Anti-Flavivirus Vaccines: Review of the Present Situation and Perspectives of Subunit Vaccines Produced in Escherichia coli.
ABSTRACT: This article aims to review the present status of anti-flavivirus subunit vaccines, both those at the experimental stage and those already available for clinical use. Aspects regarding development of vaccines to Yellow Fever virus, (YFV), Dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV) are highlighted, with particular emphasis on purified recombinant proteins generated in bacterial cells. Currently licensed anti-flavivirus vaccines are based on inactivated, attenuated, or virus-vector vaccines. However, technological advances in the generation of recombinant antigens with preserved structural and immunological determinants reveal new possibilities for the development of recombinant protein-based vaccine formulations for clinical testing. Furthermore, novel proposals for multi-epitope vaccines and the discovery of new adjuvants and delivery systems that enhance and/or modulate immune responses can pave the way for the development of successful subunit vaccines. Nonetheless, advances in this field require high investments that will probably not raise interest from private pharmaceutical companies and, therefore, will require support by international philanthropic organizations and governments of the countries more severely stricken by these viruses.
Project description:BACKGROUND: Currently existing yellow fever (YF) vaccines are based on the live attenuated yellow fever virus 17D strain (YFV-17D). Although, a good safety profile was historically attributed to the 17D vaccine, serious adverse events have been reported, making the development of a safer, more modern vaccine desirable. METHODOLOGY/PRINCIPAL FINDINGS: A gene encoding the precursor of the membrane and envelope (prME) protein of the YFV-17D strain was inserted into the non-replicating modified vaccinia virus Ankara and into the D4R-defective vaccinia virus. Candidate vaccines based on the recombinant vaccinia viruses were assessed for immunogenicity and protection in a mouse model and compared to the commercial YFV-17D vaccine. The recombinant live vaccines induced γ-interferon-secreting CD4- and functionally active CD8-T cells, and conferred full protection against lethal challenge already after a single low immunization dose of 10(5) TCID(50). Surprisingly, pre-existing immunity against wild-type vaccinia virus did not negatively influence protection. Unlike the classical 17D vaccine, the vaccinia virus-based vaccines did not cause mortality following intracerebral administration in mice, demonstrating better safety profiles. CONCLUSIONS/SIGNIFICANCE: The non-replicating recombinant YF candidate live vaccines induced a broad immune response after single dose administration, were effective even in the presence of a pre-existing immunity against vaccinia virus and demonstrated an excellent safety profile in mice.
Project description:Recent outbreaks of yellow fever virus (YFV) in West Africa and Brazil resulted in rapid depletion of global vaccine emergency stockpiles and raised concerns about being unprepared against future YFV epidemics. Here we report that a live attenuated virus similar to the Japanese encephalitis virus (JEV) vaccine JE-CVax/Imojev that consists of YFV-17D vaccine from which the structural (prM/E) genes have been replaced with those of the JEV SA14-14-2 vaccine strain confers full protection in mice against lethal YFV challenge. In contrast to the YFV-17D-mediated protection against YFV, this protection is not mediated by neutralizing antibodies but correlates with YFV-specific nonneutralizing antibodies and T cell responses against cell-associated YFV NS1 and other YFV nonstructural (NS) proteins. Our findings reveal the potential of YFV NS proteins to mediate protection and demonstrate that chimeric flavivirus vaccines, such as Imojev, could confer protection against two flaviviruses. This dual protection may have implications for the possible off-label use of JE-CVax in case of emergency and vaccine shortage during YFV outbreaks. In addition, populations in Asia that have been vaccinated with Imojev may already be protected against YFV should outbreaks ever occur on that continent, as several countries/regions in the Asia-Pacific are vulnerable to international spread of the YFV.IMPORTANCE Efficient and safe vaccines against yellow fever (e.g., YFV-17D) that provide long-lasting protection by rapidly inducing neutralizing antibody responses exist. However, the vaccine supply cannot cope with an increasing demand posed by urban outbreaks in recent years. Here we report that JE-CVax/Imojev, a YFV-17D-based chimeric Japanese encephalitis vaccine, also efficiently protects against YFV infection in mice. In case of shortage of the YFV vaccine during yellow fever outbreaks, (off-label) use of JE-CVax/Imojev may be considered. Moreover, wider use of JE-CVax/Imojev in Asia may lower the risk of the much-feared YFV spillover to the continent. More generally, chimeric vaccines that combine surface antigens and replication machineries of two distinct flaviviruses may be considered dual vaccines for the latter pathogen without induction of surface-specific antibodies. Following this rationale, novel flavivirus vaccines that do not hold a risk for antibody-dependent enhancement (ADE) of infection (inherent to current dengue vaccines and dengue vaccine candidates) could be designed.
