Zehn Jahre nach dem Ausbruch des Severe Acute Respiratory Syndrome Coronavirus, SARS-CoV, ist ein neues Betacoronavirus, das Middle East Respiratory Syndrome Coronavirus, MERS-CoV, auf der arabischen Halbinsel entdeckt worden. Seine anhaltende Ausbreitung stellt eine Bedrohung für die öffentliche Gesundheit dar. Das Spike (S) Protein der Coronaviren vermittelt den viralen Eintritt in Wirtszellen und bestimmt wesentlich den viralen Tropismus und die virale Pathogenese. Das Verständnis der Determinanten des MERS-CoV Spike (MERS-S)-vermittelnden Eintritts in Zellen könnte daher wichtige Einblicke in die MERS-CoV-Biologie liefern und war somit das erste Ziel dieser Arbeit. Um den Eintritt in die Zelle zu ermöglichen, muss das Coronavirus S-Protein durch Wirtszellproteasen aktiviert werden, welche potentielle Ziele für die therapeutischen Intervention darstellen. Daher sollten im zweiten Ziel dieser Arbeit Proteasen identifiziert werden, die MERS-S aktivieren. Das S-Protein ist das Hauptangriffsziel neutralisierender Antikörper und experimentelle Systeme zur S-Analyse können für die Diagnostik eingesetzt werden. Das letzte Ziel dieser Arbeit war es daher, die MERS-CoV Seroprävalenz in Saudi Arabien zu ermitteln. Es wurde ein lentivirales Vektorensystem etabliert, welches die Analyse des MERS-S-getriebenen Zelleintritts ermöglicht. Mit Hilfe dieses Systems konnte gezeigt werden, dass MERS-S den Eintritt in ein breites Spektrum humaner Zelllinien, wie Lungen-, Nieren- und Darmzellen vermittelt, was mit der klinischen Manifestation von MERS einhergeht. Der Wirtszelleintritt war unabhängig von bereits beschriebenen Coronavirus Eintrittsrezeptoren, wurde jedoch durch die endosomale Cysteinprotease Cathepsin L und die Transmembranserinprotease TMPRSS2 gefördert. Im Gegensatz dazu war die Aktivität von Proprotein Konvertasen für den S-Protein-vermittelnden Eintritt entbehrlich. Schließlich zeigten Neutralisationstests, dass Seren von Patienten aus der östlichen Provinz Saudi Arabiens, die zwischen 2010-2011 und 2012 entnommen wurden, keine MERS-S-neutralisierenden Antikörper enthielten. Dies deutet darauf hin, dass MERS-CoV-Infektionen vor dem Ausbruch 2012 nur selten vorkamen. Die gewonnen Ergebnisse tragen wesentlich zum Verständnis des MERS-CoV-Eintritts in Zellen bei und liefern wichtige Informationen zur MERS-CoV-Epidemiologie. Weiterhin könnte die Beobachtung, dass der Protease-Inhibitor Camostat, der für den Einsatz im Menschen zugelassen ist (in Japan), TMPRSS2 blockiert und damit den MERS-CoV Eintritt inhibiert, helfen, Behandlungsstrategien für MERS-Patienten zu etablieren.

O leite é um alimento sujeito à fraudes. As mais frequentes são a adição de água, reconstituintes, conservantes e neutralizantes. Apesar da legislação determinar a pesquisa diária dessas substâncias, a avaliação do leite pelas indústrias geralmente é realizada apenas por análises físico-químicas genéricas como densidade e crioscopia. Contudo, essas fraudes muitas vezes são calculadas para impedir sua identificação por provas de rotina não específicas. O objetivo desse trabalho foi pesquisar a ocorrência de fraudes, resíduos de antibióticos e irregularidades físico-químicas e microbiológicas em leite pasteurizado, bem como avaliar a capacidade de detecção de adulterações e a suficiência das provas oficiais específicas e inespecíficas na identificação da adição de reconstituintes, conservantes e neutralizantes. Foram avaliadas 100 amostras de leite pasteurizado, produzido no Paraná, para a presença de reconstituintes, conservantes, neutralizantes e antibióticos e demais parâmetros físico-químicos e microbiológicos determinados pela legislação. Em outro experimento, a capacidade de detecção das provas oficiais para pesquisa de sacarose, cloretos, amido, formaldeído, cloro, hipoclorito, peróxido de hidrogênio e neutralizantes da acidez foi avaliada utilizando-se de amostras adulteradas em laboratório. Foram avaliados adicionalmente testes não oficiais para avaliar a presença de inibidores do crescimento microbiano e o efeito inibidor de algumas substâncias conservantes e neutralizantes sobre a microbiota do leite cru. Os resultados mostraram que das 100 amostras de leite pasteurizado, 51% apresentaram problemas, das quais 25% estavam fora dos padrões nas análises físico-químicas e 10% nas análises microbiológicas, 14% apresentaram resíduos de antibióticos e a adição de reconstituintes foi detectada em 13% das amostras. Observou-se que a avaliação do leite apenas por análises físico-químicas de rotina, como densidade e crioscopia não é suficiente para identificar fraudes por adição de água e reconstituintes. As provas para a pesquisa específica de substâncias reconstituintes apresentaram boa capacidade de detecção, assim como a prova para pesquisa de formaldeído. A adição de sacarose em amostras adulteradas no laboratório elevou a porcentagem de