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The PfRh5-Basigin ligand–receptor interaction is an essential step in the merozoite invasion process and represents an attractive vaccine target. To reveal genotype–phenotype associations between naturally occurring allelic variants of PfRh5 and invasion inhibition, we performed ex vivo invasion inhibition assays with monoclonal antibodies targeting basigin coupled with PfRh5 next-generation amplicon sequencing. We found dose-dependent inhibition of invasion across all isolates tested, and no statistically significant difference in invasion inhibition for any single nucleotide polymorphisms. This study demonstrates that PfRh5 remains highly conserved and functionally essential, even in a highly endemic setting, supporting continued development as a strain-transcendent malaria vaccine target.

The recent stall in the global reduction of malaria deaths has made the development of a highly effective vaccine essential. A major challenge to developing an efficacious vaccine is the extensive diversity of Plasmodium falciparum antigens. While genetic diversity plays a major role in immune evasion and is a barrier to the development of both natural and vaccine-induced protective immunity, it has been under-prioritized in the evaluation of malaria vaccine candidates. This study uses genomic approaches to evaluate genetic diversity in next generation malaria vaccine candidate PfRh5. We used targeted deep amplicon sequencing to identify non-synonymous Single Nucleotide Polymorphisms (SNPs) in PfRh5 (Reticulocyte-Binding Protein Homologue 5) in 189 P. falciparum positive samples from Southern Senegal and identified 74 novel SNPs. We evaluated the population prevalence of these SNPs as well as the frequency in individual samples and found that only a single SNP, C203Y, was present at every site. Many SNPs were unique to the individual sampled, with over 90% of SNPs being found in just one infected individual. In addition to population prevalence, we assessed individual level SNP frequencies which revealed that some SNPs were dominant (frequency of greater than 25% in a polygenomic sample) whereas most were rare, present at 2% or less of total reads mapped to the reference at the given position. Structural modeling uncovered 3 novel SNPs occurring under epitopes bound by inhibitory monoclonal antibodies, potentially impacting immune evasion, while other SNPs were predicted to impact PfRh5 structure or interactions with the receptor or binding partners. Our data demonstrate that PfRh5 exhibits greater genetic diversity than previously described, with the caveat that most of the uncovered SNPs are at a low overall frequency in the individual and prevalence in the population. The structural studies reveal that novel SNPs could have functional implications on PfRh5 receptor binding, complex formation, or immune evasion, supporting continued efforts to validate PfRh5 as an effective malaria vaccine target and development of a PfRh5 vaccine.

Background:Sjögren’s disease (SjD) is a heterogenous autoimmune disease, with a wide range of symptoms, from dryness, fatigue, pain, to systemic manifestations, and an increased risk of lymphoma. Recently, three clusters of patients with SjD have been described based on unsupervised clustering analysis according to symptoms, clinical signs and biologic parameters: 1/ BA-LS (B-cell active with low symptoms); 2/ HSA (High systemic activity); 3/ LSA-HS (Low systemic activity with high symptoms). These findings suggest potential heterogeneity in pathophysiological mechanisms.Objectives:To investigate this hypothesis, we examined whether these three clusters were associated with distinct biomarkers.Methods:This study involved SjD patients meeting AECG criteria from the ASSESS cohort. The following biomarkers were measured in sera at the time of inclusion: for IFN pathways—IFN-alpha 2, IFN gamma, CXCL-10; for B cell activation—CXCL-13, BAFF, B2-microglobulin, FLT-3; for T cell activation—IL-7, CCL-19, TNF-RII. Additionally, the IFN signature was assessed using transcriptomic analysis. Kruskal-Wallis rank sum test was used to compare different clusters for continuous variables. Additionally, the risk of lymphoma and of