Dissertations / Theses on the topic 'Lassa fever'

This thesis is about the process and practice of diagnosis and the implications of new diagnostic technologies in low resource settings. As a setting and a disease which has seen significant investment in diagnostics, Lassa fever in Sierra Leone has been selected as a case study to examine these themes. In this thesis, 'new diagnostic technologies' refers to laboratory-based diagnostics which are fast, reliable, accurate and can be used in low income settings. The starting point of this thesis is a narrative surrounding such technologies which suggests that they will revolutionise low income healthcare settings by allowing accurate scientific diagnosis in places where it was not possible before. Various perspectives on diagnosis are examined and some limitations are identified in relation to their accounts of diagnostic process, context, practice and technology. To explore the case, aspects of science and technology studies, the sociology of scientific knowledge and medical anthropology are combined. A multi-sited ethnography of Lassa fever diagnosis was conducted in three settings: a rural village, a laboratory and the wards of a hospital. Documents were reviewed and interviews conducted with key actors and ex-Lassa fever patients. Analysis focused on framings (partial and subjective interpretations), narratives (persuasive storylines which make use of particular framings) and practice in relation to Lassa fever and the development of technology for its diagnosis. Assumptions about the disease, diagnostics and the process of diagnosis are identified and the conclusion considers how they compare with practice in each setting. This thesis argues that diagnosis is a complex negotiated process and that new diagnostics represent only one aspect of that process. Thus, they are not a ‘silver bullet' to transform low resource healthcare contexts. In particular, ‘improved' diagnostics do not always have the expected impacts, sometimes even introducing complexity and uncertainty. In challenging narratives about diagnostics, this thesis provides an alternative, practice-based, approach to thinking about diagnostics and innovations in health systems; this approach acknowledges the importance, and complexity, of the diverse contexts which shape innovations and technology use.

Thesis: M. Eng. in Computer Science and Molecular Biology, Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2016.
Cataloged from PDF version of thesis.
Includes bibliographical references (page 37).
Sequencing followed by metagenomic analysis is an extremely promising method for broad, unbiased disease profiling of patients for disease surveillance and diagnosis. Here, we use two popular metagenomics tools in union, k-mer-based Kraken with reads and BLAST- and LCAbased MEGAN with assembled contiguous sequence. We analyze sequence from 463 febrile and afebrile patients from Sierra Leone before and during the 2014 Ebola virus outbreak. We find that co-infection with malaria is correlated with increased survival of Ebola virus patients, from 18% survival rate to 53%. We also explore the utility of and emphasize the need for both positive and negative controls to distinguish and remove noise and contaminants from real signal, especially to keep up with increasing sensitivity in sequencing.
by Lydia A. Krasilnikova.
M. Eng. in Computer Science and Molecular Biology

Le virus Lassa entraine une fièvre hémorragique endémique en Afrique de l’Ouest et représente un problème de santé publique. Les connaissances sur la pathogénèse et les réponses immunitaires associées à la maladie sont partielles. Nous avons suivi les paramètres pathologiques, virologiques et immunologiques associés aux infections létales et non létales du LASV chez le singe cynomolgus. Le tableau clinique a été caractérisé par une dépression, une anorexie, une perte de poids et une asthénie chez les animaux survivants, tandis que ces mêmes symptômes ont été accompagnés de fièvre, de difficultés respiratoires et d’épistaxis chez les animaux infectés par une dose létale. Seuls ces derniers ont montré une perturbation des paramètres de coagulation, une