lactose detectada por equipamentos que utilizam infravermelho e ultrassom, demonstrando que estes equipamentos não quantificam especificamente a lactose. Quanto às amostras adicionadas de conservantes e neutralizantes no laboratório, a prova inespecífica da cultura de iogurte foi capaz de detectar a inibição de crescimento nas amostras adicionadas de formaldeído, peróxido de hidrogênio, hipoclorito e hidróxido de sódio, apresentando resultados próximos aos das provas oficiais para a pesquisa dessas substâncias. A adição de formaldeído, peróxido de hidrogênio e hipoclorito promoveu reduções significativas nas contagens de micro-organismos aeróbios mesófilos no leite cru adulterado no laboratório. o entanto, não foi possível detectar peróxido de hidrogênio e compostos clorados após 24 horas de refrigeração, provavelmente em consequência da degradação e /ou inativação dessas substâncias no leite. A prova para a pesquisa de neutralizantes da acidez detecta a substância apenas quando a alcalinização é excessiva. A neutralização equilibrada do ácido lático resulta na anulação da capacidade de detecção da prova. A pesquisa de inibidores microbianos, neutralizantes e reconstituintes é obrigatória apenas para leite cru, porém, a presença de grande parcela de amostras positivas para reconstituintes e antimicrobianos no leite pasteurizado demonstra falhas no controle de qualidade e pode indicar a prática de adulteração do leite também pela indústria. / Milk is one of the major targets for fraud. The most frequent are addition of water, restoratives, preservatives and neutralizers. Although legislation determines the daily survey of these substances by specific tests, milk evaluation by industries is usually performed only by generic physicochemical tests as density and freezing point. However, these adulterations are often calculated to prevent their identification by these routine non specific tests. The aim of this study was to investigate the occurrence of fraud, antibiotic residues and irregularities in physico-chemical and microbiological parameters in pasteurized milk produced in northern Paraná, as well as to evaluate the ability of adulteration detection by official tests to identify the addition of restoratives, preservatives and neutralizers in milk. An assessement was performed in 100 pasteurized milk samples to evaluate the presence of restoratives, preservatives, antibiotics and neutralizers and other physico-chemical and microbiological tests determined by legislation. In a second experiment, the detection ability of official tests for the research of sucrose, chloride, starch, formaldehyde, chlorine, hypochlorite, hydrogen peroxide and neutralizers was evaluated using samples adultered in laboratory. Additionally, unofficial tests to evaluate the presence of inhibitors of microbial growth and the inhibitory effect of some preservative and neutralizers substances on the biota of raw milk were performed. Results show that of the 100 samples of pasteurized milk evaluated, 51% had problems, from which 25% were outside parameters for physicochemical analyzes, 10% for microbiological analyzes, 14% were positive for the presence of antibiotic and in 13% adulteration by restoratives were detected. It was observed that milk evaluation only by physico-chemical routine analyzes, such as density and cryoscopy is not sufficient to identify fraud by water and restoratives addition. Specific tets for restoratives detection exhibit good detection potentials, as well as the test for formaldehyde. . The addition of sugar altered quantification of lactose detected by equipments tha use infrared and ultrasound, demonstrating that these do not detect lactose exclusively. Regarding samples added with preservatives and neutralizers substances in laboratory, the yogurt culture test was able to detect microbial grouth inhibition in samples added with formaldehyde, hydrogen peroxide, hypochlorite and sodium hydroxide. Presenting resluts similar to the official tests. The addition of formaldehyde, hydrogen peroxide and hypochlorite promoted significant reductions in the counts of mesophilic aerobic micro-organisms in raw milk adulterated in laboratory. However it was not possible to detect hydrogen peroxide and clhorated compounds after 24 hours of refrigeration, maybe due to its rapid degradation and / or inactivation of these substances in milk. Neutralizing substances detection can only dectect this substances when alcalinization is excessive. The balanced neutralization of latic acid results on the anulment of detection ability of the test. The research of preservatives, neutralizers and restoratives is not required for pasteurized milk, only for raw milk. The presence of large numbers of samples positive for these substances demonstrates flaws in quality control of raw milk or occurrence of milk adulteration by the industry itself.