new immunosuppressive drugs prescriptions were compared according to the IFN signature.Results:This analysis included 395 (94% female, median age 53 [43-63] years) patients from the ASSESS cohorts. The three clusters displayed differences in the IFN pathways (IFN signature), primarily driven by type I IFN (IFN-a2 level) elevated only in BA-LS and HSA clusters and not in the LSA-HS cluster (p=0.001). IFN gamma and CXCL-10 were not different between the 3 clusters.The same clusters that exhibit high level of type 1 IFN also had higher CXCL-13 levels (p=0.0032) reflecting B-cell activation, higher IL-7 (p=0.0042) and TNFRII (p<0.001) levels reflecting T-cell activation. Higher levels of FLT-3 were found in the HSA cluster. BAFF level was not different between the 3 clusters.Lastly, there were a trend indicating an increased risk of lymphoma in patients with positive IFN signature (HR 2.53; 95%CI 0.67–9.55), and an increased risk of immunosuppressant prescription during follow-up (HR 2.81; 95%CI 1.26-6.29).Conclusion:The two clusters BA-LS and HSA have a very distinct cytokine signature than the patients with LSA-HS. These two active clusters share a high type 1 IFN level, and elevated markers of both B-cell and T-cell activation. Patients from the BA-LS cluster being younger, it is likely that this cluster represent an earlier disease stage than HSA cluster. In order to go towards personalized medicine, work is in progress for deciphering patients in these two active clusters exhibiting one predominant pathways among type 1 IFN, B-cell and T-cell activation.REFERENCES:[1] Nguyen Y, Nocturne G, Henry J. Identification of distinct phenotypes of Sjögren disease by cluster analysis based on clinical and biological manifestations: data from the cross-sectional Paris-Saclay and the prospective ASSESS cohorts. Lancet Rheumatology 2024.Acknowledgements:The authors are indebted to all patients for their participation, and to all physicians who included patients in the Paris-Saclay and ASSESS cohorts. The Assessment of Systemic Signs and Evolution in Sjögren’s Syndrome (ASSESS) national multicenter prospective cohort was formed in 2006 with a French Ministry of Health grant (Programme Hospitalier de Recherche Clinique 2005 P060228). The ASSESS cohort is promoted by the French Society of Rheumatology and receives research grants from the French Society of Rheumatology.Disclosure of Interests:Yann Nguyen: None declared, Xavier Mariette Xavier Mariette received consulting fees from Astra Zeneca, Bristol Myer Squib, Galapagos, GSK, Novartis and Pfizer, Maxime Beydon: None declared, Divi Cornec: None declared, Jacques-Olivier Pers: None declared, Jacques MorelJacques Morel received honoraria from Abbvie, Boehringer Ingelheim, Biogen, Lilly, Mylan, Pfizer, Sanofi, Bristol Myers Squib, Fresenius Kabi, Galapagos, Medac, Novartis, Roche Chugai;,Jacques Morel received grants from Bristol Myers Squib, Fresenius Kabi, Lilly, Novartis, Pfizer, and Roche-Chugaï;, Aleth PERDRIGER: None declared, Emmanuelle Dernis Emmanuelle Dernis received consulting fees from BMS, Celgène, Lilly, MSD, Novartis, UCB; honoria for lectures from Abbvie, BMS, Janssen, Lilly, Medac, MSD, Novartis, Roche-Chugaï, Sanofi, UCB, Celgène, Amgen, Galapagos;, Valerie Devauchelle-Pensec: None declared, Damien Sene: None declared, Philippe Dieudé Philippe Dieudé received consulting fees from Pfizer, Roche Chugai, Bristol Myers Squibb, Abbvie, MSD., Philippe Dieudé received grants from Novartis, Marion Couderc: None declared, Anne-Laure Fauchais: None declared, Claire Larroche: None declared, Olivier Vittecoq: None declared, Carine Salliot Carine Salliot received Honoria from Novartis, Roche Chugaï, Eric Hachulla: None declared, Véronique Le Guern: None declared, Jacques-Eric Gottenberg Jacques-Eric Gottenberg consulting fees from Abbvie, Astra Zeneca, Sanofi, Lilly, Galapagos, Gilead, Roche Chugai, Pfizer, Bristol Myer Squib, MSD., Jacques-Eric Gottenberg received grants from Pfizer, Abbvie, Lilly, Raphaèle Seror Raphaèle Seror received consulting fees from GSK, Bristol Myer Squib, Boerhinger and Janssen; honoraria from GSK, Bristol Myer Squib, Boehringer, Amgen, Pfizer and Roche; travel fees from Amgen and GSK;, Gaetane Nocturne: None declared.