rhabdomyolyse et une hausse des marqueurs de lésions rénales. Nous avons observé un tropisme radicalement différent en fonction de la sévérité de la maladie, avec une dissémination virale dans les organes plus importante et plus rapide chez les animaux décédés, la présence de particules infectieuses plus nombreuses et des modifications anatomopathologiques plus sévères. Une réponse immunitaire innée et adaptative précoce et puissante a été associée avec le contrôle de l’infection et la survie tandis que les infections fatales ont été caractérisées par une réponse inflammatoire ressemblant au choc septique, une défaillance de la réponse immunitaire ainsi qu’une réplication virale incontrôlée. Cette étude permet d’améliorer nos connaissances de la pathogénèse de la fièvre de Lassa et d’apporter des marqueurs d’infection prédictifs de la maladie
Lassa virus causes a hemorrhagic fever endemic in West Africa and represents a threat for civilians. The pathogenesis and the immune responses associated with the disease are poorly understood. We followed pathological, virological and immunological parameters associated with fatal and non-fatal Lassa virus infection in the cynomolgus monkey. The clinical picture was characterized by depression, anorexia, weight loss and asthenia in survivors whereas the same symptoms were supported by fever, respiratory difficulties and epistaxis in animals infected with the lethal dose. Only fatalities have shown coagulation parameters dysfunction, rhabdomyolysis and an increase of renal function markers. We observed a different viral tropism in a function of the disease severity, with viral dissemination in organs that was more important and faster in fatalities, the appearance of numerous infectious particles number and more severe pathologic changes. Early and robust innate and adaptive immune response has been associated with the control of infection and recovery whereas fatal infections were characterized by a sepsis like inflammatory response, defective immune response as well as uncontrolled viral replication. This study sheds light on the pathogenesis of Lassa fever and reveals infection markers predictive of the disease outcome

Abstract

Crimean-Congo Hemorrhagic Fever virus (CCHF) and Lassa virus are giving sources illness to humans. In addition to zoonotic transmission, CCHF and Lassa virus can spread from person to person. After a short incubation period, CCHF and Lassa virus infections are characterized by a sudden onset of high fever, chills, headache and cough just like flu. Even some people are vomiting and have diarrhoea. After a few days of illness hemorrhagic manifestations occur. Treatment options for CCHF and Lassa viruses are limited, and there is no vaccine available for use in humans. The purpose of the present study was to produce recombinant nucleocapsid protein of Lassavirus and CCHF virus including an aminoterminal His-tag by a Semliki Forest Virus Replicon (pSFV 4.2). The recombinant proteins are planned to be used in future development of diagnostic methods.

La fièvre de Lassa, maladie endémique à recrudescence épidémique saisonnière, est avec 300 000 cas et 5000 décès annuels estimés à travers toute l’Afrique de l’Ouest considérée comme une priorité en terme de recherche. Ces dernières années, le Nigeria a rapporté un nombre de cas annuel en constante augmentation, dépassant désormais le millier. Mais cinquante ans après sa découverte, les connaissances sur la maladie restent parcellaires et la recherche clinique peu structurée. L’efficacité du seul traitement recommandé, la ribavirine, est aujourd’hui remise en cause. Il apparait donc urgent de pouvoir disposer de données actualisées, précises et fiables sur la maladie, son cours évolutif, sa prise en charge, son pronostic et les facteurs qui y sont associés. Il est également important que les malades qui en souffrent puissent avoir accès à un standard de soins optimisé. Il est enfin indispensable de développer les capacités de recherche clinique en zone d’endémie.Dans cette thèse, nous faisons une synthèse des connaissances accumulées sur la fièvre de Lassa depuis sa découverte en 1969. Puis nous exposons les raisons qui nous ont conduits à mettre en place un programme de réponse et de recherche sur la fièvre de Lassa. Nous expliquons la démarche suivie pour renforcer le standard de soins et mettre en place une plateforme de recherche clinique en collaboration avec l’un des principaux centres de prise en charge de la fièvre de Lassa au Nigeria. Nous présentons les premiers résultats de l’étude de cohorte prospective LASCOPE qui inaugura ce programme.Entre avril 2018 et mars 2020, ce sont 534 patients atteints de fièvre de Lassa prouvée par RT-PCR qui ont été pris en charge dont 77 (14,4%) sont décédés. 