O leite é um alimento sujeito à fraudes. As mais frequentes são a adição de água, reconstituintes, conservantes e neutralizantes. Apesar da legislação determinar a pesquisa diária dessas substâncias, a avaliação do leite pelas indústrias geralmente é realizada apenas por análises físico-químicas genéricas como densidade e crioscopia. Contudo, essas fraudes muitas vezes são calculadas para impedir sua identificação por provas de rotina não específicas. O objetivo desse trabalho foi pesquisar a ocorrência de fraudes, resíduos de antibióticos e irregularidades físico-químicas e microbiológicas em leite pasteurizado, bem como avaliar a capacidade de detecção de adulterações e a suficiência das provas oficiais específicas e inespecíficas na identificação da adição de reconstituintes, conservantes e neutralizantes. Foram avaliadas 100 amostras de leite pasteurizado, produzido no Paraná, para a presença de reconstituintes, conservantes, neutralizantes e antibióticos e demais parâmetros físico-químicos e microbiológicos determinados pela legislação. Em outro experimento, a capacidade de detecção das provas oficiais para pesquisa de sacarose, cloretos, amido, formaldeído, cloro, hipoclorito, peróxido de hidrogênio e neutralizantes da acidez foi avaliada utilizando-se de amostras adulteradas em laboratório. Foram avaliados adicionalmente testes não oficiais para avaliar a presença de inibidores do crescimento microbiano e o efeito inibidor de algumas substâncias conservantes e neutralizantes sobre a microbiota do leite cru. Os resultados mostraram que das 100 amostras de leite pasteurizado, 51% apresentaram problemas, das quais 25% estavam fora dos padrões nas análises físico-químicas e 10% nas análises microbiológicas, 14% apresentaram resíduos de antibióticos e a adição de reconstituintes foi detectada em 13% das amostras. Observou-se que a avaliação do leite apenas por análises físico-químicas de rotina, como densidade e crioscopia não é suficiente para identificar fraudes por adição de água e reconstituintes. As provas para a pesquisa específica de substâncias reconstituintes apresentaram boa capacidade de detecção, assim como a prova para pesquisa de formaldeído. A adição de sacarose em amostras adulteradas no laboratório elevou a porcentagem de lactose detectada por equipamentos que utilizam infravermelho e ultrassom, demonstrando que estes equipamentos não quantificam especificamente a lactose. Quanto às amostras adicionadas de conservantes e neutralizantes no laboratório, a prova inespecífica da cultura de iogurte foi capaz de detectar a inibição de crescimento nas amostras adicionadas de formaldeído, peróxido de hidrogênio, hipoclorito e hidróxido de sódio, apresentando resultados próximos aos das provas oficiais para a pesquisa dessas substâncias. A adição de formaldeído, peróxido de hidrogênio e hipoclorito promoveu reduções significativas nas contagens de micro-organismos aeróbios mesófilos no leite cru adulterado no laboratório. o entanto, não foi possível detectar peróxido de hidrogênio e compostos clorados após 24 horas de refrigeração, provavelmente em consequência da degradação e /ou inativação dessas substâncias no leite. A prova para a pesquisa de neutralizantes da acidez detecta a substância apenas quando a alcalinização é excessiva. A neutralização equilibrada do ácido lático resulta na anulação da capacidade de detecção da prova. A pesquisa de inibidores microbianos, neutralizantes e reconstituintes é obrigatória apenas para leite cru, porém, a presença de grande parcela de amostras positivas para reconstituintes e antimicrobianos no leite pasteurizado demonstra falhas no controle de qualidade e pode indicar a prática de adulteração do leite também pela indústria. / Milk is one of the major targets for fraud. The most frequent are addition of water, restoratives, preservatives and neutralizers. Although legislation determines the daily survey of these substances by specific tests, milk evaluation by industries is usually performed only by generic physicochemical tests as density and freezing point. However, these adulterations are often calculated to prevent their identification by these routine non specific tests. The aim of this study was to investigate the occurrence of fraud, antibiotic residues and irregularities in physico-chemical and microbiological parameters in pasteurized milk produced in northern Paraná, as well as to evaluate the ability of adulteration detection by official tests to identify the addition of restoratives, preservatives and neutralizers in milk. An assessement was performed in 100 pasteurized milk samples to evaluate the presence of restoratives, preservatives, antibiotics and neutralizers and other physico-chemical and microbiological tests determined by legislation. In