Emerging rodent-borne hantaviruses cause severe diseases in humans with no approved vaccines or t... more Emerging rodent-borne hantaviruses cause severe diseases in humans with no approved vaccines or therapeutics. We recently isolated a monoclonal broadly neutralizing antibody (nAb) from a Puumala virus–experienced human donor. Here, we report its structure bound to its target, the Gn/Gc glycoprotein heterodimer comprising the viral fusion complex. The structure explains the broad activity of the nAb: It recognizes conserved Gc fusion loop sequences and the main chain of variable Gn sequences, thereby straddling the Gn/Gc heterodimer and locking it in its prefusion conformation. We show that the nAb’s accelerated dissociation from the divergent Andes virus Gn/Gc at endosomal acidic pH limits its potency against this highly lethal virus and correct this liability by engineering an optimized variant that sets a benchmark as a candidate pan-hantavirus therapeutic.
Alphaviruses are important human and animal pathogens that can cause a range of debilitating symp... more Alphaviruses are important human and animal pathogens that can cause a range of debilitating symptoms and are found worldwide. These include arthralgic diseases caused by Old-World viruses and encephalitis induced by infection with New-World alphaviruses. Non-coding RNAs do not encode for proteins, but can modulate cellular response pathways in a myriad of ways. There are several classes of non-coding RNAs, some more well-studied than others. Much research has focused on the mRNA response to infection against alphaviruses, but analysis of non-coding RNA responses has been more limited until recently. This review covers what is known regarding host cell non-coding RNA responses in alphavirus infections and highlights gaps in the knowledge that future research should address.
Filoviruses are highly virulent pathogens which cause severe hemorrhagic fevers, including Ebola ... more Filoviruses are highly virulent pathogens which cause severe hemorrhagic fevers, including Ebola virus disease (EVD). There is currently an ongoing large outbreak in the Democratic Republic of Congo, and while investigational vaccines have been used in trials during the outbreak, there are no licensed vaccines to EVD, or the related Marburg virus disease (MVD). The glycoproteins of filoviruses are the only virally expressed proteins on the virion surface and are required for receptor binding and as such, they are the main candidate vaccine antigen. A major component of the glycoprotein are the N- and O-linked glycans, but these glycans vary in number, distribution and type depending on the cell type producing the glycoprotein. Both mammalian and insect cell lines are commonly used to produce filovirus proteins used in vaccine development, but produce proteins with very different glycosylation. Here, we describe the impact of the filovirus glycoprotein glycans on the immunogenicity o...
Filoviruses are hemorrhagic fever viruses that are some of the deadliest pathogens in the world w... more Filoviruses are hemorrhagic fever viruses that are some of the deadliest pathogens in the world with a fatality rate up to 90%. Symptoms include fever, vomiting, diarrhea, headache, weakness and can lead to hemorrhaging, multiple organ failure and death. Experimental vaccines use filovirus Surface glycoproteins (GPs) as immunogens to induce protective immune responses. Our laboratory studies the glycosylation patterns of different filovirus GPs to develop improved vaccines. Mammalian 293T cells have more complex N-linked glycosylation machinery compared to Sf9 insect cells but both are capable of O-linked glycosylation. Differences in N- and O-linked glycans on filovirus GPs may induce different immune responses upon vaccination. However, the glycosylation patterns on the different filovirus GPs have not been thoroughly elucidated. Here we report purification and glycan analysis of GPs from filoviruses produced in 293T mammalian cells and Sf9 insect cells. We are currently assessing...
Filoviruses, specifically ebola- and marburgviruses, can cause severe hemorrhagic fever in humans... more Filoviruses, specifically ebola- and marburgviruses, can cause severe hemorrhagic fever in humans. Currently, there are no US licensed vaccines available against filoviruses. An important filovirus protein required for cellular entry is glycoprotein (GP). GPs are also used as vaccine antigens that can trigger antibody production, which makes them an important factor in vaccine development. Distinct vaccine platforms are made in different cell types, such as mammalian and insect cells. Different glycosylation patterns and GP distribution in virus-like particles (VLPs) were observed by our lab in GPs expressed in mammalian cell lines, but comparative data is needed to determine how differential filovirus GP concentration in the VLP platform affects the immune response in vaccination. We hypothesize that differential filovirus GP concentration will yield diverse results in immunogenicity and will provide insight into specifically how we can utilize GP concentration to improve vaccine d...