510 participants ont été inclus. Parmi eux on dénombrait 258 hommes et 252 femmes ; 84 étaient des enfants. Le délai médian entre l’apparition des symptômes et l’admission était de 8 jours (IIQ 7-13). A l’admission, 37,8% avaient une valeur de Ct de la RT-PCR du gene GPC du virus de Lassa <30. Entre l’admission et la fin du suivi, 120 (26,5%) ont atteints un score NEWS2 ≥7, 67(13,5%) un stade KDIGO ≥2 et 41 (8,0%) ont été dialysés. Tous ont reçu de la ribavirine pendant une durée médiane de 10 jours (IIQ 9-13). 62 (12,2%) sont décédés (57 [13,4%] adultes, 5 [6,0%] enfants. Le délai médian entre l’admission et le décès était de 3 jours (IIQ 1-6). Les caractéristiques suivantes à l’admission étaient associées à la mortalité en analyse multivariable : l’âge ≥ 45 ans (Odds Ratio ajusté [aOR] 16,30 [IC95% 5,31-50,30]), un score NEWS2 ≥7 (aOR 4,79 [IC95% 1,75-13,10]), un stade KDIGO ≥2 (aOR 7,52 [IC95% 2,66-21,20]), une valeur d’ALAT ≥ 3 fois la limite supérieure de la normale (aOR 4,96 [IC95% 1,69- 14,60]) et une valeur de Ct de la RT-PCR Lassa <30 (aOR 4,65 [IC95% 1,50-14,50]). Ces résultats devraient s’avérer utiles pour la conception de futurs essais cliniques mais aussi pour l’actualisation des recommandations de prise en charge de la maladie. Le cadre de cette étude de cohorte nous a également permis de documenter et décrire une présentation atypique de la maladie, sous la forme d’une paraparésie d’apparition retardée. Cette observation originale vient compléter le spectre évolutif des manifestations neurologiques de la fièvre de Lassa.Deux études complémentaires sont nichées dans la cohorte LASCOPE, portant respectivement sur la pharmacocinétique de la ribavirine telle qu’employée dans le traitement de la fièvre de Lassa et sur la physiopathologie de l’atteinte cardio-circulatoire pouvant survenir au cours de la maladie. Le recrutement dans ces études nichées est toujours en cours.Enfin, un essai clinique de phase II évaluant la pharmacocinétique, la sécurité et la tolérance d’un nouvel antiviral, le favipiravir, pour le traitement de la fièvre de Lassa est également en cours de mise en place sur deux sites au Nigeria dont celui avec lequel nous collaborons
Lassa fever, an endemic disease with seasonal epidemic recrudescence, is considered to be responsible for 300 000 cases and 5000 death each year throughout Western Africa. As such, it has been listed as a priority for research. Over the past few years, Nigeria reported the highest and ever increasing number of cases, now exceeding a thousand per year. However, fifty years after the discovery of the disease, our knowledge is still limited and clinical research capacities remained poorly developed. The efficacy of the only available treatment, ribavirin, is still to be assessed. Hence, there is a need for up to date and reliable data on the disease course, management, mortality and prognostic factors. It is also of paramount importance that the patients suffering from Lassa fever could gain access to an optimised standard of care. Lastly, clinical research capacities need to be developed urgently in endemic areas.In this thesis, we review the knowledge gathered on Lassa fever from its discovery in 1969 till date. Then, we explain the reasons that led us to set up a response and research program on Lassa fever, as well as our approach to reinforce the standard of care and build a clinical research platform in collaboration with one of the main Lassa treatment centres in Nigeria. Next, we present the first results of the LASCOPE cohort study which represent the cornerstone of our program.From April 2018 to March 2020, 534 patients with RT-PCR proven Lassa fever were followed-up of whom 77 died (14.4%). 