Lithium salts as a treatment for COVID-19: Pre-clinical outcomes.

INTRODUCTION: Identifying effective drugs for Coronavirus disease 2019 (COVID-19) is urgently needed. An efficient approach is to evaluate whether existing approved drugs have anti-SARS-CoV-2 effects. The antiviral properties of lithium salts have been studied for many years. Their anti-inflammatory and immune-potentiating effects result from the inhibition of glycogen synthase kinase-3. AIMS: To obtain pre-clinical evidence on the safety and therapeutic effects of lithium salts in the treatment of COVID-19. RESULTS: Six different concentrations of lithium, ranging 2-12 mmol/L, were evaluated. Lithium inhibited the replication of SARS-CoV-2 virus in a dose-dependent manner with an IC50 value of 4 mmol/L. Lithium-treated wells showed a significantly higher percentage of monolayer conservation than viral control, particularly at concentrations higher than 6 mmol/L, verified through microscopic observation, the neutral red assay, and the determination of N protein in the supernatants of treated wells. Hamsters treated with lithium showed less intense disease with fewer signs. No lithium-related mortality or overt signs of toxicity were observed during the experiment. A trend of decreasing viral load in nasopharyngeal swabs and lungs was observed in treated hamsters compared to controls. CONCLUSIONS: These results provide pre-clinical evidence of the antiviral and immunotherapeutic effects of lithium against SARS-CoV-2, which supports an advance to clinical trials on COVID-19's patients.

Antifungal and immunomodulatory activity of a novel cochleate for amphotericin B delivery against Sporothrix schenckii.

INTRODUCTION: Sporotrichosis is an emergent subcutaneous mycoses caused by species of the Sporothrix schenckii complex. Amphotericin B (AmB) remains the main antifungal drug for the treatment of systemic infections, but its use is limited by toxicity reasons. AFCo3 is a novel cochleate containing detoxified LPS, which exhibits drug delivery and immunomodulating properties. Here, AFCo3 was used as the vehicle for AmB to evaluate the immunomodulatory and antifungal efficacy against S. schenckii in vitro and in vivo. METHODS AND RESULTS: The minimum inhibitory concentrations of AFCo3-AmB and AmB were 0.25 and 1µg/mL respectively. The minimum fungicidal concentration was 0.5µg/mL for AFCo3-AmB and 2µg/mL for AmB. AFCo3-AmB was less cytotoxic than AmB for peritoneal macrophages, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and reduced the AmB-induced hemolysis in murine erythrocytes. AFCo3-AmB improved the intracellular killing of phagocytized yeast and it enhanced the in vitro production of IL-1ß, TNF-α and NO in peritoneal macrophages. Moreover, AFCo3-AmB was more effective than AmB in reducing spleen and liver fungal burden after repeated (five days) intraperitoneal administration of 5mg/kg of AmB, in a Balb/c model of systemic infection, associated to a significant induction of Th1/Th17 response. Finally, blood chemistry revealed that AFCo3-AmB did not cause changes suggestive of nephrotoxicity, such as increases in total proteins, albumin, creatinine and blood urea nitrogen that were caused by free AmB. CONCLUSIONS: AFCo3-AmB exhibited a significant immunomodulator action, reduced toxicity and improved antifungal action against S. schenckii, suggesting a potential use as AmB delivery for systemic sporotrichosis treatment.

Human prophylactic vaccine adjuvants and their determinant role in new vaccine formulations

Adjuvants have been considered for a long time to be an accessory and empirical component of vaccine formulations. However, accumulating evidence of their crucial role in initiating and directing the immune response has increased our awareness of the importance of adjuvant research in the past decade. Nevertheless, the importance of adjuvants still is not fully realized by many researchers working in the vaccine field, who are involved mostly in the search for better target antigens. The choice of a proper adjuvant can be determinant for obtaining the best results for a given vaccine candidate, but it is restricted due to intellectual property and know-how issues. Consequently, in most cases the selected adjuvant continues to be the aluminum salt, which has a record of safety, but predominantly constitutes a delivery system (DS). Ideally, new strategies should combine immune potentiators (IP) and DS by mixing both compounds or by obtaining structures that contain both IP and DS. In addition, the term immune polarizer has been introduced as an essential concept in the vaccine design strategies. Here, we review the theme, with emphasis on the discussion of the few licensed new adjuvants, the need for safe mucosal adjuvants and the adjuvant/immunopotentiating activity of conjugation. A summary of toxicology and regulatory issues will also be discussed, and the Finlay Adjuvant Platform is briefly summarized.