a second experiment, the detection ability of official tests for the research of sucrose, chloride, starch, formaldehyde, chlorine, hypochlorite, hydrogen peroxide and neutralizers was evaluated using samples adultered in laboratory. Additionally, unofficial tests to evaluate the presence of inhibitors of microbial growth and the inhibitory effect of some preservative and neutralizers substances on the biota of raw milk were performed. Results show that of the 100 samples of pasteurized milk evaluated, 51% had problems, from which 25% were outside parameters for physicochemical analyzes, 10% for microbiological analyzes, 14% were positive for the presence of antibiotic and in 13% adulteration by restoratives were detected. It was observed that milk evaluation only by physico-chemical routine analyzes, such as density and cryoscopy is not sufficient to identify fraud by water and restoratives addition. Specific tets for restoratives detection exhibit good detection potentials, as well as the test for formaldehyde. . The addition of sugar altered quantification of lactose detected by equipments tha use infrared and ultrasound, demonstrating that these do not detect lactose exclusively. Regarding samples added with preservatives and neutralizers substances in laboratory, the yogurt culture test was able to detect microbial grouth inhibition in samples added with formaldehyde, hydrogen peroxide, hypochlorite and sodium hydroxide. Presenting resluts similar to the official tests. The addition of formaldehyde, hydrogen peroxide and hypochlorite promoted significant reductions in the counts of mesophilic aerobic micro-organisms in raw milk adulterated in laboratory. However it was not possible to detect hydrogen peroxide and clhorated compounds after 24 hours of refrigeration, maybe due to its rapid degradation and / or inactivation of these substances in milk. Neutralizing substances detection can only dectect this substances when alcalinization is excessive. The balanced neutralization of latic acid results on the anulment of detection ability of the test. The research of preservatives, neutralizers and restoratives is not required for pasteurized milk, only for raw milk. The presence of large numbers of samples positive for these substances demonstrates flaws in quality control of raw milk or occurrence of milk adulteration by the industry itself.

Foi investigada a existência de proteção cruzada entre bacterinas bivalentes formuladas com um de três representantes do Sorogrupo Sejroe: hardjo (bacterina A), wolffi (bacterina B) e guaricura (bacterina C), e uma estirpe do sorovar pomona, empregada por ser patogênica para hamsters (Mesocricetus auratus) e possibilitar a realização do teste de potência com desafio. Os ensaios foram efetuados em hamsters machos, comparando-se os níveis de anticorpos aglutinantes e neutralizantes, respectivamente obtidos nos testes de soroaglutinação microscópica (SAM) e inibição de leptospiras in vitro (ICL). Os animais receberam duas doses de vacinas via subcutânea; aos dez dias da segunda dose, foram inoculados com culturas não inativadas dos respectivos sorovares do Sorogrupo Sejroe. Aos 21 dias pós-infecção (d.p.i.), os animais foram sangrados, os soros (n=8) foram agrupados em pools e submetidos aos testes de SAM e ICL. O teste de potência com desafio para o sorovar pomona foi adaptado do protocolo preconizado pelo United States Department of Agriculture, mas as vacinas não foram diluídas e o esquema de imunização empregou duas aplicações de 1,0mL pela via subcutânea em intervalo de dez dias; o desafio foi efetuado aos dez dias da segunda aplicação; os óbitos por leptospirose foram registrados e, aos 21 d.p.i., os sobreviventes foram sacrificados e a condição de portadores renais foi investigada através de cultivos de tecido renal para isolamento de leptospiras. No teste de potência com o sorovar pomona, o número de doses infectantes empregado para desafio (100) situou-se dentro da faixa preconizada (10 a 100); respectivamente para as bacterinas A, B e C, as proporções de mortes por leptospirose entre os animais vacinados foram de 1/10, 0/10 e 3/10, e as de portadores renais de leptospiras entre os sobreviventes foram 2/9, 1/10 e 2/7. Os resultados do teste SAM revelaram que a bacterina A induziu reações para os sorovares hardjo e wolffi; a bacterina B, para hardjo, wolffi e guaricura, e a bacterina C, apenas para a guaricura, e do teste de ICL, que animais vacinados com as bacterinas B ou C apresentaram proteção para hardjo, wolffi e guaricura; entretanto, a bacterina A conferiu proteção apenas para wolffi. Apesar das variações no poder imunogênico segundo a estirpe de leptospira empregada para a produção das bacterinas, houve proteção cruzada entre os sorovares hardjo, wolffi