sequencing of the V3 region using a 454 Life Sciences Platform and geno2pheno (g2p) algorithm for viral tropism prediction. The false-positive rate (FPR) that defines the probability of classifying an R5 virus falsely as X4 was set at 10%. X4-specific HIV-1 viral load (VL) was calculated from sequences with an FPR of <3.75%. Virological response as defined as >1-log10 copies/ml reduction in VL was detected in 70% of patients independently of the basal tropism of the infecting virus. Viral tropism remained stable, and nonsignificant differences in FPR values before and after treatment were found for the majority of patients in both tropism groups. Only three patients (one with R5 and two with DM/X4 viruses) showed an increased (>1 log) X4 VL, and one patient harboring a DM/X4-tropic virus displayed a significant reduction in FPR values at the end of treatment. Fast changes in the composition of viral populations were observed in all patients after 10 days of maraviroc (MVC) monotherapy treatment, and a complete replacement of viral quasiespecies was found in 3/10 patients carrying R5-using viruses and 4/10 patients carrying DM/X4-using viruses.IMPORTANCE Initiation of treatment with maraviroc requires previous determination of viral tropism by genotypic or phenotypic methods because of the risk of treatment failure and selection of DM/X4-tropic variants. In this study, we confirm previous work showing that the virologic response to maraviroc is independent of basal tropism. By deep-sequencing analysis, we determined that fast changes in viral populations were due to the emergence of minority variants in some patients whereas in others generation of new strains was detected. The risk of DM/X4 selection was very low as FPR values remained stable, and only one patient showed a detrimental switch to DM/X4 variants. Our data show that some DM/X4 viruses are sensitive to maraviroc treatment probably because only a low proportion of DM/X4 viruses use preferentially the X4 receptor and contain authentically maraviroc-resistant viruses that are not accurately detected by current assays.

A single-residue change in the HIV-1 V3 loop associated with maraviroc resistance impairs CCR5 binding affinity while increasing replicative capacity.

BACKGROUND: Maraviroc (MVC) is an allosteric CCR5 inhibitor used against HIV-1 infection. While MVC-resistant viruses have been identified in patients, it still remains incompletely known how they adjust their CD4 and CCR5 binding properties to resist MVC inhibition while preserving their replicative capacity. It is thought that they maintain high efficiency of receptor binding. To date however, information about the binding affinities to receptors for inhibitor-resistant HIV-1 remains limited. RESULTS: Here, we show by means of viral envelope (gp120) binding experiments and virus-cell fusion kinetics that a MVC-resistant virus (MVC-Res) that had emerged as a dominant viral quasispecies in a patient displays reduced affinities for CD4 and CCR5 either free or bound to MVC, as compared to its MVC-sensitive counterpart isolated before MVC therapy. An alanine insertion within the GPG motif (G310_P311insA) of the MVC-resistant gp120 V3 loop is responsible for the decreased CCR5 binding affinity, while impaired binding to CD4 is due to sequence changes outside V3. Molecular dynamics simulations of gp120 binding to CCR5 further emphasize that the Ala insertion alters the structure of the V3 tip and weakens interaction with CCR5 ECL2. Paradoxically, infection experiments on cells expressing high levels of CCR5 also showed that Ala allows MVC-Res to use CCR5 efficiently, thereby improving viral fusion and replication efficiencies. Actually, although we found that the V3 loop of MVC-Res is required for high levels of MVC resistance, other regions outside V3 are sufficient to confer a moderate level of resistance. These sequence changes outside V3, however, come with a replication cost, which is compensated for by the Ala insertion in V3. CONCLUSION: These results indicate that changes in the V3 loop of MVC-resistant viruses can augment the efficiency of CCR5-dependent steps of viral entry other than gp120 binding, thereby compensating for their decreased affinity for entry receptors and improving their fusion and replication efficiencies. This study thus sheds light on unsuspected mechanisms whereby MVC-resistant HIV-1 could emerge and grow in treated patients.