Orthohantaviruses are negative-stranded RNA viruses with trisegmented genomes that can cause seve... more Orthohantaviruses are negative-stranded RNA viruses with trisegmented genomes that can cause severe disease in humans and are carried by several host reservoirs throughout the world. Old World orthohantaviruses are primarily located throughout Europe and Asia, causing hemorrhagic fever with renal syndrome, and New World orthohantaviruses are found in North, Central, and South America, causing hantavirus cardiopulmonary syndrome (HCPS). In the United States, Sin Nombre orthohantavirus (SNV) is the primary cause of HCPS with a fatality rate of ~36%. The primary SNV host reservoir is thought to be the North American deer mouse, Peromyscus maniculatus. However, it has been shown that other species of Peromyscus can carry different orthohantaviruses. Few studies have systemically surveyed which orthohantaviruses may exist in wild-caught rodents or monitored spillover events into additional rodent reservoirs. A method for the rapid detection of orthohantaviruses is needed to screen large ...
The rodent-borne hantavirus Puumala virus (PUUV) and related agents cause hemorrhagic fever with ... more The rodent-borne hantavirus Puumala virus (PUUV) and related agents cause hemorrhagic fever with renal syndrome (HFRS) in humans. Other hantaviruses, including Andes virus (ANDV) and Sin Nombre virus, cause a distinct zoonotic disease, hantavirus cardiopulmonary syndrome (HCPS). Although these infections are severe and have substantial case fatality rates, no FDA-approved hantavirus countermeasures are available. Recent work suggests that monoclonal antibodies may have therapeutic utility. We describe here the isolation of human neutralizing antibodies (nAbs) against tetrameric Gn/Gc glycoprotein spikes from PUUV-experienced donors. We define a dominant class of nAbs recognizing the “capping loop” of Gn that masks the hydrophobic fusion loops in Gc. A subset of nAbs in this class, including ADI-42898, bound Gn/Gc complexes but not Gn alone, strongly suggesting that they recognize a quaternary epitope encompassing both Gn and Gc. ADI-42898 blocked the cell entry of seven HCPS- and HF...
The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular an... more The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.
Biocompatible nanoparticles composed of poly(lactic-co-glycolic acid) (PLGA) are used as drug and... more Biocompatible nanoparticles composed of poly(lactic-co-glycolic acid) (PLGA) are used as drug and vaccine delivery systems because of their tunability in size and sustained release of cargo molecules. While the use of toxic stabilizers such as polyvinyl alcohol (PVA) limit the utility of PLGA, stabilizer-free PLGA nanoparticles are rarely used because they can be challenging to prepare. Here, we developed a tunable, stabilizer-free PLGA nanoparticle formulation capable of encapsulating plasmid DNA and demonstrated the formation of an elastin-like polymer PLGA hybrid nanoparticle with exceptional stability and biocompatibility. A suite of PLGAs were fabricated using solvent evaporation methods and assessed for particle size and stability in water. We find that under physiological conditions (PBS at 37˚C), the most stable PLGA formulation (P4) was found to contain a greater L:G ratio (65:35), lower MW, and carboxyl terminus. Subsequent experiments determined P4 nanoparticles were as stable as those made with PVA, yet significantly less cytotoxic. Variation in particle size was achieved through altering PLGA stoichiometry while maintaining the ability to encapsulate DNA and were modified with elastin-like polymers for increased immune tolerance. Overall, a useful method for tunable, stabilizer-free PLGA nanoparticle formulation was developed for use in drug and vaccine delivery, and immune targeting.
Orthohantaviruses cause severe disease in humans and can be lethal in up to 40% of cases. Sin Nom... more Orthohantaviruses cause severe disease in humans and can be lethal in up to 40% of cases. Sin Nombre orthohantavirus (SNV) is the main cause of hantavirus disease in North America.