510 participants were enrolled in the cohort: 258 men and 252 women, 84 being children. The median delay between the symptoms onset and admission was 8 days (IQR 7-13). On admission, 37.8% had a Lassa RT-PCR Ct value <30. From admission to end of follow-up, 120 (26.5%) had a NEWS2 ≥7, 67(13.5%) had a KDIGO ≥2, and 41 (8.0%) underwent dialysis. All of them received ribavirin therapy with a median duration of 10 days (IQR 9-13). 62 (12.2%) died (57 [13.4%] adults, 5 [6.0%] children. The median delay between admission and death was 3 days (IQR 1-6). The following characteristics on admission were independently associated to mortality: age ≥ 45 years (adjusted Odds Ratio [aOR] 16.30 [95%CI 5.31-50.30]), NEWS2 ≥7 (aOR 4.79 [95%CI 1.75-13.10]), KDIGO ≥2 (aOR 7.52 [95%CI 2.66-21.20]), ALAT ≥ 3 times the upper limit of normal range (aOR 4.96 [95%CI 1.69- 14.60]), a Lassa RT-PCR Ct value <30 (aOR 4.65 [95%CI 1.50-14.50]). Those results will be helpful for the conception of future therapeutic clinical trials as well as for updating existing management guidelines. The framework of this cohort also gave us the opportunity to describe an unusual presentation of the disease, namely delayed paraparesis. This original observation add to the evolving knowledge regarding the spectrum of Lassa fever-related neurological manifestations.Two complementary studies are nested in the LASCOPE cohort, concerning the pharmacokinetics of ribavirin, as it is used for the treatment of Lassa fever, as well as the pathophysiology of cardiovascular dysfunction that can occur during Lassa fever. Inclusions in these nested studies is still ongoing.Finally, an upcoming phase II clinical trial evaluating the pharmacokinetics, security and tolerability of a novel antiviral drug, favipiravir, for the treatment of Lassa fever is currently being prepared in two Nigerian sites, including the one we are collaborating with

Lassa fever virus is one of the most dangerous viruses of arenaviridae family, causing more than 500,000 infections per year in Africa. The fatality rate for hospitalized patients is as high as 20%. Due to the high fatality and lack of efficient licensed drugs and vaccines to treat and prevent, Lassa fever virus is classified as a Category A priority pathogen and biosafety level-4 agent by the Centers for Disease Control and Prevention of the USA. Cases were also found in the Americas and European countries, highlighting its potency to be a bioterrorism weapon. Like other areanaviruses, Lassa virus has developed a unique interferon suppression mechanism to evade from the host immune system, in which Lassa nucleoprotein plays the key role. To understand the LASV nucleoprotein functions, we tried to determine the first arenaviral nucleoprotein structure, LASV nucleoprotein. The LASV nucleoprotein (NP) was overexpressed and purified. The NP protein was crystallized and the structure was determined to 1.80 Å resolution. The crystals belong to space group P3, with the unit cell parameters a = b = 177.16 Å, c = 56.49 Å, α= β= 90° and γ= 120°. The LASV NP structure contains two domains, which are not similar to any reported viral nucleoprotein structures. The N-terminal domain has a novel structure with a cavity, which we proposed for cap binding, and the C-terminus is a 3'-5' ribonuclease, which is responsible for suppressing interferon production. To characterize the possible interaction between NP and other arenaviral protein, we also overexpressed and purified LASV Z. Interestingly, both NP and Z proteins have two forms and the purified NP protein and monomeric Z protein bind RNA. It is surprising that only the oligomeric Z protein interacts with NP protein but the monomeric Z protein does not as determined by Isothermal Titration Calorimetry (ITC). Our studies have reported the first arenaviral nucleoprotein structure, revealed the novel mechanism for the cap binding and immune suppression, which set up a platform for the development of novel drugs and vaccines to treat deadly arenaviral infections.

