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Das Dengue-Virus ist ein, in tropischen und subtropischen Regionen endemisches, humanpathogenes Virus, welches durch Moskitos des Genus Aedes übertragen wird. Jährlich werden ca. 95 Millionen der Infektionen klinisch manifestiert, wobei 1 % davon in der schwersten Form des Dengue-hämorrhagischen Fiebers verlaufen. Eine schnelle und einfache Diagnostik spielt dabei für die Behandlung sowie für epidemiologische Studien eine wichtige Rolle. Die serologische Diagnostik flaviviraler Infektionen im Allgemeinen und DENV im Speziellen, ist durch die Vielzahl kreuzreaktiver und infektionsverstärkender Antikörper hoch komplex. Die starke Co-Zirkulation verschiedener Flaviviren und deren ähnliche klinische Symptomatik erschwert zusätzlich die spezifische Diagnostik. Seit 2015 führte insbesondere der massive Vormarsch von ZIKV‑Infektionen in DENV-endemischen Gebieten Südamerikas zu einer noch komplexeren Sachlage, da die serologische Unterscheidung beider Infektionen kaum möglich war. Im Rahmen dieser Promotionsarbeit wurden daher rekombinante virale E-Proteine exprimiert und charakterisiert, welche die Spezifität serologischer DENV- und ZIKV‑Tests erhöhen sollen. E-Proteine sind stark immunogen, da sie die Virusoberfläche dominieren. Jedoch ist deren hochkonservierte DII-FL Region gleichzeitig das Ziel von einem Großteil kreuzreaktiver Antikörper. Studien mit WNV und JEV haben zuvor belegt, dass Mutationen in diesem Sequenzbereich, die Kreuzreaktivität mit heterologen Flaviviren herabsenken können. Deshalb wurden vier Punktmutationen in die DII-FL Region von DENV 1-4 und ZIKV E (Equad) eingefügt und in einem Insektentenzellen- basierten System exprimiert, aus dem Kulturüberstand aufgereinigt und mit Humanseren in IgM- und IgG-ELISAs untersucht. Die Ergebnisse dieser Arbeit werden in zwei Publikationen dargelegt, welche die Grundlage für diese Promotionsschrift darstellen.

Monitoring the humoral protective immune response and its durability after SARS-CoV-2 infections is essential for risk assessment of reinfections, the improvement of diagnostic methods and the evaluation of vaccine trials. We have analyzed neutralizing antibodies and IgG responses specific to different antigens, including the inactivated whole-virion of SARS-CoV-2, the spike subunit 1 protein and its receptor binding domain, as well as the nucleocapsid protein. We show the dynamic developments of the responses from the early convalescent stages up to 9 months post symptoms onset in follow-up samples from 57 COVID-19 patients with varying clinical severity. By correlating antibody signals to neutralizing titres, valid diagnostic markers for the estimation of neutralizing protection could be identified.

Monitoring the humoral protective immune response and its durability after SARS-CoV-2 infections is essential for risk assessment of reinfections, the improvement of diagnostic methods and the evaluation of vaccine trials. We have analyzed neutralizing antibodies and IgG responses specific to different antigens, including the inactivated whole-virion of SARS-CoV-2, the spike subunit 1 protein and its receptor binding domain, as well as the nucleocapsid protein. We show the dynamic developments of the responses from the early convalescent stages up to 9 months post symptoms onset in follow-up samples from 57 COVID-19 patients with varying clinical severity. By correlating antibody signals to neutralizing titres, valid diagnostic markers for the estimation of neutralizing protection could be identified.

The Latin American 2015-2016 Zika virus (ZIKV) outbreak was associated with an increase in microcephaly predominantly in northeastern Brazil. To comparatively investigate infectious causes of congenital malformations, we performed a nested case-control study in 32 mothers of cases of suspected congenital Zika syndrome (CZS) and 160 age-matched controls from Bahia, northeastern Brazil. We collected clinical and imaging data and assessed past exposure to ZIKV, Chikungunya virus (CHIKV), dengue virus, and 8 established TORCH (Toxoplasma gondii, Treponema pallidum, rubella virus, cytomegalovirus, herpes simplex virus 1 [HSV-1] and HSV-2, varicella-zoster virus, parvovirus B19) pathogens using multiple serological tests. Heterogeneous symptoms prevented unequivocal diagnosis of CZS on clinical grounds. Only ZIKV and CHIKV seroprevalence rates differed significantly between cases and controls (93.8% versus 67.8% for ZIKV [Fisher's exact text, P = 0.0021 and 20.7% versus 8.2% for CHIKV [chi(2), P = 0.0391]). High ZIKV seroprevalence rates in cases could not be explained by previous dengue virus infections potentially eliciting cross-reactive antibody responses affecting ZIKV serological tests. In conditional logistic regression analyses, only ZIKV was significantly associated with congenital malformations (P = 0.030; odds ratio, 4.0 [95% confidence interval, 1.1 to 14.1]). Our data support an association between maternal ZIKV exposure and congenital malformations. Parallels between the discrepant ZIKV and CHIKV seroprevalence rates between cases and controls and similar seroprevalence rates between cases and controls for the sexually transmitted T. pallidum and HSV-2 may suggest the occurrence of predominantly vector-borne transmission in our study population. High seroprevalence of TORCH pathogens suggests that exhaustive diagnostics will be necessary in the aftermath of the ZIKV outbreak and provides baseline data for longitudinal studies on ZIKV pathogenesis. IMPORTANCE The Latin American Zika virus (ZIKV) outbreak had a major impact on reproductive health worldwide. The reasons for the massively increased reports of neonatal microcephaly in northeastern Brazil are still unclear. Beyond the technical limitations of laboratory diagnostics, unambiguous diagnosis of ZIKV as the cause of congenital malformations is hampered by similar clinical pictures elicited by other pathogens known as TORCH pathogens. We performed a case-control study comparing mothers of children with congenital malformations to age-matched controls from Salvador, Brazil, one of the areas most extensively affected by the ZIKV outbreak. The ZIKV and Chikungunya virus seroprevalence rates differed significantly, whereas the levels of maternal exposure to TORCH pathogens were similar between cases and controls. Our data support a link between maternal ZIKV infection and congenital malformations and suggest the occurrence of predominantly vector-borne ZIKV transmission in these cases. In addition, some highly prevalent TORCH pathogens may be misinterpreted as representative of ongoing ZIKV activity in the absence of exhaustive diagnostics in northeastern Brazil.