Human prophylactic vaccine adjuvants and their determinant role in new vaccine formulations.

Adjuvants have been considered for a long time to be an accessory and empirical component of vaccine formulations. However, accumulating evidence of their crucial role in initiating and directing the immune response has increased our awareness of the importance of adjuvant research in the past decade. Nevertheless, the importance of adjuvants still is not fully realized by many researchers working in the vaccine field, who are involved mostly in the search for better target antigens. The choice of a proper adjuvant can be determinant for obtaining the best results for a given vaccine candidate, but it is restricted due to intellectual property and know-how issues. Consequently, in most cases the selected adjuvant continues to be the aluminum salt, which has a record of safety, but predominantly constitutes a delivery system (DS). Ideally, new strategies should combine immune potentiators (IP) and DS by mixing both compounds or by obtaining structures that contain both IP and DS. In addition, the term immune polarizer has been introduced as an essential concept in the vaccine design strategies. Here, we review the theme, with emphasis on the discussion of the few licensed new adjuvants, the need for safe mucosal adjuvants and the adjuvant/immunopotentiating activity of conjugation. A summary of toxicology and regulatory issues will also be discussed, and the Finlay Adjuvant Platform is briefly summarized.

Respuesta inmune sistémica y mucosal contra Neisseria meningitidis B inducida por estrategia de vacunación simultànea mucosal y parental

Immunization is one of the most successful and cost-effective health interventions ever. Immunization have been helping to reduce child mortality, improving maternal health and combating infectious diseases. In spite of its, undisputed past success and promising future, however, immunization remains an unfinished agenda because of them inadequate coverage. Several factors have been largely responsible of a difficulty to attain immunization coverage and have been recognized as a problems of current vaccines, such as: the number of dose, excessive use of parenteral route, a small number of adjuvants approve for use in human, higher reactogenicity and unavailability against intracellular pathogens, infected or altered cells and scanty feasibility to combined more than one antigen in the same formulation. For bacterial meningitis WHO estimates that 1,2 million cases occur annually and Neisseria meningitidis is the etiological agent in more than 40 percent of these cases although some meningococcal vaccines are available. To bear in mind these principals problems, a novel protocol for vaccination against N meningitidis called Single Time Vaccination Strategy (SinTimVaS) is proposed. Using female BALB/c mice, we induce systemic and mucosal immune responses against N meningitidis with only one parenteral and one mucosal dose at the same time, employing the Finlay Adjuvants derivate from N meningitidis, AFPL1 and AFCo1, respectively. In conclusion, SinTimVaS could increase the vaccination coverage and reduce the time-cost of vaccine campaigns, adding the possibility to increase the herd immunity by mucosal specific response induction(AU)