Project description:Development of a HIV-1 vaccine is a major global priority. The yellow fever virus (YFV) attenuated vaccine 17D is among the most effective of currently used vaccines. However, the stability of the YFV17D vector when carrying non-flavivirus genes has been problematic. We have constructed and expressed HIV-1 Env in YFV17D with either single transmembrane (STM) or double transmembrane (DTM) YFV E protein domains for the development of anti-HIV antibodies. Here we describe modifications of the YFV17D vector such that HIV-1 Env gp120 is expressed in up to 5 passages in Vero cells. Immunization with recombinant YFV17D vector prime followed by HIV-1 CH505 TF gp120 protein boosts were able to induce neutralizing antibodies for a HIV-1 tier 1 isolate in mice. This modified YFV vector may be a starting point for constructing HIV-1 vaccine candidate priming vectors.
Project description:Zika virus (ZIKV) infections occur in areas where dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), and other viruses of the genus Flavivirus cocirculate. The envelope (E) proteins of these closely related flaviviruses induce specific long-term immunity, yet subsequent infections are associated with cross-reactive antibody responses that may enhance disease susceptibility and severity. To gain a better understanding of ZIKV infections against a background of similar viral diseases, we examined serological immune responses to ZIKV, WNV, DENV, and YFV infections of humans and nonhuman primates (NHPs). Using printed microarrays, we detected very specific antibody responses to primary infections with probes of recombinant E proteins from 15 species and lineages of flaviviruses pathogenic to humans, while high cross-reactivity between ZIKV and DENV was observed with 11 printed native viruses. Notably, antibodies from human primary ZIKV or secondary DENV infections that occurred in areas where flavivirus is endemic broadly recognized E proteins from many flaviviruses, especially DENV, indicating a strong influence of infection history on immune responses. A predictive algorithm was used to tentatively identify previous encounters with specific flaviviruses based on serum antibody interactions with the multispecies panel of E proteins. These results illustrate the potential impact of exposure to related viruses on the outcome of ZIKV infection and offer considerations for development of vaccines and diagnostics.
Project description:Today's world is characterized by increasing population density, human mobility, urbanization, and climate and ecological change. This global dynamic has various effects, including the increased appearance of emerging infectious diseases (EIDs), which pose a growing threat to global health security.Outbreaks of EIDs, like the 2013-2016 Ebola outbreak in West Africa or the current Ebola outbreak in Democratic Republic of the Congo (DRC), have not only put populations in low- and middle-income countries (LMIC) at risk in terms of morbidity and mortality, but they also have had a significant impact on economic growth in affected regions and beyond.The Coalition for Epidemic Preparedness Innovation (CEPI) is an innovative global partnership between public, private, philanthropic, and civil society organizations that was launched as the result of a consensus that a coordinated, international, and intergovernmental plan was needed to develop and deploy new vaccines to prevent future epidemics.CEPI is focusing on supporting candidate vaccines against the World Health Organization (WHO) Blueprint priority pathogens MERS-CoV, Nipah virus, Lassa fever virus, and Rift Valley fever virus, as well as Chikungunya virus, which is on the WHO watch list. The current vaccine portfolio contains a wide variety of technologies, ranging across recombinant viral vectors, nucleic acids, and recombinant proteins. To support and accelerate vaccine development, CEPI will also support science projects related to the development of biological standards and assays, animal models, epidemiological studies, and diagnostics, as well as build capacities for future clinical trials in risk-prone contexts.
Project description:The Flaviviridae is a family of enveloped viruses with a positive-sense single-stranded RNA genome. It contains many viruses that threaten human health, such as Japanese encephalitis virus (JEV) and yellow fever virus (YFV) of the genus Flavivirus as well as hepatitis C virus of the genus Hepacivirus. Cell culture systems highly permissive for the Flaviviridae viruses are very useful for their isolation, propagation, and diagnosis, an understanding of their biology, and the development of vaccines and antiviral agents. Previously, we isolated a human hepatoma HuH-7-derived cell clone, Huh7.5.1-8, which is highly permissive to hepatitis C virus infection. Here, we have characterized flavivirus infection in the Huh7.5.1-8 cell line by comparing with that in the African green monkey kidney-derived Vero cell line, which is permissive for a wide spectrum of viruses. Upon infection with JEV, Huh7.5.1-8 cells produced a higher amount of virus particles early in infection and were more susceptible to virus-induced cell death than Vero cells. Similar outcomes were obtained when the cells were infected with another flavivirus, YFV (17D-204 strain). Quantification of cellular and extracellular viral RNA revealed that high JEV production in Huh7.5.1-8 cells can be attributed to rapid viral replication kinetics and efficient virus release early in infection. In a plaque assay, Huh7.5.1-8 cells developed JEV plaques more rapidly than Vero cells. Although this was not the case with YFV plaques, Huh7.5.1-8 cells developed higher numbers of YFV plaques than Vero cells. Sequence analysis of cDNA encoding an antiviral RNA helicase, RIG-I, showed that Huh7.5.1-8 cells expressed not only a full-length RIG-I mRNA with a known dominant-negative missense mutation but also variants without the mutation. However, the latter mRNAs lacked exon 5/6-12, indicating functional loss of RIG-I in the cells. These characteristics of the Huh7.5.1-8 cell line are helpful for flavivirus detection, titration, and propagation.