Emerging rodent-borne hantaviruses cause severe diseases in humans with no approved vaccines or t... more Emerging rodent-borne hantaviruses cause severe diseases in humans with no approved vaccines or therapeutics. We recently isolated a monoclonal broadly neutralizing antibody (nAb) from a Puumala virus–experienced human donor. Here, we report its structure bound to its target, the Gn/Gc glycoprotein heterodimer comprising the viral fusion complex. The structure explains the broad activity of the nAb: It recognizes conserved Gc fusion loop sequences and the main chain of variable Gn sequences, thereby straddling the Gn/Gc heterodimer and locking it in its prefusion conformation. We show that the nAb’s accelerated dissociation from the divergent Andes virus Gn/Gc at endosomal acidic pH limits its potency against this highly lethal virus and correct this liability by engineering an optimized variant that sets a benchmark as a candidate pan-hantavirus therapeutic.
Alphaviruses are important human and animal pathogens that can cause a range of debilitating symp... more Alphaviruses are important human and animal pathogens that can cause a range of debilitating symptoms and are found worldwide. These include arthralgic diseases caused by Old-World viruses and encephalitis induced by infection with New-World alphaviruses. Non-coding RNAs do not encode for proteins, but can modulate cellular response pathways in a myriad of ways. There are several classes of non-coding RNAs, some more well-studied than others. Much research has focused on the mRNA response to infection against alphaviruses, but analysis of non-coding RNA responses has been more limited until recently. This review covers what is known regarding host cell non-coding RNA responses in alphavirus infections and highlights gaps in the knowledge that future research should address.
Filoviruses are highly virulent pathogens which cause severe hemorrhagic fevers, including Ebola ... more Filoviruses are highly virulent pathogens which cause severe hemorrhagic fevers, including Ebola virus disease (EVD). There is currently an ongoing large outbreak in the Democratic Republic of Congo, and while investigational vaccines have been used in trials during the outbreak, there are no licensed vaccines to EVD, or the related Marburg virus disease (MVD). The glycoproteins of filoviruses are the only virally expressed proteins on the virion surface and are required for receptor binding and as such, they are the main candidate vaccine antigen. A major component of the glycoprotein are the N- and O-linked glycans, but these glycans vary in number, distribution and type depending on the cell type producing the glycoprotein. Both mammalian and insect cell lines are commonly used to produce filovirus proteins used in vaccine development, but produce proteins with very different glycosylation. Here, we describe the impact of the filovirus glycoprotein glycans on the immunogenicity o...
Filoviruses are hemorrhagic fever viruses that are some of the deadliest pathogens in the world w... more Filoviruses are hemorrhagic fever viruses that are some of the deadliest pathogens in the world with a fatality rate up to 90%. Symptoms include fever, vomiting, diarrhea, headache, weakness and can lead to hemorrhaging, multiple organ failure and death. Experimental vaccines use filovirus Surface glycoproteins (GPs) as immunogens to induce protective immune responses. Our laboratory studies the glycosylation patterns of different filovirus GPs to develop improved vaccines. Mammalian 293T cells have more complex N-linked glycosylation machinery compared to Sf9 insect cells but both are capable of O-linked glycosylation. Differences in N- and O-linked glycans on filovirus GPs may induce different immune responses upon vaccination. However, the glycosylation patterns on the different filovirus GPs have not been thoroughly elucidated. Here we report purification and glycan analysis of GPs from filoviruses produced in 293T mammalian cells and Sf9 insect cells. We are currently assessing...
Filoviruses, specifically ebola- and marburgviruses, can cause severe hemorrhagic fever in humans... more Filoviruses, specifically ebola- and marburgviruses, can cause severe hemorrhagic fever in humans. Currently, there are no US licensed vaccines available against filoviruses. An important filovirus protein required for cellular entry is glycoprotein (GP). GPs are also used as vaccine antigens that can trigger antibody production, which makes them an important factor in vaccine development. Distinct vaccine platforms are made in different cell types, such as mammalian and insect cells. Different glycosylation patterns and GP distribution in virus-like particles (VLPs) were observed by our lab in GPs expressed in mammalian cell lines, but comparative data is needed to determine how differential filovirus GP concentration in the VLP platform affects the immune response in vaccination. We hypothesize that differential filovirus GP concentration will yield diverse results in immunogenicity and will provide insight into specifically how we can utilize GP concentration to improve vaccine d...
