SHERLOCK4HAT: A CRISPR-based tool kit for diagnosis of Human African Trypanosomiasis.

BACKGROUND: To achieve elimination of Human African Trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense (gHAT), the development of highly sensitive diagnostics is needed. We have developed a CRISPR based diagnostic for HAT using SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) that is readily adaptable to a field-based setting. METHODS: We adapted SHERLOCK for the detection of T. brucei species. We targeted 7SLRNA, TgSGP and SRA genes and tested SHERLOCK against RNA from blood, buffy coat, dried blood spots (DBS), and clinical samples. FINDINGS: The pan-Trypanozoon 7SLRNA and T. b. gambiense-specific TgSGP SHERLOCK assays had a sensitivity of 0.1 parasite/µL and a limit of detection 100 molecules/µL. T. b. rhodesiense-specific SRA had a sensitivity of 0.1 parasite/µL and a limit of detection of 10 molecules/µL. TgSGP SHERLOCK and SRA SHERLOCK detected 100% of the field isolated strains. Using clinical specimens from the WHO HAT cryobank, the 7SLRNA SHERLOCK detected trypanosomes in gHAT samples with 56.1%, 95% CI [46.25-65.53] sensitivity and 98.4%, 95% CI [91.41-99.92] specificity, and rHAT samples with 100%, 95% CI [83.18-100] sensitivity and 94.1%, 95% CI [80.91-98.95] specificity. The species-specific TgSGP and SRA SHERLOCK discriminated between the gambiense/rhodesiense HAT infections with 100% accuracy. INTERPRETATION: The 7SLRNA, TgSGP and SRA SHERLOCK discriminate between gHAT and rHAT infections, and could be used for epidemiological surveillance and diagnosis of HAT in the field after further technical development. FUNDING: Institut Pasteur (PTR-175 SHERLOCK4HAT), French Government's Investissement d'Avenir program Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases (LabEx IBEID), and Agence Nationale pour la Recherche (ANR-PRC 2021 SherPa).

Age-associated gut microbiota impair hippocampus-dependent memory in a vagus-dependent manner.

Aging is known to be associated with hippocampus-dependent memory decline, but the underlying causes of this age-related memory impairment remain highly debated. Here, we show that fecal microbiota transplantation (FMT) from aged, but not young, animal donors into young mice is sufficient to trigger profound hippocampal alterations, including astrogliosis, decreased adult neurogenesis, decreased novelty-induced neuronal activation, and impairment in hippocampus-dependent memory. Furthermore, similar alterations were reported when mice were subjected to an FMT from aged human donors. To decipher the mechanisms involved in mediating these microbiota-induced effects on brain function, we mapped the vagus nerve-related (VN-related) neuronal activity patterns and report that aged FMT animals showed a reduction in neuronal activity in the ascending-VN output brain structure, whether under basal condition or after VN stimulation. Targeted pharmacogenetic manipulation of VN-ascending neurons demonstrated that the decrease in vagal activity is detrimental to hippocampal functions. In contrast, increasing vagal ascending activity alleviated the adverse effects of aged mouse FMT on hippocampal functions and had a promnesic effect in aged mice. Thus, pharmacogenetic VN stimulation is a potential therapeutic strategy to lessen microbiota-dependent age-associated impairments in hippocampal functions.

SARS-CoV-2 infection in schools in a northern French city: a retrospective serological cohort study in an area of high transmission, France, January to April 2020.