The major outer membrane protein, MOMP, is the main protein in the outer membrane of pathogenic Campylobacter bacteria. Infection with Campylobacter is the principle cause of severe enteritis and untreated may result in non-trauma related paralysis. Studies have shown, that MOMP can act as antigen and thus has the potential to provide protection by induced humoral immunity. In our study, we expressed recombinant MOMP in Escherichia1coli, developed an alternative method to extract the outer membrane protein from its lipid environment and solved and characterised its crystal structure. The information acquired through these structural studies sheds new light on the structural characteristics of this important membrane protein. The West-African Lassa virus can cause deadly haemorrhagic fever. Lassa virus only possesses five proteins, which are synergistically responsible for the virus' life cycle, and virulence. The way in which the individual proteins act with one another and with host cell proteins is not fully understood. The polymerase L is the largest of the five proteins and has multiple functions. In this study, we first divided the L protein into different domains and tested their recombinant expression in Escherichia1coli. For first time, we solved the crystal structure of the putative endonuclease domain of Lassa virus and validated its endonucleolytic function by means of RNA digestion assays and alanine point mutations.

Introduction/background: Epidemics of infectious diseases (ID) are re-occurring now more often and spreads faster into many different parts of the world due to globalization. The increasing evidence of climate change and man-made events have shown impacts to increase the emergency and re-emerging of animal- borne IDs. Studies claims that background factors of these IDs are biological, environmental and human-lifestyle related changes. The pathogen Lassa fever virus (LASV) is a zoonotic organismthat circulates in rodent reservoirs, and the animal´s hosts are rodent species (rats) of the genus Mastomys natalensis. Mastomys natalensis is primarily the reservoir species of the animal-borne disease of Lassa fever (LF) which is most prevalent in west Africa, particularly in Nigeria. Lassa fever (LF) has limited information with under-documented cases, its health effect on pregnant women especially in Nigeria is within the rural areas of Edo, Ondo, Delta, Ebony, Bauchi, Taraba and Plateau states where maternal mortalities are higher.  Aim: The overarching aim of this thesis is to analyse and discuss the health effects of Lassa fever occurrence and outcomes on pregnant women in Nigeria, with emphasis on the maternal mortality and fatality during pregnancy. Method: A statistical and systematic review was performed from retrospective studies of case series, case-control, observational and cohort studies of patients in Nigeria (pregnant women with gestation ages of pregnancy from 2 weeks –32 weeks) that tested positive to LASV. Publication status and publication date was applied for the inclusion of respective studies by electronic searches via Web of Science, Google scholar, MEDLINE and PubMed. Preferred Reporting Items for Systematic reviews and Meta-Analysis guidelines (PRISMA) was used to illustrate the flow of relevant articles in my study. R-commander and R-Studio software was used to analyze the data and to find the causal significant relationship between LF and maternal mortality using “Linear regression and linear model plot”. Result: The total number of full-text and Peer-view publications on Lassa fever virus cases was 1 609 articles. 94 articles out of the 1 609 articles were eligible for full text revision. Exclusion criteria finally yielded 6 studies that were relatively relevant to my study. However, 3 out of the 6 articles were statistically reviewed to know the influence of Lassa fever and the risk of maternal mortality during pregnancy.  Conclusion: Lassa fever occurrence have significantly shown potential increase in the severity of maternal mortality, and is predominant among pregnant women from 39 - 45 years old in Nigeria especially within the risk endemic areas of Ondo, Edo, Ebony and Bauchi states showing significant long-term diseases on LF affected pregnant women, such as encephalopathy, acute kidney dysfunction and acute kidney failure that leads to further health problems or complications like coma and sensorineural deafness.

