Sebastian Ulbert

Recent progress in West Nile virus diagnosis and vaccination

West Nile virus (WNV) is a positive-stranded RNA virus belonging to the Flaviviridae family, a large family with 3 main genera (flavivirus, hepacivirus and pestivirus). Among these viruses, there are several globally relevant human pathogens including the mosquito-borne dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV) and West Nile virus (WNV), as well as tick-borne viruses such as tick-borne encephalitis virus (TBEV). Since the mid-1990s, outbreaks of WN fever and encephalitis have occurred throughout the world and WNV is now endemic in Africa, Asia, Australia, the Middle East, Europe and the Unites States. This review describes the molecular virology, epidemiology, pathogenesis, and highlights recent progress regarding diagnosis and vaccination against WNV infections.

West Nile Virus: The Complex Biology of an Emerging Pathogen

West Nile virus (WNV) is a zoonotic virus that circulates in birds and is transmitted by mosquitoes. Incidentally, humans, horses and other mammals can also be infected. Disease symptoms caused by WNV range from fever to neurological complications, such as encephalitis or meningitis. Mortality is observed mostly in older and immunocompromised individuals. In recent years, epidemics caused by WNV in humans and horses have become more frequent in several Southern European countries, such as Italy and Greece. In 1999, WNV was introduced into the USA and spread over North America within a couple of years. The increasing number of WNV outbreaks is associated with the emergence of novel viral strains, which display higher virulence and greater epidemic potential for humans. Upon infection with WNV, the mammalian immune system counteracts the virus at several different levels. On the other side, WNV has developed elaborated escape mechanisms to avoid its elimination. This review summarizes recent findings in WNV research that help to understand the complex biology associated with this emerging pathogen.

Improvement of DNA vaccination by adjuvants and sophisticated delivery devices: vaccine-platforms for the battle against infectious diseases.

Advantages of DNA vaccination against infectious diseases over more classical immunization methods include the possibilities for rapid manufacture, fast adaptation to newly emerging pathogens and high stability at ambient temperatures. In addition, upon DNA immunization the antigen is produced by the cells of the vaccinated individual, which leads to activation of both cellular and humoral immune responses due to antigen presentation via MHC I and MHC II molecules. However, so far DNA vaccines have shown most efficient immunogenicity mainly in small rodent models, whereas in larger animals including humans there is still the need to improve effectiveness. This is mostly due to inefficient delivery of the DNA plasmid into cells and nuclei. Here, we discuss technologies used to overcome this problem, including physical means such as in vivo electroporation and co-administration of adjuvants. Several of these methods have already entered clinical testing in humans.

A DNA vaccine encoding the E protein of West Nile Virus is protective and can be boosted by recombinant domain DIII

West Nile Virus (WNV) is an emerging pathogenic flavivirus with increasing distribution worldwide. Birds are the natural host of the virus, but also mammals, including humans, can be infected. In some cases, a WNV infection can be associated with severe neurological symptoms. All currently available WNV vaccines are in the veterinary sector, and there is a need to develop safe and effective immunization technologies, which can also be used in humans. An alternative to current vaccination methods is DNA immunization. Most current DNA vaccine candidates against flaviviruses simultaneously express the viral envelope (E) and membrane (prM) proteins, which leads to the formation of virus-like particles. Here we generated a DNA plasmid, which expresses only the E-protein ectodomain. Vaccination of mice stimulated anti-WNV T-cell responses and neutralizing antibodies that were higher than those obtained after immunizing with a recombinant protein previously shown to be a protective WNV vaccine. A single dose of the plasmid was sufficient to protect animals from a lethal challenge with the virus. Moreover, immunogenicity could be boosted when DNA injection was followed by immunization with recombinant domain DIII of the E-protein. This resulted in significantly enhanced neutralizing antibody titers and a more prominent cellular immune response. The results suggest that the WNV E-protein is sufficient as a protective antigen in DNA vaccines and that protection can be significantly improved by adding a recombinant protein boost to the DNA prime.

High Zika Virus Seroprevalence in Salvador, Northeastern Brazil Limits the Potential for Further Outbreaks