Objectives:To report the results of a pilot study using rituximab combined with Peg-interferon (IFN) α2b-ribavirin in severe refractory hepatitis C virus (HCV) related mixed cryoglobulinaemia (MC) vasculitis.Methods:Sixteen consecutive patients with severe HCV-MC vasculitis that were resistant (n = 11) or relapser (n = 5) to a previous combination treatment with standard (n = 10) or Peg-IFNα2b (n = 6) plus ribavirin were included. They were treated with rituximab (375 mg/m2 intravenously weekly for 4 weeks) combined with Peg-IFNα2b (1.5 μg/kg per week subcutaneously) plus ribavirin (600–1200 mg/day orally) for 12 months.Results:Fifteen patients (93.7%) showed clinical improvement, 10 of whom (62.5%) were clinical complete responders (CR). HCV RNA and serum cryoglobulin became undetectable in all the clinical CR. Peripheral blood B cell depletion was achieved in all patients (CD19+ cells, 111 (SD 32)/mm3 at baseline versus 2(2)/mm3 after the fourth infusion of rituximab) with reconstitution starting at the end of antiviral treatment. Compared with clinical CR, the partial or non-responders had a 3.6 times longer duration of vasculitis prior to treatment and a lower rate of early virological response. Treatment was well tolerated with no infectious complications. After a mean follow-up of 19.4 (SD 3.6) months, two patients experienced clinical relapse associated with a simultaneous reappearance of HCV RNA and cryoglobulin and an increase in the number of B cells.Conclusions:Rituximab combined with Peg-IFNα2b-ribavirin represents a safe and effective treatment option in severe refractory HCV-MC vasculitis.

Background:Neurological manifestations can occur in up to half of patients with Sjögren disease (SjD) and the peripheral nervous system (PNS) is the most commonly involved (5-21%).Objectives:To analyze the frequency and phenotypic expression of PNS involvement at the time of SjD diagnosisMethods:The Big Data Project Consortium is an international, multicenter registry created in 2014. Baseline clinical information from leading centers on clinical research in SjD of the five continents was collected as a first step. The centers share a harmonized data architecture and conduct cooperative online efforts to refine collected data under the coordination of a big data statistical team. The inclusion criteria were the fulfillment of the 2002 or 2016 classification criteria.Results:By December 2023, the participant centers had included 16’703 patients from 28 countries (15’602 women, mean age at diagnosis of 51.66 years). PNS involvement at diagnosis, defined according to the ESSDAI classification, was reported in 869 (5.2%) patients. Among them, 518 (60%) showed mild active PNS involvement (pure sensory axonal polyneuropathy, V neuralgia, or proven small fibre neuropathy), 264 (30%) showed moderate involvement (axonal sensory–moto neuropathy, cryoglobulinemic pure sensory neuropathy, mild/moderate ganglionopathy, mild chronic inflammatory demyelinating polyneuropathy -CIDP-, or other cranial nerve involvements), and 87 (10%) showed highly active PNS involvement (severe motor axonal neuropathy, mononeuritis multiplex, severe ataxia due to ganglionopathy, or severe CIDP). Univariate analysis showed that patients with PNS involvement were more frequently men, diagnosed at an older age, white, had a higher frequency of activity in all the 12 ESSDAI domains and more commonly hypocomplementemia and cryoglobulinemia (all P-values <0.001). The multivariate logistic regression model adjusted for age, sex, ethnicity and the ESSDAI domains showed that systemic activity in the constitutional (OR = 1.95; 95% CI, 1.43–2.65), articular (OR = 1.35; 95% CI, 1.06–1.73), renal (OR = 1.92; 95% CI, 1.25–2.96), muscular (OR = 1.89; 95% CI, 1.11–3.24) and CNS (OR = 5.37; 95% CI, 3.18–9.06) ESSDAI domains, as well as low C3 (OR = 1.44; 95% CI, 1.05–1.98) and low C4 (OR = 1.74; 95% CI, 1.27–2.39), were independently associated with a diagnosis of PNS involvement at the time of diagnosis of SjD.Conclusion:Around 5% of patients had PNS involvement at the time of diagnosis of SjD. Patients with PNS involvement showed higher systemic activity in multiple domains (1.3-2 times higher frequency of constitutional, joint, renal, and muscular involvement), especially concerning concomitant CNS involvement (5-fold higher frequency). Hypocomplementemia was the key immunological marker independently associated with PNS involvement.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:None declared.