Le virus Lassa (LASV) induit une fièvre hémorragique chez l’homme et est responsable de 3 000 à 5 000 décès par an. Aucun vaccin ou traitement efficace contre LASV n’est disponible, et les mécanismes de pathogenèse de la fièvre de Lassa sont encore mal compris. Des études chez l’homme et le primate suggèrent que la réponse interféron de type I (IFN-I) et de la réponse T sont critiques pour la survie de l’hôte. Nous nous sommes intéressés à la réponse des cellules dendritiques (DC) à LASV, car elles peuvent à la fois produire des IFN-I et induire la réponse T. Nous avons étudié les DC plasmacytoïdes (pDC), spécialisées dans la réponse IFN-I, et les DC myéloïdes (mDC), présentatrices d’antigènes. Nous avons montré que les pDC et les mDC ne sont pas productivement infectées par MOPV et LASV. Les pDC produisent des quantités importantes d’IFN-I en réponse à MOPV, mais pas à LASV. Les mDC sont activées et produisent des IFN-I en réponse à MOPV mais aussi à LASV. Cependant, seules les mDC infectées par MOPV sont capables d’activer des lymphocytes T. De plus, la présence de lymphocytes T inhibe complètement l’activation des mDC infectées par LASV. Ces différences entre les mDC infectées par MOPV et LASV dépendent de la nucléoprotéine de LASV, qui est connue pour ses propriétés immunosuppressives, mais aussi de la glycoprotéine. En résumé, nous avons obtenu des différences de réponse à l’infection par MOPV ou LASV chez les pDC et les mDC. Ces cellules pourraient avoir un rôle essentiel in vivo dans la réponse globale à LASV, et donc dans l’issue de la fièvre de Lassa
Lassa virus (LASV) is responsible for a viral haemorrhagic fever in humans and the death of 3,000 to 5,000 people every year. There is currently no vaccine or treatmentavailable against LASV, and its pathogenesis is not completely understood yet. According to studies on humans and primates, type I interferon (IFN-I) and T cell responses appear to be critical for the host. We studied the response of dendritic cells (DC) to LASV, as DC are involved in both IFN-I production and T cell activation. We compared the response of primary human DC to LASV and Mopeia virus (MOPV), which is similar to LASV, but non-pathogenic.We focused on plasmacytoid DC (pDC), specialized in IFN-I production, and myeloid DC (mDC), specialized in antigen presentation. We showed that neither pDC nor mDC were productively infected by LASV and MOPV. pDC infected with MOPV produced large amounts of IFN-I, whereas pDC infected with LASV did not. mDC produced substantial amounts of IFN-I in response to both LASV and MOPV. However, only MOPV-infected mDC were able to activate T cells. More surprisingly, coculture with T cells completely inhibited the activation of LASV-infected mDC. These differences between LASV- and MOPV-infected mDC were mostly due to LASV nucleoprotein, which has major immunosuppressive properties, but the glycoprotein was also involved. Overall, these results showed differences in pDC and mDC response to MOPV and LASV. Therefore, both pDC and mDC may be important for the global response to LASV in vivo, and play a role in the outcome of Lassa fever

Les fièvres hémorragiques à virus Lassa (LASV) et Ebola (EBOV) représentent un important problème de santé publique en Afrique. Les réponses immunes et la pathogenèse associées à ces maladies sont peu connues. Les cellules NK ont un rôle important dans la réponse immune innée par leurs propriétés cytotoxiques, mais également dans l'induction des réponses adaptatives par leur production de cytokines et leurs interactions avec les cellules dendritiques (DC) et les macrophages. Ce projet s'attache à comprendre le rôle des cellules NK dans le contrôle de la réplication virale et dans l'induction des réponses immunitaires au cours de ces infections. Un modèle in vitro de coculture de cellules NK humaines avec des DC et macrophages autologues a été développé. L'activation des cellules NK et leurs fonctions ont été analysées après l'infection par LASV et EBOV. Par ailleurs, les réponses des cellules NK en réponse à LASV ont été comparées avec celles induites lors de l'infection par le virus Mopeia (MOPV), très proche de LASV mais non pathogène pour l'homme. Les macrophages, mais pas les DC, infectés par LASV ou MOPV induisent l'activation et l'augmentation des capacités cytotoxiques des cellules NK. Toutefois, les cellules NK ne sont pas capables de lyser les cellules infectées et ne produisent pas d'IFN-γ. Les cellules NK s'activent et sont capables de lyser les cellules infectées en présence de macrophages mais également de DC infectés par des LASV mutants. Cependant, les IFN de type I sécrétés en grande quantité en réponse à ces virus ne sont pas impliqués dans l'activation des cellules NK. L'infection par EBOV n'induit qu'une très faible activation des cellules NK en présence de DC ou macrophages et ne conduit pas à la sécrétion de cytokines, ni à la modification du potentiel cytotoxique.Ces résultats permettent d'améliorer la compréhension des réponses immunes et des mécanismes de pathogenèse mis en place lors des fièvres hémorragiques Lassa et Ebola.