ABSTRACT During 2015 to 2016, Brazil reported more Zika virus (ZIKV) cases than any other country, yet population exposure remains unknown. Serological studies of ZIKV are hampered by cross-reactive immune responses against heterologous viruses. We conducted serosurveys for ZIKV, dengue virus (DENV), and Chikungunya virus (CHIKV) in 633 individuals prospectively sampled during 2015 to 2016, including microcephaly and non-microcephaly pregnancies, HIV-infected patients, tuberculosis patients, and university staff in Salvador in northeastern Brazil using enzyme-linked immunosorbent assays (ELISAs) and plaque reduction neutralization tests. Sera sampled retrospectively during 2013 to 2015 from 277 HIV-infected patients were used to assess the spread of ZIKV over time. Individuals were georeferenced, and sociodemographic indicators were compared between ZIKV-positive and -negative areas and areas with and without microcephaly cases. Epidemiological key parameters were modeled in a Bayesian framework. ZIKV seroprevalence increased rapidly during 2015 to 2016, reaching 63.3% by 2016 (95% confidence interval [CI], 59.4 to 66.8%), comparable to the seroprevalence of DENV (75.7%; CI, 69.4 to 81.1%) and higher than that of CHIKV (7.4%; CI, 5.6 to 9.8%). Of 19 microcephaly pregnancies, 94.7% showed ZIKV IgG antibodies, compared to 69.3% of 257 non-microcephaly pregnancies (P = 0.017). Analyses of sociodemographic data revealed a higher ZIKV burden in low socioeconomic status (SES) areas. High seroprevalence, combined with case data dynamics allowed estimates of the basic reproduction number R0 of 2.1 (CI, 1.8 to 2.5) at the onset of the outbreak and an effective reproductive number Reff of <1 in subsequent years. Our data corroborate ZIKV-associated congenital disease and an association of low SES and ZIKV infection and suggest that population immunity caused cessation of the outbreak. Similar studies from other areas will be required to determine the fate of the American ZIKV outbreak. IMPORTANCE The ongoing American Zika virus (ZIKV) outbreak involves millions of cases and has a major impact on maternal and child health. Knowledge of infection rates is crucial to project future epidemic patterns and determine the absolute risk of microcephaly upon maternal ZIKV infection during pregnancy. For unknown reasons, the vast majority of ZIKV-associated microcephaly cases are concentrated in northeastern Brazil. We analyzed different subpopulations from Salvador, a Brazilian metropolis representing one of the most affected areas during the American ZIKV outbreak. We demonstrate rapid spread of ZIKV in Salvador, Brazil, and infection rates exceeding 60%. We provide evidence for the link between ZIKV and microcephaly, report that ZIKV predominantly affects geographic areas with low socioeconomic status, and show that population immunity likely caused cessation of the outbreak. Our results enable stakeholders to identify target populations for vaccination and for trials on vaccine efficacy and allow refocusing of research efforts and intervention strategies. IMPORTANCE The ongoing American Zika virus (ZIKV) outbreak involves millions of cases and has a major impact on maternal and child health. Knowledge of infection rates is crucial to project future epidemic patterns and determine the absolute risk of microcephaly upon maternal ZIKV infection during pregnancy. For unknown reasons, the vast majority of ZIKV-associated microcephaly cases are concentrated in northeastern Brazil. We analyzed different subpopulations from Salvador, a Brazilian metropolis representing one of the most affected areas during the American ZIKV outbreak. We demonstrate rapid spread of ZIKV in Salvador, Brazil, and infection rates exceeding 60%. We provide evidence for the link between ZIKV and microcephaly, report that ZIKV predominantly affects geographic areas with low socioeconomic status, and show that population immunity likely caused cessation of the outbreak. Our results enable stakeholders to identify target populations for vaccination and for trials on vaccine efficacy and allow refocusing of research efforts and intervention strategies.

Isolation of West Nile Virus from Urine Samples of Patients with Acute Infection

ABSTRACT This study demonstrated that West Nile virus (WNV) excreted in the urine of patients with acute infection can be isolated in cell cultures. In addition, the protocols for WNV isolation from urine samples were standardized, and factors that may affect the efficiency of WNV isolation were identified.

Matrix-M™ adjuvanted envelope protein vaccine protects against lethal lineage 1 and 2 West Nile virus infection in mice

West Nile virus (WNV) is a mosquito-transmitted flavivirus and an emerging pathogen in many parts of the world. In the elderly and immunosuppressed, infection can progress rapidly to debilitating and sometimes fatal neuroinvasive disease. Currently, no WNV vaccine is approved for use in humans. As there have been several recent outbreaks in the United States and Europe, there is an increasing need for a human WNV vaccine. In this study, we formulated the ectodomain of a recombinant WNV envelope (E) protein with the particulate saponin-based adjuvant Matrix-M™ and studied the antigen-specific immune responses in mice. Animals immunized with Matrix-M™ formulated E protein developed higher serum IgG1 and IgG2a and neutralizing antibody titers at antigen doses ranging from 0.5 to 10 μg compared to those immunized with 3 or 10 μg of E alone, E adjuvanted with 1% Alum, or with the inactivated virion veterinary vaccine, Duvaxyn(®) WNV. This phenotype was accompanied by strong cellular recall responses as splenocytes from mice immunized with Matrix-M™ formulated vaccine produced high levels of Th1 and Th2 cytokines. Addition of Matrix-M™ prolonged the duration of the immune response, as elevated humoral and cellular responses were maintained for more than 200 days. Importantly, mice vaccinated with Matrix-M™ formulated E protein were protected from lethal challenge with both lineage 1 and 2 WNV strains. In summary, Matrix-M™ adjuvanted E protein elicited potent and durable immune responses that prevented lethal WNV infection, and thus is a promising vaccine candidate for humans.