Background:Central nervous system (CNS) involvement is one of the numerous extra-glandular manifestations of primary Sjögren disease (SjD) and it has been described in 8-70% of patients at various stages of the disease.Objectives:To characterize how CNS involvement presents at the diagnosis of SjD.Methods:The Big Data Sjögren Consortium is an international, multicentre registry created in 2014. Baseline clinical information from leading centers on clinical research in SjD of the five continents was collected as a first step. The centers share a harmonized data architecture and conduct cooperative online efforts to refine collected data under the coordination of a big data statistical team. The inclusion criteria were the fulfillment of the 2002 or 2016 classification criteria. CNS involvement at diagnosis was defined according to the EULAR Sjögren syndrome disease activity index (ESSDAI) classification.Results:By December 2023, 16,703 patients from 28 countries (15’602 women, mean age at diagnosis of 51.66 years) were included. Among them, 284 (1.7%) showed CNS involvement at the rime of SjD diagnosis, including 173 with moderate involvement (cranial nerve involvement of central origin, optic neuritis, or multiple sclerosis-like syndrome with symptoms restricted to pure sensory impairment or proven cognitive impairment) and 111 with highly active CNS involvement (symptomatic cerebral vasculitis, seizures, transverse myelitis, lymphocytic meningitis, or multiple sclerosis-like syndrome with motor deficit). Univariate analysis showed that patients with CNS involvement were more frequently men and white, had the constitutional, renal, muscular, and peripheral nervous system ESSDAI domains more frequently involved, and more often low C3 values (all P-values <0.001). The multivariate logistic regression model adjusted for the epidemiological variables (age, sex, ethnicity) and the above-mentioned ESSDAI variables with p values <0.001 showed that activity in the renal (OR = 1.88; 95% CI, 1.07–3.30) and PNS (OR = 5.47; 95% CI, 3.75–7.99) ESSDAI domains were independently associated with a concomitant diagnosis of CNS involvement at the time of diagnosis of SjD.Conclusion:In a large sample of worldwide SjD patients, CNS involvement was present at disease diagnosis in less than 2% of patients, being one of the less frequent systemic involvements of the disease. Patients with CNS involvement at SjD diagnosis showed a 2-fold higher frequency of concomitant renal involvement and a 5-fold higher frequency of having concomitant PNS involvement. No serological marker was identified with the risk of being diagnosed with SjD-related CNS involvement.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:None declared.

