Immunogenicity and Tolerance of BNT162b2 mRNA Vaccine in Allogeneic Hematopoietic Stem Cell Transplant Patients.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (ASCT) induces acquired immunodeficiency, potentially altering vaccine response. Herein, we aimed to explore the clinical tolerance and the humoral and cellular immune responses following anti-SARS-CoV-2 vaccination in ASCT recipients. METHODS: A prospective, non-randomized, controlled study that involved 43 ASCT subjects and 31 healthy controls. Humoral response was investigated using the Elecsys® test anti-SARS-CoV-2. Cellular response was assessed using the QFN® SARS-CoV-2 test. The lymphocyte cytokine profile was tested using the LEGENDplex™ HU Th Cytokine Panel Kit (12-plex). RESULTS: Adverse effects (AE) were observed in 69% of patients, encompassing pain at the injection site, fever, asthenia, or headaches. Controls presented more side effects like pain in the injection site and asthenia with no difference in the overall AE frequency. Both groups exhibited robust humoral and cellular responses. Only the vaccine transplant delay impacted the humoral response alongside a previous SARS-CoV-2 infection. Noteworthily, controls displayed a Th1 cytokine profile, while patients showed a mixed Th1/Th2 profile. CONCLUSIONS: Pfizer-BioNTech® anti-SARS-CoV-2 vaccination is well tolerated in ASCT patients, inducing robust humoral and cellular responses. Further exploration is warranted to understand the impact of a mixed cytokine profile in ASCT patients.

Immunogenicity of Mix-and-Match CoronaVac/BNT162b2 Regimen versus Homologous CoronaVac/CoronaVac Vaccination: A Single-Blinded, Randomized, Parallel Group Superiority Trial.

(1) Background: This study aimed to compare the immunogenicity of the mix-and-match CoronaVac/BNT162b2 vaccination to the homologous CoronaVac/CoronaVac regimen. (2) Methods: We conducted a simple-blinded randomized superiority trial to measure SARS-CoV-2 neutralization antibodies and anti-spike receptor binding domain (RBD) IgG concentrations in blood samples of participants who had received the first dose of CoronaVac vaccine followed by a dose of BNT162b2 or CoronaVac vaccine. The primary endpoint for immunogenicity was the serum-neutralizing antibody level with a percentage of inhibition at 90% at 21-35 days after the boost. A difference of 25% between groups was considered clinically relevant. (3) Results: Among the 240 eligible participants, the primary endpoint data were available for 100 participants randomly allocated to the mix-and-match group versus 99 participants randomly allocated to the homologous dose group. The mix-and-match regimen elicited significantly higher levels of neutralizing antibodies (median level of 96%, interquartile range (IQR) (95-97) versus median level of 94%, IQR (81-96) and anti-spike IgG antibodies (median level of 13,460, IQR (2557-29,930) versus median level of 1190, IQR (347-4964) compared to the homologous group. Accordingly, the percentage of subjects with a percentage of neutralizing antibodies > 90% was significantly higher in the mix-and-match group (90.0%) versus the homologous (60.6%). Interestingly, no severe events were reported within 30 days after the second dose of vaccination in both groups. (4) Conclusions: Our data showed the superiority of the mix-and-match CoronaVac/BNT162b2 vaccination compared to the CoronaVac/CoronaVac regimen in terms of immunogenicity, thus constituting a proof-of-concept study supporting the use of inactivated vaccines in a mix-and-match strategy while ensuring good immunogenicity and safety.

Imaging Leishmania major Antigens in Experimentally Infected Macrophages and Dermal Scrapings from Cutaneous Leishmaniasis Lesions in Tunisia.

Leishmania major cutaneous leishmaniasis (CL) lesions are characterized by an intense process of parasite destruction and antigen processing that could limit microscopic amastigote detection. The aim of our study was to develop a direct immunofluorescence (DIF) assay for in situ visualization of L. major antigens and access its reliability in the routine diagnosis of CL. The developed DIF assay used IgG polyclonal antibodies produced in rabbits by intravenous injections of live L. major metacyclic promastigotes chemically coupled to fluorescein isothiocyanate. Applied to L. major infected RAW macrophages, corresponding macrophage-derived amastigotes and dermal scrapings from CL lesions, the immunofluorescence assay stained specifically Leishmania amastigotes and showed a diffuse Leishmania antigen deposit into cytoplasm of phagocytic cells. Reliability of DIF in CL diagnosis was assessed on 101 methanol-fixed dermal smears from 59 positive and 42 negative CL lesions diagnosed by direct microscopy and/or kDNA real-time PCR. Sensitivity and specificity of DIF was 98.3% and 100%, respectively, being more sensitive than microscopy (p < 0.001) and as sensitive as ITS1-PCR. ITS1-PCR-RFLP allowed Leishmania species identification in 56 out of the 58 DIF-positive smears, identifying 52 L. major, two L. infantum and two L. tropica cases, which indicates antigenic cross-reactivity between Leishmania species.