e guaricura. / The existence of cross-protection among bivalent bacterins, produced with one of three leptospires belonging to Serogroup Sejroe: hardjo (bacterin A), wolffi (bacterin B) and guaricura (bacterin C), and a strain of serovar pomona (included because of its pathogenicity to hamsters and the possibility of performing potency assay with challenge), was investigated in male hamsters (Mesocricetus auratus) by comparison of agglutinating and neutralizing antibodies titers, respectively measured by microscopic agglutination (MAT) and in vitro growth inhibition (GIT) tests. All animals received two doses of bacterins by subcutaneous route; after ten days from the second dose, they were inoculated with non-inactivated cultures of respective serovars of Serogroup Sejroe. At 21 post-challenge day (p.c.d.), all animals were bled and their sera (n=8) were joined in pools and tested by MAT and GIT. The potency assay with challenge performed only with serovar pomona was modified from protocol of USDA, but vaccines were not diluted and the immunization schedule employed two 1.0 mL vaccine doses by subcutaneous route with 10?day interval; the challenge was performed after ten days from the second dose; the number of deaths due to leptospirosis was registered; at 21 p.c.d., the survivors were sacrificed and their renal carrier state was investigated by culture of renal tissue for leptospires isolation. In the potency assay with serovar pomona, the number of infectious doses employed for challenge (100 infective units) was in accordance with the recommended range (10-1,000 infective units); respectively to bacterins A, B and C, proportions of deaths due to leptospirosis among vaccinated animals were 1/10, 0/10 and 3/10, and proportions of leptospires renal carrier among survivors were 2/9, 1/10 and 2/7. Results of MAT showed that bacterin A induced reactions against serovars hardjo and wolffi; bacterin B, against hardjo, wolffi and guaricura, and bacterin C, against guaricura; results of GIT revealed that vaccinated animals with bacterins B or C presented protection against serovar hardjo, wolffi and guaricura; however, bacterin A induced protection only against wolffi. A cross?protection was observed among serovars hardjo, wolffi and guaricura, although variations exist in the immunogenic capacity according to the strain of leptospires used for the bacterins production.

O leite é um alimento sujeito à fraudes. As mais frequentes são a adição de água, reconstituintes, conservantes e neutralizantes. Apesar da legislação determinar a pesquisa diária dessas substâncias, a avaliação do leite pelas indústrias geralmente é realizada apenas por análises físico-químicas genéricas como densidade e crioscopia. Contudo, essas fraudes muitas vezes são calculadas para impedir sua identificação por provas de rotina não específicas. O objetivo desse trabalho foi pesquisar a ocorrência de fraudes, resíduos de antibióticos e irregularidades físico-químicas e microbiológicas em leite pasteurizado, bem como avaliar a capacidade de detecção de adulterações e a suficiência das provas oficiais específicas e inespecíficas na identificação da adição de reconstituintes, conservantes e neutralizantes. Foram avaliadas 100 amostras de leite pasteurizado, produzido no Paraná, para a presença de reconstituintes, conservantes, neutralizantes e antibióticos e demais parâmetros físico-químicos e microbiológicos determinados pela legislação. Em outro experimento, a capacidade de detecção das provas oficiais para pesquisa de sacarose, cloretos, amido, formaldeído, cloro, hipoclorito, peróxido de hidrogênio e neutralizantes da acidez foi avaliada utilizando-se de amostras adulteradas em laboratório. Foram avaliados adicionalmente testes não oficiais para avaliar a presença de inibidores do crescimento microbiano e o efeito inibidor de algumas substâncias conservantes e neutralizantes sobre a microbiota do leite cru. Os resultados mostraram que das 100 amostras de leite pasteurizado, 51% apresentaram problemas, das quais 25% estavam fora dos padrões nas análises físico-químicas e 10% nas análises microbiológicas, 14% apresentaram resíduos de antibióticos e a adição de reconstituintes foi detectada em 13% das amostras. Observou-se que a avaliação do leite apenas por análises físico-químicas de rotina, como densidade e crioscopia não é suficiente para identificar fraudes por adição de água e reconstituintes. As provas para a pesquisa específica de substâncias reconstituintes apresentaram boa capacidade de detecção, assim como a prova para pesquisa de formaldeído. A adição de sacarose em amostras adulteradas no laboratório elevou a porcentagem de lactose detectada por equipamentos que utilizam infravermelho e ultrassom, demonstrando que estes equipamentos