Vaccines to the Plasmodium falciparum reticulocyte binding-like protein homologue 5 (PfRH5) target the blood-stage of the parasite life cycle. PfRH5 has the potential to trigger the production of strain-transcendent antibodies and has proven its efficacy both in pre-clinical and early clinical studies. Vaccine-induced monoclonal antibodies (mAbs) to PfRH5 showed promising outcomes against cultured P. falciparum laboratory strains from distinct geographic areas. Here, we assessed the functional impact of vaccine-induced anti-PfRH5 mAbs on more genetically diverse P. falciparum clinical isolates. We used mAbs previously isolated from single B cells of UK adult PfRH5 vaccinees and used ex-vivo growth inhibition activity (GIA) assays to assess their efficacy against P. falciparum clinical isolates. Next-generation sequencing (NGS) was used to assess the breadth of genetic diversity in P. falciparum clinical isolates and to infer the genotype/phenotype relationship involved in antibody susceptibility. We showed a dose-dependent inhibition of clinical isolates with three main GIA groups: high, medium and low. Except for one isolate, our data show no significant differences in the mAb GIA profile between P. falciparum clinical isolates and the 3D7 reference strain, which harbors the vaccine allele. We also observed an additive relationship for mAb combinations, whereby the combination of GIA-low and GIA-medium antibodies resulted in increased GIA, having important implications for the contribution of specific clones within polyclonal IgG responses. While our NGS analysis showed the occurrence of novel mutations in the pfrh5 gene, these mutations were predicted to have little or no functional impact on the antigen structure or recognition by known mAbs. Our present findings complement earlier reports on the strain transcendent potential of anti-PfRH5 mAbs and constitute, to our knowledge, the first report on the susceptibility of P. falciparum clinical isolates from natural infections to vaccine-induced human mAbs to PfRH5.

Objectives/Goals: Transmission-blocking vaccines hold promise for malaria elimination by reducing community transmission. But a major challenge that limits the development of efficacious vaccines is the vast parasite’s genetic diversity. This work aims to assess the genetic diversity of the Pfs25 vaccine candidate in complex infections across African countries. Methods/Study Population: We employed next-generation amplicon deep sequencing to identify nonsynonymous single nucleotide polymorphisms (SNPs) in 194 Plasmodium falciparum samples from four endemic African countries: Senegal, Tanzania, Ghana, and Burkina Faso. The individuals aged between 1 and 74 years, but most of them ranged from 1 to 19 years, and all presented symptomatic P. falciparum infection. The genome amplicon sequencing was analyzed using Geneious software and P. falciparum 3D7 as a reference. The SPNs were called with a minimum coverage of 500bp, and for this work, we used a very sensitive threshold of 1% variant frequency to determine the frequency of SNPs. The identified SNPs were threaded to the crystal structure of the Pfs25 protein, which allowed us to predict the impact of the novel SNP in the protein or antibody binding. Results/Anticipated Results: We identified 26 SNPs including 24 novel variants, and assessed their population prevalence and variant frequency in complex infections. Notably, five variants were detected in multiple samples (L63V, V143I, S39G, L63P, and E59G), while the remaining 21 were rare variants found in individual samples. Analysis of country-specific prevalence showed varying proportions of mutant alleles, with Ghana exhibiting the highest prevalence (44.6%), followed by Tanzania (12%), Senegal (11.8%), and Burkina Faso (2.7%). Moreover, we categorized SNPs based on their frequency, identifying dominant variants (>25%), and rare variants ( Discussion/Significance of Impact: We identified additional SNPs in the Pfs25 gene beyond those previously reported. However, the majority of these newly discovered display low variant frequency and population prevalence. Further research exploring the functional implications of these variations will be important to elucidate their role in malaria transmission.

