Engineering modular platforms to control biomolecular architecture can advance both the understanding and the manipulation of biological systems. Icosahedral particles uniformly displaying single antigens stimulate potent immune activation and have been successful in various licensed vaccines. However, it remains challenging to display multiple antigens on a single particle and to induce broader immunity protective across strains or even against distinct diseases. Here, we design a dually addressable synthetic nanoparticle by engineering the multimerizing coiled-coil IMX313 and two orthogonally reactive split proteins. SpyCatcher protein forms an isopeptide bond with SpyTag peptide through spontaneous amidation. SnoopCatcher forms an isopeptide bond with SnoopTag peptide through transamidation. SpyCatcher-IMX-SnoopCatcher provides a modular platform, whereby SpyTag-antigen and SnoopTag-antigen can be multimerized on opposite faces of the particle simply upon mixing. We demonstrate efficient derivatization of the platform with model proteins and complex pathogen-derived antigens. SpyCatcher-IMX-SnoopCatcher was expressed in Escherichia coli and was resilient to lyophilization or extreme temperatures. For the next generation of malaria vaccines, blocking the transmission of the parasite from human to mosquito is an important goal. SpyCatcher-IMX-SnoopCatcher multimerization of the leading transmission-blocking antigens Pfs25 and Pfs28 greatly enhanced the antibody response to both antigens in comparison to the monomeric proteins. This dual plug-and-display architecture should help to accelerate vaccine development for malaria and other diseases.
The threat posed by pandemics grows alongside increased globalization and technological innovation. Distant cultures can now be connected in a day’s time, and international trade links global health and economic prosperity. This report details nine priority areas and accompanying action items that will help to address current pandemic response problems.
Developing centralized leadership; coordinating existing agencies and departments; reforming WHO; and providing adequate funding to establish sufficient supplies, infrastructure, expertise ,and institutions are paramount to success in pandemic response.
The reports stresses that Foreign aid for global health and related investments has never been more important to international security and US national security
Schmallenberg virus (SBV) is transmitted by insect vectors, and therefore vaccination is one of the most important tools of disease control. In our study, novel subunit vaccines on the basis of an amino-terminal domain of SBV Gc of 234 amino acids (“Gc Amino”) first were tested and selected using a lethal small animal challenge model and then the best performing formulations also were tested in cattle. We could show that neither E. coli expressed nor the reduced form of “Gc Amino” protected from SBV infection. In contrast, both, immunization with “Gc Amino”-encoding DNA plasmids and “Gc-amino” expressed in a mammalian system, conferred protection in up to 66% of the animals. Interestingly, the best performance was achieved with a multivalent antigen containing the covalently linked Gc domains of both, SBV and the related Akabane virus. All vaccinated cattle and mice were fully protected against SBV challenge infection. Furthermore, in the absence of antibodies against the viral N-protein, differentiation between vaccinated and field-infected animals allows an SBV marker vaccination concept. Moreover, the presented vaccine design also could be tested for other members of the Simbu serogroup and might allow the inclusion of additional immunogenic domains.
Synthetic biology uses living cells as molecular foundries for the biosynthesis of drugs, therapeutic proteins, and other commodities. However, the need for specialized equipment and refrigeration for production and distribution poses a challenge for the delivery of these technologies to the field and to low-resource areas. Here, we present a portable platform that provides the means for on-site, on-demand manufacturing of therapeutics and biomolecules. This flexible system is based on reaction pellets composed of freeze-dried, cell-free transcription and translation machinery, which can be easily hydrated and utilized for biosynthesis through the addition of DNA encoding the desired output. We demonstrate this approach with the manufacture and functional validation of antimicrobial peptides and vaccines and present combinatorial methods for the production of antibody conjugates and small molecules. This synthetic biology platform resolves important practical limitations in the production and distribution of therapeutics and molecular tools, both to the developed and developing world
Coronaviruses (CoVs) have a remarkable potential to change tropism. This is particularly illustrated over the last 15 years by the emergence of two zoonotic CoVs, the severe acute respiratory syndrome (SARS)- and Middle East respiratory syndrome (MERS)-CoV. Due to their inherent genetic variability, it is inevitable that new cross-species transmission events of these enveloped, positive-stranded RNA viruses will occur. Research into these medical and veterinary important pathogens—sparked by the SARS and MERS outbreaks—revealed important principles of inter- and intraspecies tropism changes. The primary determinant of CoV tropism is the viral spike (S) entry protein. Trimers of the S glycoproteins on the virion surface accommodate binding to a cell surface receptor and fusion of the viral and cellular membrane. Recently, high-resolution structures of two CoV S proteins have been elucidated by single-particle cryo-electron microscopy. Using this new structural insight, we review the changes in the S protein that relate to changes in virus tropism. Different concepts underlie these tropism changes at the cellular, tissue, and host species level, including the promiscuity or adaptability of S proteins to orthologous receptors, alterations in the proteolytic cleavage activation as well as changes in the S protein metastability. A thorough understanding of the key role of the S protein in CoV entry is critical to further our understanding of virus cross-species transmission and pathogenesis and for development of intervention strategies
Porcine enteric coronaviruses (CoVs) cause severe disease in the porcine herds worldwide, leading to important economic losses. Despite the knowledge of these viruses since the 1970s, vaccination strategies have not been implemented, leading to continuous re-emergence of novel virulent strains. Live attenuated vaccines historically have been the most efficient. We consider that the new trend is the development of recombinant vaccines by using reverse genetics systems to engineer attenuated viruses, which could be used as effective and safe modified live vaccine candidates. To this end, host cell signaling pathways influencing porcine CoV virulence should be identified. Similarly, the identity of viral proteins involved in the modulation of host cell pathways influencing CoV pathogenesis should be analyzed. With this information, and using reverse genetics systems, it is possible to design viruses with modifications in the viral proteins acting as virulence factors, which may lead to attenuated viruses and, therefore, vaccine candidates. In addition, novel antiviral drugs may be developed once the host cell pathways and the molecular mechanism affecting porcine CoV replication and virulence are known. This review is focused in the host cell responses to enteric porcine CoV infection and the viral proteins involved in pathogenesis.