Orthohantaviruses are negative-stranded RNA viruses with trisegmented genomes that can cause seve... more Orthohantaviruses are negative-stranded RNA viruses with trisegmented genomes that can cause severe disease in humans and are carried by several host reservoirs throughout the world. Old World orthohantaviruses are primarily located throughout Europe and Asia, causing hemorrhagic fever with renal syndrome, and New World orthohantaviruses are found in North, Central, and South America, causing hantavirus cardiopulmonary syndrome (HCPS). In the United States, Sin Nombre orthohantavirus (SNV) is the primary cause of HCPS with a fatality rate of ~36%. The primary SNV host reservoir is thought to be the North American deer mouse, Peromyscus maniculatus. However, it has been shown that other species of Peromyscus can carry different orthohantaviruses. Few studies have systemically surveyed which orthohantaviruses may exist in wild-caught rodents or monitored spillover events into additional rodent reservoirs. A method for the rapid detection of orthohantaviruses is needed to screen large ...
The rodent-borne hantavirus Puumala virus (PUUV) and related agents cause hemorrhagic fever with ... more The rodent-borne hantavirus Puumala virus (PUUV) and related agents cause hemorrhagic fever with renal syndrome (HFRS) in humans. Other hantaviruses, including Andes virus (ANDV) and Sin Nombre virus, cause a distinct zoonotic disease, hantavirus cardiopulmonary syndrome (HCPS). Although these infections are severe and have substantial case fatality rates, no FDA-approved hantavirus countermeasures are available. Recent work suggests that monoclonal antibodies may have therapeutic utility. We describe here the isolation of human neutralizing antibodies (nAbs) against tetrameric Gn/Gc glycoprotein spikes from PUUV-experienced donors. We define a dominant class of nAbs recognizing the “capping loop” of Gn that masks the hydrophobic fusion loops in Gc. A subset of nAbs in this class, including ADI-42898, bound Gn/Gc complexes but not Gn alone, strongly suggesting that they recognize a quaternary epitope encompassing both Gn and Gc. ADI-42898 blocked the cell entry of seven HCPS- and HF...
The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular an... more The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.
Biocompatible nanoparticles composed of poly(lactic-co-glycolic acid) (PLGA) are used as drug and... more Biocompatible nanoparticles composed of poly(lactic-co-glycolic acid) (PLGA) are used as drug and vaccine delivery systems because of their tunability in size and sustained release of cargo molecules. While the use of toxic stabilizers such as polyvinyl alcohol (PVA) limit the utility of PLGA, stabilizer-free PLGA nanoparticles are rarely used because they can be challenging to prepare. Here, we developed a tunable, stabilizer-free PLGA nanoparticle formulation capable of encapsulating plasmid DNA and demonstrated the formation of an elastin-like polymer PLGA hybrid nanoparticle with exceptional stability and biocompatibility. A suite of PLGAs were fabricated using solvent evaporation methods and assessed for particle size and stability in water. We find that under physiological conditions (PBS at 37˚C), the most stable PLGA formulation (P4) was found to contain a greater L:G ratio (65:35), lower MW, and carboxyl terminus. Subsequent experiments determined P4 nanoparticles were as stable as those made with PVA, yet significantly less cytotoxic. Variation in particle size was achieved through altering PLGA stoichiometry while maintaining the ability to encapsulate DNA and were modified with elastin-like polymers for increased immune tolerance. Overall, a useful method for tunable, stabilizer-free PLGA nanoparticle formulation was developed for use in drug and vaccine delivery, and immune targeting.
Orthohantaviruses cause severe disease in humans and can be lethal in up to 40% of cases. Sin Nom... more Orthohantaviruses cause severe disease in humans and can be lethal in up to 40% of cases. Sin Nombre orthohantavirus (SNV) is the main cause of hantavirus disease in North America.
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Papers by Steven Bradfute