Distinguishing West Nile virus infection using a recombinant envelope protein with mutations in the conserved fusion-loop

BackgroundWest Nile Virus (WNV) is an emerging mosquito-transmitted flavivirus that continues to spread and cause disease throughout several parts of the world, including Europe and the Americas. Specific diagnosis of WNV infections using current serological testing is complicated by the high degree of cross-reactivity between antibodies against other clinically relevant flaviviruses, including dengue, tick-borne encephalitis (TBEV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses. Cross-reactivity is particularly problematic in areas where different flaviviruses co-circulate or in populations that have been immunized with vaccines against TBEV, JEV, or YFV. The majority of cross-reactive antibodies against the immunodominant flavivirus envelope (E) protein target a conserved epitope in the fusion loop at the distal end of domain II.MethodsWe tested a loss-of-function bacterially expressed recombinant WNV E protein containing mutations in the fusion loop and an adjacent loop domain as a possible diagnostic reagent. By comparing the binding of sera from humans infected with WNV or other flaviviruses to the wild type and the mutant E proteins, we analyzed the potential of this technology to specifically detect WNV antibodies.ResultsUsing this system, we could reliably determine WNV infections. Antibodies from WNV-infected individuals bound equally well to the wild type and the mutant protein. In contrast, sera from persons infected with other flaviviruses showed significantly decreased binding to the mutant protein. By calculating the mean differences between antibody signals detected using the wild type and the mutant proteins, a value could be assigned for each of the flaviviruses, which distinguished their pattern of reactivity.ConclusionsRecombinant mutant E proteins can be used to discriminate infections with WNV from those with other flaviviruses. The data have important implications for the development of improved, specific serological assays for the detection of WNV antibodies in regions where other flaviviruses co-circulate or in populations that are immunized with other flavivirus vaccines.

AUF1 p45 promotes West Nile virus replication by an RNA chaperone activity that supports cyclization of the viral genome

ABSTRACT A central aspect of current virology is to define the function of cellular proteins (host factors) that support the viral multiplication process. This study aimed at characterizing cellular proteins that assist the RNA replication process of the prevalent human pathogen West Nile virus (WNV). Using in vitro and cell-based approaches, we defined the p45 isoform of AU-rich element RNA-binding protein 1 (AUF1) as a host factor that enables efficient WNV replication. It was demonstrated that AUF1 p45 has an RNA chaperone activity, which aids the structural rearrangement and cyclization of the WNV RNA that is required by the viral replicase to initiate RNA replication. The obtained data suggest the RNA chaperone activity of AUF1 p45 is an important determinant of the WNV life cycle. IMPORTANCE In this study, we identified a cellular protein, AUF1 (also known as heterogeneous ribonucleoprotein D [hnRNPD]), acting as a helper (host factor) of the multiplication process of the important human pathogen West Nile virus. Several different variants of AUF1 exist in the cell, and one variant, AUF1 p45, was shown to support viral replication most significantly. Interestingly, we obtained a set of experimental data indicating that a main function of AUF1 p45 is to modify and thus prepare the West Nile virus genome in such a way that the viral enzyme that generates progeny genomes is empowered to do this considerably more efficiently than in the absence of the host factor. The capability of AUF1 p45 to rearrange the West Nile virus genome was thus identified to be an important aspect of a West Nile virus infection.

Vaccination of mice using the West Nile virus E-protein in a DNA prime-protein boost strategy stimulates cell-mediated immunity and protects mice against a lethal challenge.

West Nile virus (WNV) is a mosquito-borne flavivirus that is endemic in Africa, the Middle East, Europe and the United States. There is currently no antiviral treatment or human vaccine available to treat or prevent WNV infection. DNA plasmid-based vaccines represent a new approach for controlling infectious diseases. In rodents, DNA vaccines have been shown to induce B cell and cytotoxic T cell responses and protect against a wide range of infections. In this study, we formulated a plasmid DNA vector expressing the ectodomain of the E-protein of WNV into nanoparticles by using linear polyethyleneimine (lPEI) covalently bound to mannose and examined the potential of this vaccine to protect against lethal WNV infection in mice. Mice were immunized twice (prime – boost regime) with the WNV DNA vaccine formulated with lPEI-mannose using different administration routes (intramuscular, intradermal and topical). In parallel a heterologous boost with purified recombinant WNV envelope (E) protein was evaluated. While no significant E-protein specific humoral response was generated after DNA immunization, protein boosting of DNA-primed mice resulted in a marked increase in total neutralizing antibody titer. In addition, E-specific IL-4 T-cell immune responses were detected by ELISPOT after protein boost and CD8+ specific IFN-γ expression was observed by flow cytometry. Challenge experiments using the heterologous immunization regime revealed protective immunity to homologous and virulent WNV infection.