Use of Immunomagnetic Separation tool in Leishmania promastigotes capture.

Immunomagnetic Separation (IMS) assay has been used for isolation of viable whole organisms. The objective of our work is to produce anti-Leishmania magnetic beads and to assess the efficiency of the IMS technique on Leishmania promastigote capture in culture media. Polyclonal anti-Leishmania antibodies were produced by intravenous injection of viable metacyclic promastigotes of Leishmania (L.) major to rabbit. Purified anti-Leishmania IgG was assessed for their reactivity against both L. major and L. infantum promastigotes then covalently conjugated to magnetic beads and used for IMS. This latter was applied on either L. major promastigote cultures of known concentrations or early stage (24h, 48h, 72h) Novy-MacNeal-Nicolle (NNN) cultures of tissue fluid obtained from cutaneous leishmaniasis (CL) lesions. Promastigotes capture was assessed by either microscopy or qPCR after sample boiling. Indirect immunofluorescence assay showed that polyclonal antibodies reacted against both L. major and L. infantum promastigotes. In 50 µL solution, immunomagnetic beads were able to capture 5 live promastigotes out of 20 and 1050 out of 2500, giving an estimated efficiency of 25-42%. The efficiency of the IMS was lower for a lower number of parasites but still repeatable. On the other hand, IMS-qPCR applied to 14 NNN cultures of confirmed Leishmania lesions showed a higher sensitivity to detect live parasites than routine microscopy observation of promastigotes growth (93% positivity at 72h versus 50% positivity within 2-4 weeks incubation). The estimated number of captured parasites at 72h ranged from 1 to more than 100 parasites / 50 µL liquid phase of culture. These preliminary results open the way for interesting perspectives in the use of cultures for leishmaniasis diagnosis and also for other applications such as Leishmania detection in cultures taken from reservoir animals or sandflies.

Diagnosis of Mediterranean visceral leishmaniasis by detection of Leishmania-related antigen in urine and oral fluid samples.

Implementation of simple diagnostic tests using non-invasive collection of biological specimens is of great importance in the diagnosis of pediatric visceral leishmaniasis caused by Leishmania infantum. Latex agglutination kit (KAtex®) is widely used in the diagnosis mainly in L. donovani endemic areas. However its utilization in L. infantum endemic regions remains limited and its use on noninvasive biological specimen apart urine was not reported. In this study, KAtex® kit was used to detect Leishmania-related antigen in urine and oral fluid of 35 L. infantum visceral leishmaniasis cases and 62 controls including non-infectious disease and infectious disease controls (34 and 28 respectively). Sensitivity and specificity of urine based KAtex® were 51.4% and 98.3% respectively, whereas, sensitivity and specificity of oral-fluid based KAtex® were 80% and 88.3% respectively. Although, sensitivity of oral-fluid KAtex® was high, its specificity varied significantly according to the presence or the absence of an infectious disease (71.4% versus 97%, p=0.01).

The sera from adult patients with suggestive signs of autoimmune diseases present antinuclear autoantibodies that cross-react with Leishmania infantum conserved proteins: crude Leishmania histone and Soluble Leishmania antigens [corrected].

Visceral leishmaniasis has been associated with hyper-gammaglobulinemia and antinuclear antibodies and may simulate systemic lupus erythematosus. Sera from patients with visceral leishmaniasis have been shown to strongly react against conserved proteins from the parasite, such as ribosomal and histones. Some of these proteins have also been described as immunogenic in several auto-immune syndromes, and the detection of antibodies against them is considered to be indicative of disorder in the immune system. This study aimed to assess by enzyme-linked immunosorbent assay, test routinely employed in visceral leishmaniasis diagnosis, the recognition of Crude Leishmania histone and Soluble Leishmania antigens proteins from Leishmania infantum by adult patients with suggestive signs of autoimmune diseases. Our results show that the humoral response generated during autoimmune diseases cross-reacts with the parasitic Crude Leishmania histone and Soluble Leishmania antigens. In these cases, higher precautions must be taken to confirm the presence of visceral leishmaniasis in front of positive serology in antinuclear antibodies positive sera, in order to avoid wrong diagnosis.

Hd86 mRNA expression profile in Hyalomma scupense life stages, could it contribute to explain anti-tick vaccine effect discrepancy between adult and immature instars?