Characterisation of Legionella Clinical Isolates in Spain from 2012 to 2022.

Although cases of Legionnaires' disease are notifiable, data on the phenotypic and genotypic characterisation of clinical isolates are limited. This retrospective study aims to report the results of the characterisation of Legionella clinical isolates in Spain from 2012 to 2022. Monoclonal antibodies from the Dresden panel were used for phenotypic identification of Legionella pneumophila. Genotypic characterisation and sequence type assignment were performed using the Sequence-Based Typing scheme. Of the 1184 samples, 569 were identified as Legionella by culture. Of these, 561 were identified as L. pneumophila, of which 521 were serogroup 1. The most common subgroups were Philadelphia (n = 107) and Knoxville (n = 106). The SBT analysis revealed 130 different STs, with the most common genotypes being ST1 (n = 87), ST23 (n = 57), ST20 (n = 30), and ST42 (n = 29). Knoxville has the highest variability with 32 different STs. ST23 is mainly found in Allentown/France (n = 46) and ST42 in Benidorm (n = 18), whereas ST1 is widely distributed. The results demonstrate that clinical isolates show high genetic diversity, although only a few sequence types (STs) are responsible for most cases. However, outbreaks can also occur with rare genotypes. More data on LD and associated epidemiological studies are needed to establish the risk of an isolate causing outbreak in the future.

Fulminant septic shock due to community-acquired pneumonia caused by Legionella pneumophila SG1 Olda OLDA ST1. Case report.

Legionellers' desease accounts for 1-8 % of cases of severe community-acquired pneumonia (CAP). Legionella spp. Is the causative organism that can result in respiratory failure, multi-organ dysfunction, sepsis, and death. Therefore, rapid diagnosis and efficient treatment are crucial. We report the clinical and microbiology study of a patient with community-acquired pneumonia caused by Legionella pneumophila, with fatal outcome. After death, the strain causing the infection was identified as Legionella pneumophila serogroup 1, Olda OLDA phenotype and sequence-type 1. This is the first reported case of septic shock and death associated with an isolate of these characteristics.

Immunogenicity of a third dose with mRNA-vaccines in the ChAdOx1-S/BNT162b2 vaccination regimen against SARS-CoV-2 variants.

CombiVacS study has demonstrated a strong immune response of the heterologous ChAdOx1-S/BNT162b2 vaccine combination. The primary outcomes of the study were to assess the humoral immune response against SARS-CoV-2, 28 days after a third dose of a mRNA vaccine, in subjects that received a previous prime-boost scheme with ChAdOx1-S/BNT162b2. Secondary outcomes extended the study to 3 and 6 months. The third vaccine dose of mRNA-1273 in naive participants previously vaccinated with ChAdOx1-S/BNT162b2 regimen reached higher neutralizing antibodies titers against the variants of concern Delta and BA.1 lineage of Omicron compared with those receiving a third dose of BNT162b2 at day 28. These differences between BNT162b2 and mRNA-1273 arms were observed against the ancestral variant G614 at day 90. Suboptimal neutralizing response was observed against BQ.1.1, XBB.1.5/XBB.1.9, and JN.1 in a relevant proportion of individuals 180 days after the third dose, even after asymptomatic Omicron breakthrough infections. EudraCT (2021-001978-37); ClinicalTrials.gov (NCT04860739).

Cellular and humoral functional responses after BNT162b2 mRNA vaccination differ longitudinally between naive and subjects recovered from COVID-19.

We have analyzed BNT162b2 vaccine-induced immune responses in naive subjects and individuals recovered from coronavirus disease 2019 (COVID-19), both soon after (14 days) and later after (almost 8 months) vaccination. Plasma spike (S)-specific immunoglobulins peak after one vaccine shot in individuals recovered from COVID-19, while a second dose is needed in naive subjects, although the latter group shows reduced levels all along the analyzed period. Despite how the neutralization capacity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mirrors this behavior early after vaccination, both groups show comparable neutralizing antibodies and S-specific B cell levels late post-vaccination. When studying cellular responses, naive individuals exhibit higher SARS-CoV-2-specific cytokine production, CD4+ T cell activation, and proliferation than do individuals recovered from COVID-19, with patent inverse correlations between humoral and cellular variables early post-vaccination. However, almost 8 months post-vaccination, SARS-CoV-2-specific responses are comparable between both groups. Our data indicate that a previous history of COVID-19 differentially determines the functional T and B cell-mediated responses to BNT162b2 vaccination over time.