Enfoques mucosales en vacunologia de Neisseria

Meningococcal B strains accounts for some 72 percent and 28 percent of meningococcal diseases in infants and toddlers in Europe and the USA, respectively. Nevertheless, meningococcal diseases are rare in Cuba owing to the wide spread program on antimeningococcal vaccination in the country. Finlay Institute is one of the pioneering organizations in Neisseria Vaccinology mainly by its contribution to N. meningitidis serogroup B outer membrane-based bivalent vaccine, VA-MENGOC-BC™. This vaccine was given intramuscularly in more than 60 million doses corresponding 10,7 millions of them to Cuban young adults, children, and infants. However, most dangerous or commensally Neisseria strains enter and establish in the mucosa, where the secretory (S) IgA is the main specific guardian and is mainly induced by mucosal routes. However, few mucosal vaccines exist principally due to the absent of mucosal adjuvants. We develop a Finlay Adjuvant (AF) platform based in outer membrane vesicles (Proteoliposome, PL) and its derivate Cochleate (Co). AFPL1 derived from serogroup B N meningitidis is a potent Th1/CTL driving parenteral adjuvant. AFCo1 is a potent mucosal adjuvant. Therefore, we sought to go deeper in the possible mucosal cross recognition between N. meningitidis serogroups and Neisseria species and explore a concurrent mucosal and parenteral immunization strategy (SinTimVaS) in order to develop suitable mucosal vaccines. Experiments were conducted in Balb/c or C57Bl6 mice with mucosal and systemic immunization using AFCo1 and AFPL1. Human sera and saliva were also analyzed for cross cognition. Mucosal cross recognition at SIgA level in human saliva between N. meningitidis serogroups B, A, C, Y, and W135 were observed. This SIgA cross recognition response was also observed between pathogenic (N meningitidis serogroup B, N gonorrhoeae) and non-pathogenic strains (N flava, N lactamica). The possible influence of meningococcal vaccination ...(AU)

A vaccine against S. pyogenes: design and experimental immune response.

Streptococcus pyogenes causes severe invasive infections: the post-streptococcal sequelae of acute rheumatic fever (RF) and rheumatic heart disease (RHD), acute glomerulonephritis, and uncomplicated pharyngitis and pyoderma. Efforts to produce a vaccine against S. pyogenes began several decades ago, and different models have been proposed. Here, we describe the methodology used in the development of a new vaccine model, consisting of both T and B protective epitopes constructed as synthetic peptides and recombinant proteins. Two adjuvants were tested in an experimental inbred mouse model: a classical Freund's adjuvant and a new adjuvant (AFCo1) that induces mucosal immune responses and is obtained by calcium precipitation of a proteoliposome derived from the outer membrane of Neisseria meningitides B. The StreptInCor vaccine epitope co-administrated with AFCo1 adjuvant induced mucosal (IgA) and systemic (IgG) antibodies as preferential Th1-mediated immune responses. No autoimmune reactions were observed, suggesting that the vaccine epitope is safe.

New vaccines require potent adjuvants like AFPL1 and AFCo1.

Neisseria meningitidis B proteoliposome (AFPL1 when used as adjuvant) and its derivative-Cochleate (AFCo1) contain immunopotentiating and immunomodulating properties and delivery system capacities required for a good adjuvant. Additionally, they contain meningococcal protective antigens and permit packaging of other antigens and pathogen-associated molecular patterns (PAMP). Consequently, we hypothesized that they would function as good vaccine adjuvants for their own antigens and also for non-related antigens. AFPL1 is a detergent-extracted outer membrane vesicle of N. meningitidis B transformed into AFCo1 in calcium environment. Both are produced at Finlay Institute under good manufacture practices (GMP) conditions. We show their exceptional characteristics: combining in the same structure, the potentiator activity, polarizing agents and delivery system capacities; presenting multimeric protein copies; containing multiprotein composition and multi and synergistic PAMP components; acting with incorporated or co-administrated antigens; inducing type I IFN-gamma and IL-12 cytokines suggesting the stimulation of human plasmocytoid precursor and conventional dendritic cells, respectively, inducing a preferential Th1 immune response with TCD4(+), TCD8(+), cross-presentation and cytotoxic T-lymphocyte (CTL) in vivo responses; and functioning by parenteral and mucosal routes. AFPL1-AFCo1 protective protein constitutions permit per se their function as a vaccine. In addition to Phase IV Men BC vaccine, AFPL1 has ended the preclinical stage in an allergy vaccine and is concluding the preclinical stage of a nasal meningococcal vaccine. In conclusion, AFPL1 and AFCo1 induced signal 1, 2 and 3 polarizing to a Th1 (including CTL) response when they acted directly as vaccines or were used as adjuvants with incorporated or co-administered antigens by parenteral or mucosal routes. Both are very promising adjuvants.