Bm86 midgut protein has been used in order to control ticks of the Hyalomma genus. Previous studies demonstrated the inefficacity of this antigen in the control of Hyalomma scupense, whereas recombinant Hd86 antigen, the Bm86 ortholog in H. scupense produced in Pichia pastoris, was protective against larval H. scupense tick stage infestations but ineffective in the control of the adult stage. One possible explanation for this result is the variation in Hd86 expression levels between these two developmental stages. To test this hypothesis, Hd86 mRNA levels were characterized in H. scupense developmental stages. The expression profile of Hd86 demonstrated a significant variation between tick life stages and showed a significant reduction in the number of transcripts during feeding and, particularly after molting to adults. The most interesting result was noted after molting of engorged nymphs in unfed adults where the expression levels decreased significantly by 12.78 (10.77-17.39) (p<0.001) and 9.25 (5.77-15.72)-fold (p<0.001) in unfed males and unfed females, respectively. Comparing unfed nymphs to unfed adult ticks, the Hd86 expression levels decreased by 13.82 (5.39-24.45) (p=0.035) and 9.93 (2.87-22.08)-fold (p=0.038) in males and females respectively. Lower Hd86 mRNA levels in adult ticks should result in lower protein levels and thus less antibody-antigen interactions necessary for vaccine efficacy in ticks fed on vaccinated animals. Thus, the observed differences in Hd86 expression profile between immature and adult stages might explain, in part, the discrepancy of the Hd86 vaccine efficacy against these two life stages of H. scupense.

Contribution of specific anti-hydatid IgG subclasses in the diagnosis of echinococcosis primary infection and relapses.

BACKGROUND: Serological diagnosis of hydatid disease still faces problems of sensitivity, limiting its use to either diagnosis or post-surgical monitoring. The use of IgG subclasses seems to overcome these difficulties. The contribution of IgG subclasses was evaluated in the diagnosis of primary infested and hydatid cyst relapse patients. METHODS: A group of patients (n = 34) diagnosed for the first time with liver cystic echinococcosis (CE) and a group of patients with CE surgical recurrence were included. Enzyme-linked immunosorbent assay anti-hydatid antigens (HA) specific IgG1, 2, 3, and 4 subclasses were analyzed by ROC curves. RESULTS: ROC curve analyses demonstrated that IgG4 had the ability to discriminate between primary infested and relapsed groups whereas IgG2 was not discriminatory. The sensitivity of IgG4 was statistically higher in the relapsed cases group (97.1% versus 70.6%, p = 0.008). CONCLUSIONS: anti-HA specific IgG2 was the best marker of primary infestation whereas IgG4 was the best marker of relapse.

Efficacy of Hyalomma scupense (Hd86) antigen against Hyalomma excavatum and H. scupense tick infestations in cattle.

The Rhipicephalus microplus recombinant Bm86-based tick vaccines have shown their efficacy for the control of several Hyalomma cattle ticks genera, namely H. dromedarii and H. anatolicum. However, H. scupense species, the most important tick in North Africa has never been studied. Vaccination trials using either a recombinant Bm86-based vaccine or a recombinant Hd86-based vaccine (the Bm86 ortholog in H. scupense) were conducted in cattle against immature and adult H. scupense ticks and adult H. excavatum ticks. The results showed a 59.19% reduction in the number of scupense nymphs engorging on Hd86 vaccinated cattle. However, cattle vaccination with Bm86 or Hd86 did not have an effect on H. scupense or H. excavatum adult ticks infestations. These results showed that Hd86 vaccines are selectively effective against H. scupense immature instars and emphasize on an integrated anti-tick vaccine control in North Africa.

Molecular characterization of Bm86 gene orthologs from Hyalomma excavatum, Hyalomma dromedarii and Hyalomma marginatum marginatum and comparison with a vaccine candidate from Hyalomma scupense.

The ixodid ticks from the Hyalomma genus are important pests of livestock, having major medical and veterinary significance in Northern Africa. Beside their direct pathogenic effects, these species are vectors of important diseases of livestock and in some instances of zoonoses. Anti-tick vaccines developed in Australia and Cuba based on the concealed antigen Bm86 have variable efficacy against H. anatolicum and H. dromedarii. This variation in vaccine efficacy could be explained by the variability in protein sequence between the recombinant Bm86 vaccine and Bm86 orthologs expressed in different Hyalomma species. Bm86 orthologs from three Hyalomma tick species were amplified in two overlapping fragments and sequenced. The rate of identity of the amino acid sequence of Hm86, He86 and Hdr86, the orthologs of Bm86, respectively, in H. marginatum marginatum, H. excavatum and H. dromedarii, with the Bm86 proteins from Rhipicephalus microplus (Australia, Argentina and Mozambique) ranged between 60 and 66%. The obtained amino-acid sequences of Hmm86, He86 and Hdr86 were compared with the Hd86-A1 sequence from H. scupense used as an experimental vaccine. The results showed an identity of 91, 88 and 87% for Hmm86, He86 and Hdr86, respectively. A specific program has been used to predict B cells epitopes sites. The comparison of antigenic sites between Hd86-A1 and Hm86/Hdr86/He86 revealed a diversity affecting 4, 8 and 12 antigenic peptides out of a total of 28 antigenic peptides, respectively. When the Bm86 orthologs amplification protocol adopted in this study was applied to H. excavatum, two alleles named He86p2a1 and He86p2a2 were detected in this species. This is the first time that two different alleles of Bm86 gene are recorded in the same tick specimen. He86p2a1 and He86p2a2 showed an amino acid identity of 92%. When He86p2a1 and He86p2a2 were compared to the corresponding sequence of Hd86-A1 protein, an identity of 86.4 and 91.0% was recorded, respectively. When compared to He86, Hdr86 and Hm86, Bm86 used in commercial and experimental vaccines, showed a greater extent of diversity than noted when the same Hyalomma orthologs were compared to Hd86-A1. Although significant, these variations were less extensive within the Hyalomma genus. Accordingly, thus suggesting that Hd86-A1 vaccine candidate might be more appropriate to target Hyalomma tick species than corresponding Bm86 commercial vaccines. However, vaccination trials with both antigens are required to validate this hypothesis.