BackgroundChildren's role in SARS-CoV-2 epidemiology remains unclear. We investigated an initially unnoticed SARS-CoV-2 outbreak linked to schools in northern France, beginning as early as mid-January 2020.AimsThis retrospective observational study documents the extent of SARS-CoV-2 transmission, linked to an affected high school (n = 664 participants) and primary schools (n = 1,340 study participants), in the context of unsuspected SARS-CoV-2 circulation and limited control measures.MethodsBetween 30 March and 30 April 2020, all school staff, as well as pupils and their parents and relatives were invited for SARS-CoV-2 antibody testing and to complete a questionnaire covering symptom history since 13 January 2020.ResultsIn the high school, infection attack rates were 38.1% (91/239), 43.4% (23/53), and 59.3% (16/27), in pupils, teachers, and non-teaching staff respectively vs 10.1% (23/228) and 12.0% (14/117) in the pupils' parents and relatives (p < 0.001). Among the six primary schools, three children attending separate schools at the outbreak start, while symptomatic, might have introduced SARS-CoV-2 there, but symptomatic secondary cases related to them could not be definitely identified. In the primary schools overall, antibody prevalence in pupils sharing classes with symptomatic cases was higher than in pupils from other classes: 15/65 (23.1%) vs 30/445 (6.7%) (p < 0.001). Among 46 SARS-CoV-2 seropositive pupils < 12 years old, 20 were asymptomatic. Whether past HKU1 and OC43 seasonal coronavirus infection protected against SARS-CoV-2 infection in 6-11 year olds could not be inferred.ConclusionsViral circulation can occur in high and primary schools so keeping them open requires consideration of appropriate control measures and enhanced surveillance.

Interferon-γ, a valuable surrogate marker of Plasmodium falciparum pre-erythrocytic stages protective immunity.

Immunity against the pre-erythrocytic stages of malaria is the most promising, as it is strong and fully sterilizing. Yet, the underlying immune effectors against the human Plasmodium falciparum pre-erythrocytic stages remain surprisingly poorly known and have been little explored, which in turn prevents any rational vaccine progress. Evidence that has been gathered in vitro and in vivo, in higher primates and in humans, is reviewed here, emphasizing the significant role of IFN-γ, either as a critical immune mediator or at least as a valuable surrogate marker of protection. One may hope that these results will trigger investigations in volunteers immunized either by optimally irradiated or over-irradiated sporozoites, to quickly delineate better surrogates of protection, which are essential for the development of a successful malaria vaccine.

Antibody-mediated and cellular immune responses induced in naive volunteers by vaccination with long synthetic peptides derived from the Plasmodium vivax circumsporozoite protein.

Plasmodium vivax circumsporozoite (CS) protein is a leading malaria vaccine candidate. We describe the characterization of specific immune responses induced in 21 malaria-naive volunteers vaccinated with long synthetic peptides derived from the CS protein formulated in Montanide ISA 720. Both antibody- and cell-mediated immune responses were analyzed. Antibodies were predominantly of IgG1 and IgG3 isotypes, recognized parasite proteins on the immunofluorescent antibody test, and partially blocked sporozoite invasion of hepatoma cell lines in vitro. Peripheral blood mononuclear cells from most volunteers (94%) showed IFN-γ production in vitro upon stimulation with both long signal peptide and short peptides containing CD8+ T-cell epitopes. The relatively limited sample size did not allow conclusions about HLA associations with the immune responses observed. In summary, the inherent safety and tolerability together with strong antibody responses, invasion blocking activity, and the IFN-γ production induced by these vaccine candidates warrants further testing in a phase II clinical trial.

Evidence for multiple B- and T-cell epitopes in Plasmodium falciparum liver-stage antigen 3.

Liver-stage antigen 3 (LSA-3) is a new vaccine candidate that can induce protection against Plasmodium falciparum sporozoite challenge. Using a series of long synthetic peptides (LSP) encompassing most of the 210-kDa LSA-3 protein, a study of the antigenicity of this protein was carried out in 203 inhabitants from the villages of Dielmo (n = 143) and Ndiop (n = 60) in Senegal (the level of malaria transmission differs in these two villages). Lymphocyte responses to each individual LSA-3 peptide were recorded, some at high prevalences (up to 43%). Antibodies were also detected to each of the 20 peptides, many at high prevalence (up to 84% of responders), and were directed to both nonrepeat and repeat regions. Immune responses to LSA-3 were detectable even in individuals of less than 5 years of age and increased with age and hence exposure to malaria, although they were not directly related to the level of malaria transmission. Thus, several valuable T- and B-cell epitopes were characterized all along the LSA-3 protein, supporting the antigenicity of this P. falciparum vaccine candidate. Finally, antibodies specific for peptide LSP10 located in a nonrepeat region of LSA-3 were found significantly associated with a lower risk of malaria attack over 1 year of daily clinical follow-up in children between the ages of 7 and 15 years, but not in older individuals.