BackgroundSince 2002, the European American Consensus Group criteria required the presence of 4 criteria among whom the presence either anti-SSA/anti-SSB antibodies or a focus score upper or equal to 1 at the labial salivary gland biopsy for the diagnosis of pSS. The direct consequence of such classification criteria is the constitution of two different immunological patterns in pSS: one with positive anti-SSA or anti-SSB antibodies, representing 60 to 70% of patients (called seropositive pSS) and the second with negative anti-SSA and anti-SSB antibodies (seronegative pSS).ObjectivesTo analyze the clinical and biological differences between anti-SSA seropositive patients and seronegative patients from a cohort of 176 pSS patients.MethodsWe included 176 patients fulfilling 2002-AECG criteria for pSS and retrospectively analyzed their clinical and biological features according to the presence or absence of anti-SSA antibodies.ResultsThe median age at pSS diagnosis was 54 years; 79% were female; 68% Caucasian and 13% Black. The median follow-up time was 41 months.pSS criteria included xerostomia for 89% of patients, xerophtalmia 96%; a positive minor salivary glands biopsy 83% of 151 patients and anti-SSA antibodies 62% (109 patients) and anti-SSB antibodies 32%. Cumulative extraglandular manifestations included arthralgia in 66%, purpura 13%, lung involvement 10%, peripheral neuropathy 36%, central nervous system involvement 6% and B-NHL 6%. Peripheral neuropathy was small fiber neuropathy (24%), large fiber sensory neuropathy (6%) and sensorimotor neuropathy (4.5%).Seropositive patients (109 patients, 62%), when compared to seronegative patients (67 patients, 38%), were younger at pSS diagnosis (49 vs 58 years; P=0.00015), more frequently Black (18% vs 4.5%; P=0.010) and had less frequently xerostomia (84% vs 97%; P=0.006), peripheral neuropathy (28% vs 51%; P=0.002), mainly small fiber neuropathy (16% vs 39%; P=0.001) but more often purpura (20% vs 3%; P=0.01). There was no significant difference regarding, articular or lung involvement or B-NHL. Biologically, they were hallmarked by a higher prevalence of 1) B-cell chronic activation markers which included hypergammaglobulinemia (61% vs 25%; P<10–5), rheumatoid factor (65% vs 32%; P<10–5), and abnormal FLC ratio (31% vs 0%; P<10–5), 2) peripheral cytopenia including anemia (27% vs 8%; P=0.003), leucopenia (17% vs 5%; P=0.03) and lymphopenia (50% vs 29%; P=0.01). There was no difference regarding mixed cryoglobulin (11% vs 6%) and monoclonal gammopathy (16%vs 16%).ConclusionsOur results showed that the presence or the absence of anti-SSA antibodies are associated with two distinct clinical and biological faces of the “same disease”. Anti-SSA positive patients are younger at diagnosis with a higher prevalence of purpura and B cell activation markers and cytopenia; inversely, anti-SSA negative patients are older at diagnosis with a lower prevalence of B cell activation and are more prompt to develop sensory neuropathies mainly small fiber neuropathies. These findings question the pSS clinical and biological homogeneity and the selection of patients for B cell targeting therapies.Disclosure of InterestNone declared

Sero-surveillance can monitor and project disease burden and risk. However, SARS-CoV-2 antibody test results can produce false positive results, limiting their efficacy as a sero-surveillance tool. False positive SARS-CoV-2 antibody results are associated with malaria exposure, and understanding this association is essential to interpret sero-surveillance results from malaria-endemic countries. Here, pre-pandemic samples from eight malaria endemic and non-endemic countries and four continents were tested by ELISA to measure SARS-CoV-2 Spike S1 subunit reactivity. Individuals with acute malaria infection generated substantial SARS-CoV-2 reactivity. Cross-reactivity was not associated with reactivity to other human coronaviruses or other SARS-CoV-2 proteins, as measured by peptide and protein arrays. ELISAs with deglycosylated and desialated Spike S1 subunits revealed that cross-reactive antibodies target sialic acid on N-linked glycans of the Spike protein. The functional activity of cross-reactive antibodies measured by neutralization assays showed that cross-reactive antibodies did not neutralize SARS-CoV-2 in vitro. Since routine use of glycosylated or sialated assays could result in false positive SARS-CoV-2 antibody results in malaria endemic regions, which could overestimate exposure and population-level immunity, we explored methods to increase specificity by reducing cross-reactivity. Overestimating population-level exposure to SARS-CoV-2 could lead to underestimates of risk of continued COVID-19 transmission in sub-Saharan Africa.