Hd86, the Bm86 tick protein ortholog in Hyalomma scupense (syn. H. detritum): expression in Pichia pastoris and analysis of nucleotides and amino acids sequences variations prior to vaccination trials.

The genus Hyalomma includes the most frequent tick species infesting livestock in North Africa, one of these species, Hyalomma scupense (syn. H. detritum) is particularly important due to its role in the transmission of tropical theileriosis to cattle (Theileria annulata infection). We have cloned and characterized the orthologs of the Bm86 gene from H. scupense strains collected over Tunisia in 2006 and 2009. The recombinant protein rHd86 was expressed in Pichia pastoris for vaccination purpose using a transcript from the 2006 strain. The rHd86 was then purified from the yeast culture supernatant by a filtration and a size exclusion process. It was recognized by specific anti-Bm86 antisera. An important extent of inter-specific diversity ranging from 35 to 40% was recorded between Hd86 and Bm86/Bm95 proteins whilst a very limited level of intra-specific diversity (1.7%) occurred between the Hd86 vaccine candidate protein and its homologues from H. scupense strains collected in 2009. These results emphasise the need for assessing the efficacy against H. scupense and others important cattle Hyalomma species in Tunisia of our Hd86 vaccine candidate alongside with a Bm86 vaccine.

Diagnosis of mediterranean visceral leishmaniasis by detection of leishmania antibodies and leishmania DNA in oral fluid samples collected using an Oracol device.

Current methods for diagnosis of visceral leishmaniasis (VL) require invasive sampling procedures such as visceral aspiration and/or blood drawing. The use of diagnostic tests using oral fluid, which is easier to collect, would be more simple and practical for VL diagnosis, especially under field conditions. Oral fluids from 37 VL patients and 40 healthy controls were collected using Oracol devices. Blood samples and oral fluid specimens from both groups were analyzed by recombinant protein K39 (rK39) enzyme-linked immunosorbent assay and quantitative real-time PCR. Detection of antibodies in the oral fluid had a sensitivity of 100% and a specificity of 97.5%. Antibody levels measured in serum and oral fluid showed a significant positive correlation (ρ = 0.655 and P = 0.01). Detection of Leishmania DNA in oral fluid had a sensitivity of 94.6% and a specificity of 90%. The median parasite load estimated in blood was 133 parasites/ml (interquartile range [IR], 10 to 1,048), whereas that in oral fluid specimens was 3 parasites/ml (IR, 0.41 to 92). However, there was no significant linear relationship between parasite loads assessed in the two biological samples (ρ = 0.31 and P = 0.06). VL diagnosis based on specific antibody detection and Leishmania DNA identification using oral fluid samples was equivalent in accuracy to that using blood and therefore is promising for clinical use.

ELISA on saliva samples for the detection of anti-hydatid cyst antibodies.

BACKGROUND: The detection of antibodies in saliva samples proved to be effective in the diagnosis of several microbial diseases. These antibodies were screened in saliva samples of patients with hydatid cysts. METHODS: Anti-hydatid fluid antigen IgG and IgA antibodies were screened in saliva and sera of patients with hydatid cysts (n=37) as well as in healthy controls (n=30) using an in-house developed immunoenzymatic assay. RESULTS: Salivary anti-hydatid fluid antigen IgG showed a sensitivity of 86.5% and a specificity of 80%. A positive correlation was observed between anti-hydatid fluid antigen IgG in saliva and in serum (r = 0.364; p = 0.02). CONCLUSIONS: The detection of anti-hydatid fluid antigen IgG antibodies in saliva using ELISA promises to be interesting for the diagnosis of cystic echinococcosis.