Protection against Plasmodium falciparum challenge induced in Aotus monkeys by liver-stage antigen-3-derived long synthetic peptides.

The vaccine potential of Plasmodium falciparum liver stage antigen-3 (LSA3) was investigated in Aotus monkeys using two long synthetic peptides corresponding respectively to an N-terminal non-repeat peptide (NRP) and repeat 2 (R2) region of the LSA3, adjuvanted by ASO2. Both 100-222 (NRP) and 501-596 repeat peptides induced effector B- and T-cell responses in terms of antigen-driven antibodies and/or specific IFN-gamma secretion. Animals challenged with P. falciparum sporozoites were protected following immunization with either the NRP region alone or the NRP combined with the R2 repeat region, as compared with controls receiving the adjuvant alone. These results indicate that the NRP may be sufficient to induce full, sterile protection and confirm the vaccine potential of LSA3 previously demonstrated in chimpanzees and in Aotus.

Genetic immunisation by liver stage antigen 3 protects chimpanzees against malaria despite low immune responses.

BACKGROUND: The true interest of genetic immunisation might have been hastily underestimated based on overall immunogenicity data in humans and lack of parallelism with other, more classical immunisation methods. PRINCIPAL FINDINGS: Using malaria Liver Stage Antigen-3 (LSA-3), we report that genetic immunization induces in chimpanzees, the closest relative of humans, immune responses which are as scarce as those reported using other DNA vaccines in humans, but which nonetheless confer strong, sterile and reproducible protection. The pattern was consistent in 3/4 immunized apes against two high dose sporozoite challenges performed as late as 98 and 238 days post-immunization and by a heterologous strain. CONCLUSIONS: These results should, in our opinion, lead to a revisiting of the value of this unusual means of immunisation, using as a model a disease, malaria, in which virulent challenges of volunteers are ethically acceptable.

Safety and elicitation of humoral and cellular responses in colombian malaria-naive volunteers by a Plasmodium vivax circumsporozoite protein-derived synthetic vaccine.

Substantial experimental evidence indicates that the Plasmodium circumsporozoite (CS) protein has great potential as a vaccine candidate. We tested the safety and immunogenicity of vaccines composed of P. vivax CS-derived synthetic peptides. Sixty-nine healthy, malaria-naive volunteers were randomized to receive three injections of placebo or synthetic proteins N, R, or C (10, 30, or 100 microg/dose) in a double-blinded fashion. Vaccines were well tolerated and no serious adverse events were observed. Peptides N and R elicited humoral responses at all doses; peptide C elicicted these responses only at doses of 30 and 100 microg. The N peptide at a dose of 100 microg elicited the greatest antibody response. Antibodies to the three peptides recognized P. vivax sporozoites in an immunofluorescent antibody test. Peripheral blood mononuclear cells from most immunized volunteers also produced interferon-gamma upon peptide in vitro stimulation. These vaccines appear safe, well tolerated, and immunogenic in malaria-naive volunteers. Further optimization and development of this vaccine is being attempted to conduct phase II clinical trials.

Proceso para el desarrollo de una vacuna contra la fase hepática de Plasmodium vivax / Process to development a vaccine against to hepatic phase of Plasmodium vivax