Lassa hemorrhagic fever is a disease endemic to Western and Central Africa. It is also the most commonly imported hemorrhagic fever disease with numerous cases reported in travelers to the region throughout the forty one years since its first characterization in Nigeria. Lassa fever afflicts an estimated 300,000 or more people in Western Africa each year, with approximately 5,000 fatalities. These statistics may underreport the actual number of infections and overall fatality rate from the disease; in that modern, highly sensitive, specific, and rapid recombinant protein-based diagnostics recently implemented in Sierra Leone have demonstrated a higher prevalence amongst native populations. Despite significant advances in the diagnosis of Lassa fever control of the disease in resource poor and settings with vastly different social practices from those of western societies is both impractical and ecologically undesirable. The development of a robust vaccine platform that would confer long term protective immunity against Lassa fever and related Old world arenaviral agents is a highly desirable, more readily implementable and manageable means to control the disease. Such a vaccine would have applications both in the global health and biodefense arenas. To this end, critical aspects of Lassa virus gene expression were studied with the end goal of designing a non-infectious, non-replicative virus-like particle (VLP platform expressing all relevant immunogenic and protective arenaviral components. LASV VLP were immunogenic in mice, but ongoing and future studies will be aimed at determining their protective efficacy in susceptible animal models. A human vaccine comprised of LASV VLP is the ultimate endpoint
acase@tulane.edu

Tese de Doutoramento em Medicina
Lassa fever (LF), is a viral hemorrhagic fever caused by Lassa virus (LASV), for which neither an approved vaccine or effective treatment is available. LASV is an endemic virus in western Africa and a major health and economic burden, causing an estimate 100,000-300,000 infections yearly, with the number of reported infections increasing in the last years. This thesis describes the development of LASSARAB, a dual LF and rabies vaccine based on recombinant rabies vector. Rabies is another equally important disease in Africa that is estimated to cause thousands of deaths every year, despite vaccination being available. LASSARAB uses a codon optimized version of LASV’s glycoprotein (GPC) as its LASV immunogen. After confirming that LASSARAB expresses and incorporates GPC in the virions, LASSARAB’s potential as an LF vaccine was tested using several LASSARAB based vaccine candidates: inactivated LASSARAB formulated in PBS only; inactivated LASSARAB formulated with GLA-SE adjuvant (a TLR-4 agonist); live-LASSARAB; and live-LASSARAB-ΔG, a variant of LASSARAB that lacks the Rabies G gene. Inactivated LASSARAB formulations induced a significant GPC specific IgG response above background with LASSARAB+GLA-SE inducing higher IgG titers than LASSARAB alone as well as a lower IgG1/IgG2c ratio. Neither live- LASSARAB vector induced a significant LASV GPC immune response. In LASV challenge studies, in both guinea pig and mouse, inactivated LASSARAB+GLA-SE, significantly protected 80% of the animals against LF disease. Higher titers of anti-GPC IgGs were correlated with protection independently of LASV neutralizing titers. Instead, non-neutralizing LASV GPC-specific antibodies, through antibody-dependent cellular functions (ADCC and ADCP) appear to the main drivers of protection against LF as demonstrated by in vitro and in vivo studies. Overall, these findings demonstrate an effective inactivated LF and rabies vaccine and suggest a novel correlate of protection for LF. Currently, further LASSARAB immunogenicity studies using NHPs are underway determine its eligibility for clinical phase 1 trials.