A sensitive phenotypic assay for the determination of human immunodeficiency virus type 1 tropism.

OBJECTIVES: To develop a sensitive phenotypic assay based on recombinant viruses (RVs) for characterizing HIV-1 tropism. METHODS: Viral tropism was assessed in 159 plasma samples. The env gene was amplified and ligated into pNL-lacZ/env-Ren, which carries a luciferase reporter gene. Resulting constructs were transfected into HEK293T cells to generate RVs. To assess co-receptor tropism, U87.CD4.CXCR4/CCR5 cells were infected and luciferase activity was measured. RESULTS: RVs containing env from different HIV-1 subtypes were replication competent. Minor variants were detectable in 1% of the viral population. Tropism was determined in 65% of samples with a viral load of <1000 copies/mL. The phenotypic assay described here was validated with the Trofile™ and Trofile™ES assays. Considering the Trofile™ assay as a reference, the sensitivity for R5 and R5X4/X4 detection was 90% and 77%, and the specificity was 77% and 90%, respectively. Our assay was 86% concordant with Trofile™ (90% for R5 and 77% for R5X4/X4). When our system was compared with Trofile™ES, the concordance was 89% (86% for R5 and 92% for R5X4/X4), the sensitivity for R5 was 86% and for R5X4/X4 was 92%, and the specificity for R5 was 92% and for R5X4/X4 was 86%. The phenotypic results were compared with those obtained using the following V3 genotypic prediction tools: position-specific scoring matrix; geno2pheno[coreceptor]; C4.5; C4.5 using positions 8 and 12; PART; support vector machines; and the charge rule. CONCLUSIONS: We describe a system to assess co-receptor tropism based on the generation of chimeric replication-competent viruses with high sensitivity in the detection of minor populations. A good correlation of our results with Trofile™ assays was found.

Evolution of T-cell responses to hepatitis C virus (HCV) during pegylated interferon plus ribavirin treatment in HCV-monoinfected and in HCV/HIV-coinfected patients.

BACKGROUND: The role of T-cell immunity in chronic hepatitis C virus (HCV) infection remains controversial. As in HIV infection, virus replication could drive or be contained by T-cell immunity. We have examined the effect of HIV coinfection and of suppression of HCV replication with therapy on HCV-specific T-cell responses. PATIENTS AND METHODS: Thirty-five patients with chronic hepatitis C (17 coinfected with HIV) initiating anti-HCV therapy were analysed. HCV-specific responses were assessed at different time points using intracellular interferon-gamma staining in response to a panel of overlapping peptides comprising E2, NS3, NS5a and NS5b HCV proteins. RESULTS: At baseline, HCV-specific responses were significantly lower in HIV-coinfected patients. At week 12 of therapy, CD8+ T-cell responses against all HCV proteins significantly decreased in HCV-monoinfected patients and this was maintained throughout the follow-up period. Although the same trend occurred in the HIV-coinfected group, differences were not significant. CD4+ T-cell responses against NS3 significantly diminished in the HCV-monoinfected group, whereas in coinfected patients CD4+ T-cell responses were low at baseline and did not experience any significant variation. CONCLUSIONS: HCV-specific T-cell responses are lower in HIV-coinfected patients and vanish following complete suppression of HCV replication under successful HCV therapy, suggesting that they are dependent on continuous antiqenic stimulation.

Coinfection with hepatitis C virus increases lymphocyte apoptosis in HIV-infected patients.