Project description:Even in countries that are currently not facing a flavivirus epidemic, the spread of mosquito-borne flaviviruses presents an increasing public threat, owing to climate change, international travel, and other factors. Many of these countries lack the resources (viral strains, clinical specimens, etc.) needed for the research that could help cope with the threat imposed by flaviviruses, and therefore, an alternative approach is needed. Using an in silico approach to global databases, we aimed to design and develop flavivirus NS1 recombinant proteins with due consideration towards antigenic variation. NS1 genes analyzed in this study included a total of 6,823 sequences, from Dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), Zika virus (ZIKV), and Yellow fever virus (YKV). We extracted and analyzed 316 DENV NS1 sequence types (STs), 59 JEV STs, 75 WNV STs, 30 YFV STs, and 43 ZIKV STs using a simple algorithm based on phylogenetic analysis. STs were reclassified according to the variation of the major epitope by MHC II binding. 78 DENV epitope type (EpT), 29 JEV EpTs, 29 WNV EpTs, 12 YFV EpTs, and 5 ZIKV EpTs were extracted according to their major epitopes. Also, frequency results showed that there were dominant EpTs in all flavivirus. Fifteen STs were selected and purified for the expression of recombinant antigen in Escherichia coli by sodium dodecyl sulfate extraction. Our study details a novel in silico approach for the development of flavivirus diagnostics, including a simple way to screen the important peptide regions.
Project description:Yellow fever virus (YFV)-17D is an empirically developed, highly effective live-attenuated vaccine that has been administered to human beings for almost a century. YFV-17D has stood as a paradigm for a successful viral vaccine, and has been exploited as a potential virus vector for the development of recombinant vaccines against other diseases. In this study, a DNA-launched YFV-17D construct (pBeloBAC-FLYF) was explored as a new modality to the standard vaccine to combine the commendable features of both DNA vaccine and live-attenuated viral vaccine. The DNA-launched YFV-17D construct was characterized extensively both in cell culture and in mice. High titres of YFV-17D were generated upon transfection of the DNA into cells, whereas a mutant with deletion in the capsid-coding region (pBeloBAC-YF/?C) was restricted to a single round of infection, with no release of progeny virus. Homologous prime-boost immunization of AAD mice with both pBeloBAC-FLYF and pBeloBAC-YF/?C elicited specific dose-dependent cellular immune response against YFV-17D. Vaccination of A129 mice with pBeloBAC-FLYF resulted in the induction of YFV-specific neutralizing antibodies in all vaccinated subjects. These promising results underlined the potential of the DNA-launched YFV both as an alternative to standard YFV-17D vaccination and as a vaccine platform for the development of DNA-based recombinant YFV vaccines.
Project description:Zika virus (ZIKV) is a mosquito-borne flavivirus that emerged as a global threat following the most recent outbreak in Brazil in 2015. ZIKV infection of pregnant women is associated with fetal abnormalities such as microcephaly, and infection of adults can lead to Guillain-Barré syndrome, an autoimmune disease characterized by neurological deficits. Although there are currently licensed vaccines for other flaviviruses, there remains an urgent need for preventative vaccines against ZIKV infection. Herein we describe the current efforts to accelerate the development of ZIKV vaccines using various platforms, including live attenuated virus, inactivated virus, DNA and RNA, viral vectors, and in silico-predicted immunogenic viral epitopes. Many of these approaches have leveraged lessons learned from past experience with Dengue and other flavivirus vaccines.
Project description:Dengue viruses (DENV) cause countless human deaths each year, whilst West Nile virus (WNV) has re-emerged as an important human pathogen. There are currently no WNV or DENV vaccines licensed for human use, yet vaccines exist against other flaviviruses. To investigate flavivirus cross-reactivity, sera from a human cohort with a history of vaccination against tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV) and yellow fever virus (YFV) were tested for antibodies by plaque reduction neutralization test. Neutralization of louping ill virus (LIV) occurred, but no significant neutralization of Murray Valley encephalitis virus was observed. Sera from some individuals vaccinated against TBEV and JEV neutralized WNV, which was enhanced by YFV vaccination in some recipients. Similarly, some individuals neutralized DENV-2, but this was not significantly influenced by YFV vaccination. Antigenic cartography techniques were used to generate a geometric illustration of the neutralization titres of selected sera against WNV, TBEV, JEV, LIV, YFV and DENV-2. This demonstrated the individual variation in antibody responses. Most sera had detectable titres against LIV and some had titres against WNV and DENV-2. Generally, LIV titres were similar to titres against TBEV, confirming the close antigenic relationship between TBEV and LIV. JEV was also antigenically closer to TBEV than WNV, using these sera. The use of sera from individuals vaccinated against multiple pathogens is unique relative to previous applications of antigenic cartography techniques. It is evident from these data that notable differences exist between amino acid sequence identity and mapped antigenic relationships within the family Flaviviridae.