A febre de Lassa (LF), é uma febre hemorrágica viral causada pelo vírus Lassa (LASV), para a qual não existe nem vacina ou tratamento aprovado. O LASV é um vírus presente na África ocidental que incute um pesado encargo na saúde pública regional, e estima-se que causa 100,000 a 300,000 infeções por ano. Esta tese é sobre o desenvolvimento de LASSARAB, uma potencial vacina contra a LF e Raiva baseada num vetor do vírus da Raiva recombinante. A Raiva, é outra doença igualmente importante em África que causa milhares de mortes todos anos apesar de haver vacina disponível. LASSARAB usa uma versão codon-optimized da glicoproteína de LASV (GPC) como o imunogénio de base contra LASV. Após confirmar que LASSARAB expressa e incorpora o LASV GPC nas partículas virais, o potencial de LASSARAB foi testado através de vários candidatos incluindo: LASSARAB inativado formulado em PBS apenas; LASSARAB inativado formulado com o adjuvante GLA-SE (um agonista TLR4); LASSARAB vivo; e LASSARAB-ΔG vivo, uma variante de LASSARAB que não expressa o gene da glicoproteína de raiva. Ambos os candidatos LASSARAB inativados induziram uma elevada resposta imune contra GPC, com LASSARAB+GLA-SE induzindo níveis de IgGs contra GPC mais elevados assim como um ratio IgG1/IgG2c mais baixo. Em estudos de exposição com LASV em porquinhos da Índia e ratinhos, o LASSARAB+GLA-SE inativo foi capaz de proteger em 80% os animais contra LF. Níveis mais elevados de IgGs específicos para GPC, foram correlacionados com proteção independentemente dos níveis de anticorpos neutralizantes contra LASV. Pelo contrário, IgGs específicos contra GPC não neutralizantes, através de funções celulares dependentes de anticorpos (ADCC e ADCP) parecem ser os principais intervenientes na proteção contra LF. Em conjunto, estes resultados demonstram uma vacina inativada eficaz contra LF e Raiva e sugerem uma correlação de proteção contra LF, baseado na resposta de IgGs específicos contra GPC. Atualmente, estudos em primatas estão a decorrer para testar a imunogenicidade de LASSARAB neste modelo em preparação para clinical phase 1 trials.
Financial support for this PhD was provided by FCT (Fundação para Ciência e Tecnologia), by the fellowship PD/BD/105847/2014, awarded through the MD/PhD program of University of Minho.

M.A.
Two species of multimammate mouse, Mastomys coucha and M. natalensis are common, and widely distributed in southern Africa, occurring sympatrically in some areas, and allopatrically in others. The limits of their distribution are only provisional so far. As they share a high degree of morphological similarity, they are, as yet, impossible to identify with certainty in the field. Each species of multimammate mouse carries important diseases: with M. coucha being a carrier for the bacterium causing plague, and M. natalensis carrying the virus causing Lassa fever. In many areas, multimammate mice, being highly adaptable and ecological generalists, have become co-habitants with humans. This fact, coupled to the medical significance of both species, lends importance to being able to identify each species where it occurs, especially in areas where they occur sympatrically. Thus, a total of 40 specimens of M. natalensis were trapped from Richards Bay and La Lucia ridge in KwaZulu-Natal, and 43 specimens of M. coucha from Montgomery Park in Johannesburg and from the shores of the Vaal Dam in the Free State with the aim of comparing these two species via gel electrophoresis. These specimens were from allopatric populations from the centres of their provisional distributions. It was expected that there would be genetic differences between the two sibling species. Blood, liver, and muscle samples were taken, either in the field from dead specimens caught in snap-traps, or back in the laboratory from live-trapped specimens. Fifteen proteins or enzymes provided interpretable results at a total of 39 loci. Nineteen of these were polymorphic