Anticuerpos IgA anti-lipopolisacárido inducidos por candidato vacunal oral 638 vivo / Mucosal IgA anti-lipopolysaccharide antibodies induced by 638 oral live attenuated candidate vaccine

La cepa de Cólera atenuada 638 ha inducido una buena respuesta en modelos animales y en un estudio piloto humano ha probado ser segura e inmunogénica. Sin embargo, no ha sido evaluada la IgA específica en mucosas ni tampoco se ha comparado la respuesta inducida por la cepa 638 con aquélla inducida por la conocida cepa reactogénica JBK70. Por ello, fueron evaluadas las células secretoras de anticuerpos (ASC) anti-lipopolisacárido (LPS) sanguíneas y los anticuerpos antiLPS en saliva como indicadores de respuestas mucosas de voluntarios inoculados con las cepas 638 o JBK70. Con vistas a determinar la producción local o no de la IgA específica, la cinética de los anticuerpos IgA anti-LPS séricos y salivares fueron comparados. La respuesta vibriocida sérica fue también medida. Tres grupos con 638 (109, 108 y 107 unidades form adoras de colonias, CFU), uno con JBK70 (109 CFU) y otro con placebo fueron enrolados. La respuesta sérica de ASC IgA+ fue mayor que la de ASC IgG+.La IgA anti-LPS en saliva tuvo valores máximos a los 9 días y decayó hasta valores negativos en el día 14 después de la inoculación. La IgA anti-LPS sérica permanece elevada entre los 7 y 28 días después de la inoculación lo que sugiere que la IgA en saliva es localmente y transitoriamente producida. La respuesta vibriocida sérica fue incrementada después de la inoculación. Respuestas similares fueron obtenidas con las cepas 638 y JBK70 (AU)

Larga duración de la respuesta inmunitaria celular inducida en lactantes y niños inmunizados con vacuna proteoliposómica antimeningocócica BC / Long-lasting cellular immune response in babies, children, and pre-teenagers vaccinated with a proteoliposome based anti-meningococcal BC vaccine

VA-MENGOC-BC® es una vacuna contra los sero grupos B y C de Neisseria meningitidis. La respuesta humoral ha sido extensivamente evaluada pero no la respuesta celular. Estudios prospectivos y retrospectivos fueron realizados para especificar la inducción y duración de la respuesta inmunitaria. El estudio prospectivo fue llevado a cabo en 62 lactantes usando un test de hipersensibilidad retardada (DTH) antes de la primera (3,5 meses de edad), junto a la segunda (42 días después) y 28 días después de la segunda dosis. En los lactantes, la DTH fue negativa antes y 100 por ciento positiva después de la vacunación. El estudio retrospectivo incluyó 535 niños que habían sido vacunados entre 2 y 7 años antes. La positividad de la DTH fue de 100 por ciento en todos los grupos. En los niños vacunados 5 ó 7 años antes, las técnicas de linfoproliferación (LP) y las células secretoras de anticuerpos (ASC) fueron también determinadas. La LP fue positiva en el 26 y 34 por ciento antes de la dosis de re fuerzo en los niños vacunados de 5 y 7 años, respectivamente y decreció posteriormente. Los ASC fueron negativos antes de la dosis de refuerzo y generalmente positivos 7 días después de ésta. No obstante, en los niños vacunados hacía 7 años el 12 por ciento tuvo una pequeña cantidad de ASC antes del refuerzo, los cuales pudieran estar relacionados con la alta frecuencia de circulación de Neisseria en la población o de microorganismos con reactividad cruzada. El mayor incremento en los ASC después del refuerzo (desde 0,73 hasta 166,24 x 106 PBMC) fue observado en aquellos negativos que tenían bajos números de ASC antes del refuerzo al compararlos con los totalmente negativos (desde 0 hasta 67,7 x 106 PBMC). Estos resultados muestran claramente la inducción de respuesta celular en lactantes, la persistencia de respuesta celular en los grupos vacunados hacía tiempo y la memoria de larga duración detectada por una tercera dosis (AU)

Immune response induction and new effector mechanisms possibly involved in protection conferred by the Cuban anti-meningococcal BC vaccine.