Introducción: Anualmente se producen en el mundo entre 80 y 100 millones de casos de malaria ocasionada por Plasmodium vivax, segunda especie de Plasmodium en importancia a nivel mundial y primera en el continente americano. Ante la falla de los métodos clásicos de control de la malaria, derivada de la creciente resistencia de los mosquitos a los insecticidas y de los parásitos a los medicamentos disponibles, se ha trabajado intensamente en la búsqueda de vacunas que puedan prevenir completamente la infección o limitar los efectos patológicos de la enfermedad. Objetivos: Este trabajo describe el proceso de desarrollo de una vacuna experimental dirigida contra las formas pre-eritrocíticas del parásito, para lo cual se ha seleccionado la proteína circumesporozoito (CS) que se expresa de forma abundante en la superficie del parásito y que se halla comprometida en el proceso de invasión hepática. Metodología: El proceso consistió en una exhaustiva caracterización inmunológica de la proteína, mediante péptidos sintéticos de diferente longitud, seguida de pruebas de toxicidad e inmunogenicidad en animales con los tres péptidos largos que cubren las regiones N, R y C de la CS. Como etapa inicial de la prueba en humanos, se hizo un ensayo clínico fase I que probó la seguridad e inmunogenicidad, de cada uno de los péptidos formulados en el adyuvante Montanide ISA-720. El ensayo fue al azar, doble ciego y comprometió a 23 voluntarios sanos, hombres y mujeres entre 18 y 33 años de edad, sin historia de malaria. Conclusiones: La vacuna fue muy bien tolerada y demostró buena seguridad e inmunogenicidad en los ensayos preclínicos así como en todos los voluntarios, facilitando el avance a ulteriores fases de investigaciónclinica

Use of long synthetic peptides to study the antigenicity and immunogenicity of the Plasmodium vivax circumsporozoite protein.

Three long synthetic peptides corresponding to amino (N), repeat (R) and carboxyl (C) regions of the Plasmodium vivax circumsporozoite (CS) protein were synthesised and used to assess their potential as vaccine candidates. Antigenicity studies were carried out using human blood samples from residents of a malaria-endemic area of Colombia, and immunogenicity was tested in Aotus monkeys. The N and C peptides spanned the total native amino and carboxyl flanking regions, whereas the R peptide corresponded to a construct based on the first central nona-peptide repeated in tandem three times and colinearly linked to a universal T-cell epitope (ptt-30) derived from tetanus toxin. All three peptides had been shown previously to contain several B-, T-helper (Th) and Cytotoxic T Lymphocytes (CTL) epitopes. Sixty-one percent of the human sera reacted with the R region, whereas 35 and 39% of the samples had antibodies against the N and C peptides, respectively. Human Peripheral Blood Mononuclear Cells (PBMC) showed higher levels of IFN-gamma than IL-4 when stimulated with peptides containing Th epitopes. Aotus monkeys immunised with the peptides formulated in either Montanide ISA720 or Freund's adjuvants produced strong antibody responses that recognised the peptide immunogens and the native circumsporozoite protein on sporozoites. Additionally, high IFN-gamma production was induced when Aotus lymphocytes were stimulated in vitro with each of the three peptides. We observed boosting of antibody responses and IFN-gamma production by exposure to live sporozoites. These results confirm the high antigenicity and immunogenicity of such synthetic polypeptides and underline their vaccine potential.

Immunogenicity and protective efficacy of Plasmodium falciparum liver-stage Ag-3 in Aotus lemurinus griseimembra monkeys.

Three recombinant proteins spanning the Plasmodium falciparum liver-stage Ag-3 (LSA-3) were used to immunize Aotus monkeys. The proteins were delivered subcutaneously without adjuvant, adsorbed onto polystyrene 0.5 microm particles at a concentration of 2 microg per immunization. Control animals received glutathione-S-transferase formulated similarly. Animals were challenged as late as 5 months after the last immunization, by intravenous inoculation of 100,000 P. falciparum sporozoites of a strain heterologous to the one from which the immunogens were derived. Sterile protection was achieved in three of the five immunized monkeys but in none of four controls. Antibodies were at low titer, but reacted with the native parasite protein and were boosted by parasite challenge. Ag-specific IFN-gamma secretion was detectable in all LSA-3-immunized animals in response to the LSA-3-derived Ag. The protection was apparently associated with high levels of IFN-gamma production in response to in vitro recall Ag. These results lend support to the vaccine potential of LSA-3 indicated by previous results obtained in chimpanzees, as well as the value of yet another Ag-delivery system. They also support the value of the Aotus model for the pre-clinical development of pre-erythrocytic-stage vaccines.