não quantificam especificamente a lactose. Quanto às amostras adicionadas de conservantes e neutralizantes no laboratório, a prova inespecífica da cultura de iogurte foi capaz de detectar a inibição de crescimento nas amostras adicionadas de formaldeído, peróxido de hidrogênio, hipoclorito e hidróxido de sódio, apresentando resultados próximos aos das provas oficiais para a pesquisa dessas substâncias. A adição de formaldeído, peróxido de hidrogênio e hipoclorito promoveu reduções significativas nas contagens de micro-organismos aeróbios mesófilos no leite cru adulterado no laboratório. o entanto, não foi possível detectar peróxido de hidrogênio e compostos clorados após 24 horas de refrigeração, provavelmente em consequência da degradação e /ou inativação dessas substâncias no leite. A prova para a pesquisa de neutralizantes da acidez detecta a substância apenas quando a alcalinização é excessiva. A neutralização equilibrada do ácido lático resulta na anulação da capacidade de detecção da prova. A pesquisa de inibidores microbianos, neutralizantes e reconstituintes é obrigatória apenas para leite cru, porém, a presença de grande parcela de amostras positivas para reconstituintes e antimicrobianos no leite pasteurizado demonstra falhas no controle de qualidade e pode indicar a prática de adulteração do leite também pela indústria. / Milk is one of the major targets for fraud. The most frequent are addition of water, restoratives, preservatives and neutralizers. Although legislation determines the daily survey of these substances by specific tests, milk evaluation by industries is usually performed only by generic physicochemical tests as density and freezing point. However, these adulterations are often calculated to prevent their identification by these routine non specific tests. The aim of this study was to investigate the occurrence of fraud, antibiotic residues and irregularities in physico-chemical and microbiological parameters in pasteurized milk produced in northern Paraná, as well as to evaluate the ability of adulteration detection by official tests to identify the addition of restoratives, preservatives and neutralizers in milk. An assessement was performed in 100 pasteurized milk samples to evaluate the presence of restoratives, preservatives, antibiotics and neutralizers and other physico-chemical and microbiological tests determined by legislation. In a second experiment, the detection ability of official tests for the research of sucrose, chloride, starch, formaldehyde, chlorine, hypochlorite, hydrogen peroxide and neutralizers was evaluated using samples adultered in laboratory. Additionally, unofficial tests to evaluate the presence of inhibitors of microbial growth and the inhibitory effect of some preservative and neutralizers substances on the biota of raw milk were performed. Results show that of the 100 samples of pasteurized milk evaluated, 51% had problems, from which 25% were outside parameters for physicochemical analyzes, 10% for microbiological analyzes, 14% were positive for the presence of antibiotic and in 13% adulteration by restoratives were detected. It was observed that milk evaluation only by physico-chemical routine analyzes, such as density and cryoscopy is not sufficient to identify fraud by water and restoratives addition. Specific tets for restoratives detection exhibit good detection potentials, as well as the test for formaldehyde. . The addition of sugar altered quantification of lactose detected by equipments tha use infrared and ultrasound, demonstrating that these do not detect lactose exclusively. Regarding samples added with preservatives and neutralizers substances in laboratory, the yogurt culture test was able to detect microbial grouth inhibition in samples added with formaldehyde, hydrogen peroxide, hypochlorite and sodium hydroxide. Presenting resluts similar to the official tests. The addition of formaldehyde, hydrogen peroxide and hypochlorite promoted significant reductions in the counts of mesophilic aerobic micro-organisms in raw milk adulterated in laboratory. However it was not possible to detect hydrogen peroxide and clhorated compounds after 24 hours of refrigeration, maybe due to its rapid degradation and / or inactivation of these substances in milk. Neutralizing substances detection can only dectect this substances when alcalinization is excessive. The balanced neutralization of latic acid results on the anulment of detection ability of the test. The research of preservatives, neutralizers and restoratives is not required for pasteurized milk, only for raw milk. The presence of large numbers of samples positive for these substances demonstrates flaws in quality control of raw milk or occurrence of milk adulteration by the industry itself.