To test the role of hepatitis C virus (HCV) in CD4 cell depletion in human immunodeficiency virus (HIV)-coinfected patients, T cell apoptosis was measured by annexin V labeling in 31 HIV-infected and 30 HIV-HCV-coinfected patients who were not receiving antiretroviral therapy. Apoptosis in naive CD4(+) T cells and in naive and memory CD8(+) T cells was significantly higher in HIV-HCV-coinfected than in monoinfected patients.

Suppression of viral replication with highly active antiretroviral therapy has no impact on the functional profile of HIV-specific CD8(+) T cells.

A better control of viral replication in long-term non-progressors has been associated with polyfunctional CD8(+) T cell responses. However, low levels of HIV replication could be the cause rather than the consequence of enhanced immune responses in long-term non-progressors. The functional profile and the expansion ability of HIV-Gag- and HIV-Nef-specific CD8 responses were analysed measuring the production of MIP-1beta, IL-2, TNF-alpha and expression of CD107, using polychromatic flow cytometry, in 36 HIV-infected patients at baseline and after 12 months of highly active antiretroviral therapy (HAART) and complete viral suppression. Most patients presented detectable Gag and Nef responses both at baseline and after 1 year of HAART, with a significant decline after achieving viral suppression. At baseline, the majority of CD8(+) response was due to cells producing only MIP-1beta or simultaneously MIP-1beta and CD107. The functional profile did not significantly change after achieving complete viral suppression with HAART. Therefore, control of HIV-1 replication after 1 year of HAART had no significant impact on the quality of HIV-1-specific CD8 response, but the effects of treatment in long-term, or of early HAART are not known. Thus, it is still uncertain whether multifunctional CD8 responses are the cause or consequence of low plasma viremia.

No major differences in the functional profile of HIV Gag and Nef-specific CD8+ responses between long-term nonprogressors and typical progressors.

The mechanism explaining the failure of HIV-specific CD8(+) T cell responses to successfully control HIV replication remains elusive. A total of 83 drug-naive HIV-infected individuals, 27 of whom were long-term nonprogressors (LTNP), was examined. The ability of CD8(+) T lymphocytes to produce three different cytokines (MIP-1beta, TNF-alpha, IL-2) in response to HIV Gag and Nef peptides and to polyclonal stimuli and the ability of HIV-specific CD8(+) T cells to expand in vitro were evaluated by multiparameter flow cytometry. In response to polyclonal stimulation, LTNP presented significantly higher levels of several CD8(+) T cell subsets than progressors. While most patients presented detectable Gag and Nef-specific CD8(+) responses, no significant differences in any of the CD8(+) functional T cell subsets were recognized when comparing LTNP and progressors. HIV responses were dominated by cells producing only MIP-1beta or TNF-alpha, being similar in LTNP and progressors. However, expansion of HIV-specific CD8(+) T cells was more frequent in LTNP than progressors, especially for cells producing MIP-1beta. LTNP show higher levels of CD8(+) responses against polyclonal stimuli than progressors. However, HIV-specific CD8(+) responses do not differ between them except for a more preserved ability of cells from LTNP to expand in vitro.

Influence of HCV genotype and co-infection with human immunodeficiency virus on CD4(+) and CD8(+) T-cell responses to hepatitis C virus.

The role of T-cells in clearance of hepatitis C virus (HCV) during acute infection is critical. The relevance of the immunological response in the control of HCV replication is less clear in chronic HCV infection. HCV-specific T-cell responses were examined in 92 interferon-naive individuals with chronic hepatitis C. A panel of 441 overlapping peptides spanning all expressed HCV proteins was used to measure HCV-specific T-cell responses, using flow cytometry after stimulating peripheral blood mononuclear cells (PBMCs) with different pools of these peptides. Most patients showed responses to at least one HCV protein, with NS5B for CD8(+) responses and E2 for CD4(+) responses identified most frequently. Both the prevalence and breadth of CD4(+) and CD8(+) responses were lower in co-infected patients, independently of the HCV genotype.