Aotus monkeys: their great value for anti-malaria vaccines and drug testing.

Non-human primates represent a valuable resource for testing potential vaccines candidates and drugs for human use. Malaria remains one of the greatest burdens for the humanity represented by approximately 500 million new clinical cases per year worldwide and at least two million deaths caused annually. Additional control measures such as vaccines and new anti-malarial compounds are therefore urgently needed. Safety and protective efficacy studies in animal models are critical steps for vaccines and drugs development and primate models are probably the most appropriate for this purpose. Although Aotus genus provides several species susceptible to both Plasmodium falciparum and Plasmodium vivax, having different susceptibility to malaria, Aotus lemurinus griseimembra represents the best current malaria primate model because of its high susceptibility to infection by blood forms and sporozoites of both species of Plasmodium. Although the ultimate validation of this model depends upon human trials, over the past two decades these monkeys have proved very useful to test multiple malaria vaccine candidates prior to trials in humans. A good correlation between the B- and T-cell epitopes recognised by humans and by immunised monkeys has been documented, and cross reactivity between reagents for human and Aotus cytokines and lymphocyte markers have been identified and are facilitating the selection of vaccine candidates for clinical trials. Aotus also represents a good model for the screening of anti-malarial drugs and the understanding of malaria pathogenesis as well. In view of the decreasing availability of these primates, breeding programs and biomedical research facilities must be improved in countries of primate origin.

Reproducible infection of intact Aotus lemurinus griseimembra monkeys by Plasmodium falciparum sporozoite inoculation.

Aotus lemurinus griseimembra is considered one of the best nonhuman primate species for malarial studies because of its susceptibility to infection by Plasmodium falciparum asexual blood stages. However, reproducible transmission of infective P. falciparum sporozoites by mosquito inoculation has been difficult to achieve even in splenectomized monkeys. Characterization of an Aotus-P. falciparum cyclical transmission model has become a top priority as a result of the significant progress toward the development of preerythrocytic malaria vaccines. Herein, we describe a reproducible model developed using intact A. lemurinus griseimembra monkeys intravenously inoculated with sporozoites from a monkey-adapted P. falciparum (Santa Lucia) strain and a wild Falciparum-Cali-Colombia-4 (FCC-4) strain. Sporozoites were obtained by salivary gland dissection of laboratory-reared Anopheles albimanus mosquitoes. Parasitemia was monitored by thick-smear microscopy, parasite lactate dehydrogenase (pLDH) determination, and mosquito xenodiagnosis. The last method proved to be the most sensitive method for monitoring parasitemias. Infection with the Santa Lucia strain showed a mean prepatent period of 16 days (range 6-21 days), whereas infection with the wild FCC-4 strain resulted in a 24-day prepatent period. Mean peak parasite density was approximately 900 parasites/microliter for both parasite strains. The prepatent period, the peak of parasitemia, and the duration of patency were independent of the size of the sporozoite inoculum and the presence of spleen in the host. This model is being successfully used to test the protective efficacy of P. falciparum preerythrocytic vaccine candidates.

Capacidad inhibidora del crecimiento intraeritrocitico del Plasmodium falciparum en sueros de areas malaricas / Inhibitory capability of intraeritrocitic growth of Plasmodium falciparum in serum from malaric areas

Se describe el fenomeno de inhibicion del crecimiento intraeritrocitico de Plasmodium falciparum in vitro producido por factores solubles de tipo no anticuerpo presentes en el suero de individuos de areas malaricas de la costa pacifica colombiana, fenomeno denominado "crisis". Se determinaron en sueros de 80 individuos, los titulos de anticuerpos antimalaricos por la tecnica de inmunofluorescencia indirecta y la capacidad inhibitora del crecimiento intraeritrocitico del parasito por el metodo de incorporacion de hipoxantina radiomarcada