Les infections chroniques de l’Immunodéficience Humaine et de l’Hépatite C (VIH et VHC) sont à l’origine de pandémies. Malgré des traitements avancés, leurs relations avec le système immunitaire ne sont pas résolues et restent nécessaires pour établir de nouvelles stratégies thérapeutiques. Les cellules Natural Killer (NK) sont des effecteurs majeurs antiviraux et sont importants pour l’immunité innée et adaptative. Ils contrôlent leur cytotoxicité et leur fonction immunorégulatrice via de multiples récepteurs activateurs et inhibiteurs qui sont enclenchés par interaction avec leurs ligands respectifs. Parmi tous les récepteurs, je me suis particulièrement intéressé aux Natural Cytotoxicity Receptors NKp30 et NKp44. Il est intéressant de noter que le laboratoire a précédemment identifié un épitope conservé de la gp41 du VIH-1 qui induit l’expression du ligand de NKp44 sur des LT CD4+ les rendant susceptibles à la lyse par des cellules NK-NKp44+. Après plusieurs études, le laboratoire a mis en place une stratégie vaccinale basée sur un peptide de l’épitope conservé de la gp41 qui induit chez la souris des Anticorps Neutralisants (AcNs W614A-3S) contre l’infection VIH-1. Alors que le VIH-2 est considéré comme un modèle unique d’une infection VIH contrôlée, les données sur les cellules NK y sont très limitées. Nous avons observé une dérégulation du récepteur NKp30 et une augmentation de ses ligands qui conduisent à des déficiences de leurs fonctions et représentent un nouveau mécanisme de persistance virale. Ensuite, nous avons observé pendant l’infection chronique par le VHC une forte proportion de cellules NK intra-hépatiques exprimant NKp44 qui corrèle avec la fibrose et la charge virale. De plus, nous avons identifié un épitope conservé de la protéine Core du VHC qui induit le ligand de NKp44 sur des lignées hépatiques. Ces données suggèrent que la déplétion des hépatocytes passe par un mécanisme similaire à celui observé pendant l’infection VIH-1. Enfin, une étude sur des patients VIH-1 contrôleurs nous a permis d’identifier la présence d’AcNs W614A-3S qui sont associées au contrôle viral et au maintien de LT CD4+ fonctionnelles. Ces données ont confirmé le potentiel de ces AcNs et, dont leur production par vaccination, ont été confirmée chez le lapin et le singe. Ainsi, ces études apportent de nouvelles données dans les relations entre les cellules NK et le VIH ou le VHC ainsi que de nouvelles stratégies thérapeutiques. Ces études ont notamment confirmé le pouvoir des AcNs W614A-3S dans un vaccin contre le VIH-1. / Human Immunodeficiency and Hepatitis C (HIV and HCV) chronic infections are at the origin of pandemics. Despite advance drug treatments, their relationship with the immune system is not resolved and is still required to establish new therapeutic strategies. Natural Killer (NK) cells are major antiviral effectors of the immune system and are important for innate and adaptive immune processes. They mediate cytotoxicity and immunoregulation via various activator and inhibitor receptors that are triggered upon interaction with their cognate ligands. Among all receptors, I particularly took an interest in Natural Cytotoxicity Receptors NKp30 and NKp44. Interestingly, the lab previously identified a conserved HIV-1 gp41 épitope that induce expression of NKp44 ligand on CD4+ T cells making them susceptible to lyses by NK-NKp44+ cells. After various studies, the lab established a vaccine strategy based on a peptide from the conserved gp41 épitope that induced in mice Neutralizing Antibodies (Nab W614A-3S) against HIV-1 infection. Whereas HIV-2 infection could be considered as a HIV control infection unique model, data on NK cells are very limited. We found a down-modulation of NKp30 receptor and an increased of its ligands that lead to functional impairments of NK cells and could represent a new viral persistence mechanism. Then, during the HCV chronic infection we found a high proportion of intrahepatic NK cells expressing NKp44 that correlates with fibrosis and viral load. Furthermore we identified a conserved épitope of HCV core protein that induced NKp44 ligand on hepatic cell lines. These data suggest that destruction of hepatocyte might occur by a similar mechanism observed during HIV-1 infection. Finally, a study on HIV-1 controllers patients allow us to identify the presence of Nab W614A-3S that correlates with viral control and the preservation of functional CD4+ T cells. These data confirm the potency of this Nab and their induction by vaccination has been also confirmed in rabbit and macaques. Thus, these studies highlight new data regarding relationship between NK cells and HIV or HCV that could represent new therapeutic approaches. These studies especially confirm the potency of Nab W614A-3S to implement a vaccine against HIV-1.

Oncolytic viruses promise to significantly improve current cancer treatments through their tumour-selective replication and multimodal attack against cancer cells. However, one of the biggest setbacks for oncolytic virus therapies is the intravenous delivery of the virus, as it can be cleared by neutralizing antibodies (nAbs) from the bloodstream before it reaches the tumour cells. In our group, we have succeeded in developing aptamers to vesicular stomatitis virus (VSV), as well as to rabbit anti-VSV polyclonal neutralizing antibodies (nAbs). We tested these aptamers’ biological activity with a cell-based plaque forming assay and found that the aptamers prevented in vitro neutralization of VSV by nAbs and increased the virus infection rate of transformed cells up to 77%. In line with this approach, we enhanced the delivery of oncolytic viruses by selecting aptamers to the CT26 colon carcinoma cell line. The binding of aptamer pools has been tested on flow cytometry and the best pools were subjected to high throughput sequencing. Selected aptamers were linked to anti-VSV aptamers and applied for target delivery of the virus to cancer cells. Development of this aptamer-based technology aims to improve viral anti-cancer therapies, with a potential to be applied as treatment for patients affected with cancer. Finally, in collaboration with a group from Erlangen University, we performed an aptamer selection using capillary electrophoresis and cell-SELEX. The target, the extracellular domain of human CD83, is a maturation marker for dendritic cells and is involved in the regulation of the immune system. Selected aptamer sequences bound selectively to mature dendritic cells, in comparison to immature dendritic cells, and thus hold promise to be applied for further studies leading to a better understanding of CD83’s mechanism of action.