Coronavirus (CoV) nonstructural protein 14 (nsp14) is a 60-kDa protein encoded by the replicase gene that is part of the replication-transcription complex. It is a bifunctional enzyme bearing 3′-to-5′ exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) activities. ExoN hydrolyzes single-stranded RNAs and double-stranded RNAs (dsRNAs) and is part of a proofreading system responsible for the high fidelity of CoV replication. nsp14 N7-MTase activity is required for viral mRNA cap synthesis and prevents the recognition of viral mRNAs as “non-self” by the host cell. In this work, a set of point mutants affecting different motifs within the ExoN domain of nsp14 was generated, using transmissible gastroenteritis virus as a model of Alphacoronavirus. Mutants lacking ExoN activity were nonviable despite being competent in both viral RNA and protein synthesis. A specific mutation within zinc finger 1 (ZF-C) led to production of a viable virus with growth and viral RNA synthesis kinetics similar to that of the parental virus. Mutant recombinant transmissible gastroenteritis virus (TGEV) ZF-C (rTGEV-ZF-C) caused decreased cytopathic effect and apoptosis compared with the wild-type virus and reduced levels of dsRNA accumulation at late times postinfection. Consequently, the mutant triggered a reduced antiviral response, which was confirmed by evaluating different stages of the dsRNA-induced antiviral pathway. The expression of beta interferon (IFN-β), tumor necrosis factor (TNF), and interferon-stimulated genes in cells infected with mutant rTGEV-ZF-C was reduced compared to the levels seen with the parental virus. Overall, our data revealed a potential role for CoV nsp14 in modulation of the innate immune response.
Serial passage of viruses in cell culture has been traditionally used to attenuate virulence and identify determinants of viral pathogenesis. In a previous study, we found that a strain of Schmallenberg virus (SBV) serially passaged in tissue culture (termed SBVp32) unexpectedly displayed increased pathogenicity in suckling mice compared to wild-type SBV. In this study, we mapped the determinants of SBVp32 virulence to the viral genome M segment. SBVp32 virulence is associated with the capacity of this virus to reach high titers in the brains of experimentally infected suckling mice. We also found that the Gc glycoprotein, encoded by the M segment of SBVp32, facilitates host cell protein shutoff in vitro. Interestingly, while the M segment of SBVp32 is a virulence factor, we found that the S segment of the same virus confers by itself an attenuated phenotype to wild-type SBV, as it has lost the ability to block the innate immune system of the host. Single mutations present in the Gc glycoprotein of SBVp32 are sufficient to compensate for both the attenuated phenotype of the SBVp32 S segment and the attenuated phenotype of NSs deletion mutants. Our data also indicate that the SBVp32 M segment does not act as an interferon (IFN) antagonist. Therefore, SBV mutants can retain pathogenicity even when they are unable to fully control the production of IFN by infected cells. Overall, this study suggests that the viral glycoprotein of orthobunyaviruses can compensate, at least in part, for the function of NSs. In addition, we also provide evidence that the induction of total cellular protein shutoff by SBV is determined by multiple viral proteins, while the ability to control the production of IFN maps to the NSs protein.
Middle East respiratory syndrome coronavirus (MERS-CoV) is not efficiently transmitted between humans, but it is highly prevalent in dromedary camels. Here we report that the MERS-CoV receptor—dipeptidyl peptidase 4 (DPP4)—is expressed in the upper respiratory tract epithelium of camels but not in that of humans. Lack of DPP4 expression may be the primary cause of limited MERS-CoV replication in the human upper respiratory tract and hence restrict transmission.
Middle East respiratory syndrome coronavirus (MERS-CoV) infections have led to an ongoing outbreak in humans, which was fueled by multiple zoonotic MERS-CoV introductions from dromedary camels. In addition to the implementation of hygiene measures to limit further camel-to-human and human-to-human transmissions, vaccine-mediated reduction of MERS-CoV spread from the animal reservoir may be envisaged. Here we show that a modified vaccinia virus Ankara (MVA) vaccine expressing the MERS-CoV spike protein confers mucosal immunity in dromedary camels. Compared with results for control animals, we observed a significant reduction of excreted infectious virus and viral RNA transcripts in vaccinated animals upon MERS-CoV challenge. Protection correlated with the presence of serum neutralizing antibodies to MERS-CoV. Induction of MVA-specific antibodies that cross-neutralize camelpox virus would also provide protection against camelpox