This report explores the participation of some afferent mechanisms in the immune response induced by the Cuban anti-meningococcal vaccine VA-MENGOC-BC. The induction of delayed-type hypersensitivity in nursing babies and lymphocyte proliferation after immunization is demonstrated. The presence of gamma interferon IFN-gamma and interleukin-2 (IL-2) mRNAs but absence of IL-4, IL-5, and IL-10 mRNAs were observed in peripheral blood mononuclear cells from immunized subjects after in vitro challenge with outer membrane vesicles. In addition, some effector functions were also explored. The presence of opsonic activity was demonstrated in sera from vaccinees. The role of neutrophils as essential effector cells was shown. In conclusion, we have shown that, at least in the Cuban adult population, VA-MENGOC-BC induces mechanisms with a T-helper 1 pattern in the afferent and effector branches of the immune response.

Nitric oxide participates in the immune response against Neisseria meningitidis serogroup B.

The present report explores the role of nitric oxide into the immune response against Neisseria meningitidis serogroup B. Here we show that NO mediates the alphaTNF increase induced by N. meningitidis derived lipopolysaccharides (LPS), at the same time that participates in the bactericidal activity of resting or gammaIFN activated macrophages and plays a role in the specific DTH and IgG response induced by a commercial anti-meningococcal vaccine. Our findings suggest a positive role for NO at the final effector mechanisms and in the early events driving the immunity against N. meningitidis, suggesting also an insight into its role in endotoxic shock.

Preliminary assessment of the safety and immunogenicity of a new CTXPhi-negative, hemagglutinin/protease-defective El Tor strain as a cholera vaccine candidate.

Vibrio cholerae 638 (El Tor, Ogawa), a new CTXPhi-negative hemagglutinin/protease-defective strain that is a cholera vaccine candidate, was examined for safety and immunogenicity in healthy adult volunteers. In a double-blind placebo-controlled study, no significant adverse reactions were observed in volunteers ingesting strain 638. Four volunteers of 42 who ingested strain 638 and 1 of 14 who received placebo experienced loose stools. The strain strongly colonized the human small bowel, as evidenced by its isolation from the stools of 37 of 42 volunteers. V. cholerae 638, at doses ranging from 4 x 10(7) to 2 x 10(9) vibrios, elicited significant serum vibriocidal antibody and anti-Ogawa immunoglobulin A antibody secreting cell responses.

Evaluation of the immune response in symptomatic and asymptomatic human giardiasis.

To better understand the common association of Giardia lamblia infection and allergic reactivity, total and specific IgE values were evaluated and different manifestations of symptomatic and asymptomatic infected human hosts were analyzed. The humoral, cellular, and nonspecific immune responses were evaluated in Cuban adults. Increased total serum IgE levels were significantly higher (p < 0.01) in Giardia patients than in negative controls; cure of giardiasis was characterized by a decrease in IgE levels and some patients regained normal IgE values. The skin test was positive in 91% (103/123) of chronic patients and only in 23% (20/123) of negative controls (p < 0.05). A positive test was seen in patients with antecedents of recent giardiasis (< 4 months). Specific IgE was higher in patients than in control sera, and in the former it decreased with sera dilution. During the follow-up period of cured patients, the proportion of IgE decreased and the opposite occurred in noncured patients. The cellular response evaluated by LIF was positive in 92% (11/12) of carriers and significantly higher (p < 0.05) than in symptomatic patients 8% (1/12); the same occurred with IgG and IgA antibody response; titers mainly of IgA were higher in asymptomatic carriers than in patients; all carriers were negative to the skin test. These results indicate the presence of total and specific IgE responses in humans infected with Giardia, but the response in symptomatic cases (patients) is different from that in asymptomatic cases (carriers).(ABSTRACT TRUNCATED AT 250 WORDS)

Fasciolasis humana epidémica. Cuba 1983. VI. Estudio clínico de 40 niños del Hospital provincial de Sagua la Grande / Epidemic human fascioliasis. Cuba 1983. VI. Clinical study of 40 children in the Hospital Provincial de Sagua la Grande

Se estudiaron 40 niños con "Síndrome Eosinofílico Febril" en los cuales se confirmó la infección por Fasciola Hepática por presentar la triada clásica: fiebre, eosinofilia y dolor abdominal; tener antecedentes de ingestión de berro (82%), laparoscopia y biopsias positivas (100%) y pruebas de intradermoreacción inmediata (100%) y electrocinéresis (100%) positivas a antígenos de Fasciola. Este trabajo demuestra la afectación de los niños durante el brote epidémico de Cuba en 1983

[Epidemic human fascioliasis. Cuba 1983. VI. Clinical study of 40 children in the Hospital Provincial of Sagua la Grande]. / Fascioliasis humana epidémica. Cuba 1983. VI. Estudio clínico de 40 niños del Hospital Provincial de Sagua la Grande.