Passive immunization with neutralizing antibodies (nAbs) may prevent mother-to-child transmission (MTCT) of HIV-1 and/or impact HIV-1 exposed infant outcomes. This study compared plasma neutralizing activity against heterologous HIV-1 variants and the quasispecies present in the infected mothers among exposed uninfected infants (HEU) to infants that eventually acquired infection and between transmitting versus non-transmitting mothers. HEU (n = 42), compared to those that eventually acquired infection (n = 21), did not possess higher nAb responses against heterologous envelopes (p = 0.46) or their mothers’ variants (p = 0.45). Transmitting as compared to non-transmitting mothers, however, had significantly higher plasma neutralizing activity against heterologous envelopes (p = 0.03), although these two groups did not have significant differences in their ability to neutralize autologous strains (p = 0.39). Furthermore, infants born to mothers with greater neutralizing breadth and potency were significantly more likely to have a serious adverse event (p = 0.03). These results imply that pre-existing anti-HIV-1 neutralizing activity does not prevent breast milk transmission. Additionally, high maternal neutralizing breadth and potency may adversely influence both frequency of breast milk transmission and subsequent infant morbidity. In addition to neutralization, passively acquired maternal antibodies that mediate antibody dependent cellular cytotoxicity (ADCC) may impact both breast milk transmission and infant outcomes. To date, no study has rigorously compared ADCC activity against a broad panel of heterologous strains or circulating maternal viruses among HEU versus infants that eventually acquire infection and among transmitting versus non-transmitting mothers. We developed a high-throughput assay that measures the lysis of infected reporter target cells in the presence and absence of antibodies. This assay yields similar results as other methods that use decreases in reporter signal from target cells or lysis of primary cells estimated by flow cytometry. In contrast to other ADCC methods, we show that our assay allows assessment of ADCC breadth and potency in a relatively high throughput manner because it uses novel target cells that are susceptible to infection by diverse HIV-1 variants. Estimating ADCC breadth and potency and activity against maternal strains will have important insights about the impact of passively acquired maternal antibodies on MTCT.

HIV-1 primarily utilizes the CCR5 receptor as a co-receptor, but over time, viruses can evolve to use the CXCR4 protein. Changes in the viral envelope V3 loop mediate this switch. The emergence of CXCR4-utilizing viruses has been presumed to occur as a consequence of decreased humoral immunity. We show that exclusively CXCR4-using (X4) viruses contain a 2 to 3 amino acid insertion in the V3 loop. Structural modeling revealed that this insertion caused a protrusion in the V3 loop, which impacts CCR5 receptor interaction. These genotypic and structural motifs affected neutralization susceptibility because X4, as compared to co-circulating CCR5-utilizing (R5) viruses, were less neutralization sensitive to autologous contemporaneous and heterologous plasma. Individuals with co-circulating X4 and R5, as compared to those with only R5, viruses had similar neutralization breadth and potency indicating that the emergence of X4 viruses is not associated with decreased humoral immunity. These results suggest that X4 viruses are neutralization escape variants and arise due to humoral selective pressure. This work has implications for future antibody-based therapeutics. Along with providing a framework for developing an HIV-1 vaccine, broadly neutralizing antibodies (bnAbs) are also being investigated as a potential therapeutic. BnAbs target a limited number of conserved HIV-1 envelope structures, including glycans in and around the V1/V2 and V3 domains. Along with the V3 loop, changes in V1/V2 are also known to impact co-receptor usage. We show that viruses that exclusively use the CXCR4 co-receptor, as compared to variants that only utilize CCR5, were less neutralization sensitive to V1/V2 and V3 directed bnAbs. In contrast, R5 and X4 viruses did not demonstrate neutralization differences to bnAbs that target non-V1/V2 and V3 envelope regions, such as the CD4 binding site and the membrane proximal external region. Structural modeling revealed that the predicted orientation of the V1/V2 loop among diverse HIV-1 variants predicts susceptibility to V3 loop directed bnAbs. In aggregate, our results suggest that viruses with different co-receptor usage have differing bnAb susceptibility. Furthermore, structural modeling may be used as a tool to predict neutralization susceptibility to bnAbs against regions associated with co-receptor usage. / 2020-06-12T00:00:00Z

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