We studied forty children with "Febrile-eosinophilic-Syndrome". They presented Fasciola Hepatic infection confirmed by: Fever, high eosinophils and abdominal pain. 82% used to eat watercress. All of them had positive biopsies-laparoscopies-intradermo-reaction and also reaction to Fasciola antigen. We present the results of and epidemic in Cuba during 1983.

[A group of African students parasitized with Mansonella perstans in Cuba (a non-endemic area) from 1979 to 1982]. / Estudio de un grupo de estudiantes Africanos parasitados con Manzonella perstans en Cuba (área no endémica) durante 1979 a 1982.

Sixty cases of Manzonella perstans were studied during a three year period by clinical and blood parasitologic studies. It was observed that 31.2 was negative and that mean microfilaremia decreased from 49.3, at the beginning of the study, to 32.9 at the end of it, in those cases yet positive. According to the persistent microfilaremia, which means a relative longevity of adult parasites, the surveillance of individuals suffering it is recommended at their arrival to a non-endemic area where the vectors of such disease are present.

[Indirect immunofluorescence in filariasis. II. Humoral response in a population living close to a patient with confirmed Wuchereria bancrofti (Nematoda: Filarioidea), Cuba, 1983]. / Inmunofluorescencia indirecta en filariasis. II. Respuesta humoral en una población alrededor de un paciente confirmado con Wuchereria bancrofti (Nematoda: Filarioidea), Cuba, 1983.

This paper deals with the study by Knott technique, membrane filter and indirect immunofluorescence, of 256 individuals living in Pinar del Rio City, neighbors of a patient suffering elephantiasis produced by Wuchereria bancrofti, 111 donors of the blood bank in the same city, and 10 patients coming from endemic areas who were hospitalized at the "Pedro Kouri" Tropical Medicine Institute, with certainty diagnosis of bancroftiasis. Circulating microfilariae were not found in the neighbors of the positive case, however, a high antifilaria antibody titer (1:512) was observed in the neighbors, suggesting it that in any time this population could have been in contact with this parasite.

[Indirect immunofluorescence in filariasis. I. Standardization of the technique. Cuba, 1983]. / Inmunofluorescencia indirecta en filariasis. I. Normalizacion de la tecnica. Cuba, 1983.

Indirect immunofluorescence was standardized for the diagnosis of human filiariae, using antigens of Dipetalonema viteae. Due to the determined sensibility and specificity, the tier of 1:512 was recommended for aiding clinicians in the individual diagnosis of suspicious patients and that of 1:256 for epidemiologic studies. The titers of cross reactions were less than 1:128.

[Indirect immunofluorescence in filariasis. III. Comparison of microfilaremia and treatment. Cuba, 1983]. / Inmunofluorescencia indirecta en filariasis. III. Comparacion de la microfilaremia y el tratamiento. Cuba, 1983.

Twenty six patients coming from endemic areas, with diagnosis of filariasis caused by Wuchereria bancrofti, Loa loa and Manzonella (Dipetalonema perstans) and 29 patients suspicious of suffering filariasis were studied by means of Knott, membrane filter and indirect immunofluorescence techniques. Results of microfilaremia were compared with antibody titers and it was verified that there was not relationship between these parameters. The suspicious patients presented higher antibody levels than those of the verified patients. In additions, a preliminary study was carried out on the serologic behaviour of some patients treated with diethylcarbamazine (6 mg/kg daily, during 14 days) and increase of antibody titers was observed in most of the cases after three days and a decrease after the first month of treatment.