9 research outputs found
Epitope mapping of Japanese encephalitis virus neutralizing antibodies by native mass spectrometry and hydrogen/deuterium exchange
Japanese encephalitis virus (JEV) remains a global public health concern due to its epidemiological distribution and the existence of multiple strains. Neutralizing antibodies against this infection have shown efficacy in in vivo studies. Thus, elucidation of the epitopes of neutralizing antibodies can aid in the design and development of effective vaccines against different strains of JEV. Here, we describe a combination of native mass spectrometry (native-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) to complete screening of eight mouse monoclonal antibodies (MAbs) against JEV E-DIII to identify epitope regions. Native-MS was used as a first pass to identify the antibodies that formed a complex with the target antigen, and it revealed that seven of the eight monoclonal antibodies underwent binding. Native mass spectra of a MAb (JEV-27) known to be non-binding showed broad native-MS peaks and poor signal, suggesting the protein is a mixture or that there are impurities in the sample. We followed native-MS with HDX-MS to locate the binding sites for several of the complex-forming antibodies. This combination of two mass spectrometry-based approaches should be generally applicable and particularly suitable for screening of antigen-antibody and other protein-protein interactions when other traditional approaches give unclear results or are difficult, unavailable, or need to be validated
Broadly neutralizing monoclonal antibodies protect against multiple tick-borne flaviviruses
Although Powassan virus (POWV) is an emerging tick-transmitted flavivirus that causes severe or fatal neuroinvasive disease in humans, medical countermeasures have not yet been developed. Here, we developed a panel of neutralizing anti-POWV mAbs recognizing six distinct antigenic sites. The most potent of these mAbs bind sites within domain II or III of the envelope (E) protein and inhibit postattachment viral entry steps. A subset of these mAbs cross-react with other flaviviruses. Both POWV type-specific and cross-reactive neutralizing mAbs confer protection in mice against POWV infection when given as prophylaxis or postexposure therapy. Several cross-reactive mAbs mapping to either domain II or III also protect in vivo against heterologous tick-transmitted flaviviruses including Langat and tick-borne encephalitis virus. Our experiments define structural and functional correlates of antibody protection against POWV infection and identify epitopes targeted by broadly neutralizing antibodies with therapeutic potential against multiple tick-borne flaviviruses
Isolation of a potently neutralizing and protective human monoclonal antibody targeting yellow fever virus
Yellow fever virus (YFV) causes sporadic outbreaks of infection in South America and sub-Saharan Africa. While live-attenuated yellow fever virus vaccines based on three substrains of 17D are considered some of the most effective vaccines in use, problems with production and distribution have created large populations of unvaccinated, vulnerable individuals in areas of endemicity. To date, specific antiviral therapeutics have not been licensed for human use against YFV or any other related flavivirus. Recent advances in monoclonal antibody (mAb) technology have allowed the identification of numerous candidate therapeutics targeting highly pathogenic viruses, including many flaviviruses. Here, we sought to identify a highly neutralizing antibody targeting the YFV envelope (E) protein as a therapeutic candidate. We used human B cell hybridoma technology to isolate mAbs from circulating memory B cells from human YFV vaccine recipients. These antibodies bound to recombinant YFV E protein and recognized at least five major antigenic sites on E. Two mAbs (designated YFV-136 and YFV-121) recognized a shared antigenic site and neutralized the YFV-17D vaccine strai
A broadly reactive antibody targeting the N-terminal domain of SARS-CoV-2 spike confers Fc-mediated protection
Most neutralizing anti-SARS-CoV-2 monoclonal antibodies (mAbs) target the receptor binding domain (RBD) of the spike (S) protein. Here, we characterize a panel of mAbs targeting the N-terminal domain (NTD) or other non-RBD epitopes of S. A subset of NTD mAbs inhibits SARS-CoV-2 entry at a post-attachment step and avidly binds the surface of infected cells. One neutralizing NTD mAb, SARS2-57, protects K18-hACE2 mice against SARS-CoV-2 infection in an Fc-dependent manner. Structural analysis demonstrates that SARS2-57 engages an antigenic supersite that is remodeled by deletions common to emerging variants. In neutralization escape studies with SARS2-57, this NTD site accumulates mutations, including a similar deletion, but the addition of an anti-RBD mAb prevents such escape. Thus, our study highlights a common strategy of immune evasion by SARS-CoV-2 variants and how targeting spatially distinct epitopes, including those in the NTD, may limit such escape
Modulation of the Extent of Cooperative Structural Change During Protein Folding by Chemical Denaturant
Protein
folding and unfolding reactions invariably appear to be
highly cooperative reactions, but the structural and sequence determinants
of cooperativity are poorly understood. Importantly, it is not known
whether cooperative structural change occurs throughout the protein,
or whether some parts change cooperatively and other parts change
noncooperatively. In the current study, hydrogen exchange mass spectrometry
has been used to show that the mechanism of unfolding of the PI3K
SH3 domain is similar in the absence and presence of 5 M urea. The
data are well described by a four state N ↔ I<sub>N</sub> ↔
I<sub>2</sub> ↔ U model, in which structural changes occur
noncooperatively during the N ↔ I<sub>N</sub> and I<sub>N</sub> ↔ I<sub>2</sub> transitions, and occur cooperatively during
the I<sub>2</sub> ↔ U transition. The nSrc-loop and RT-loop,
as well as β strands 4 and 5 undergo noncooperative unfolding,
while β strands 1, 2, and 3 unfold cooperatively in the absence
of urea. However, in the presence of 5 M urea, the unfolding of β
strand 4 switches to become cooperative, leading to an increase in
the extent of cooperative structural change. The current study highlights
the relationship between protein stability and cooperativity, by showing
how the extent of cooperativity can be varied, using chemical denaturant
to alter protein stability
Chemical Denaturants Smoothen Ruggedness on the Free Energy Landscape of Protein Folding
To
characterize experimentally the ruggedness of the free energy
landscape of protein folding is challenging, because the distributed
small free energy barriers are usually dominated by one, or a few,
large activation free energy barriers. This study delineates changes
in the roughness of the free energy landscape by making use of the
observation that a decrease in ruggedness is accompanied invariably
by an increase in folding cooperativity. Hydrogen exchange (HX) coupled
to mass spectrometry was used to detect transient sampling of local
energy minima and the global unfolded state on the free energy landscape
of the small protein single-chain monellin. Under native conditions,
local noncooperative openings result in interconversions between Boltzmann-distributed
intermediate states, populated on an extremely rugged “uphill”
energy landscape. The cooperativity of these interconversions was
increased by selectively destabilizing the native state via mutations,
and further by the addition of a chemical denaturant. The perturbation
of stability alone resulted in seven backbone amide sites exchanging
cooperatively. The size of the cooperatively exchanging and/or unfolding
unit did not depend on the extent of protein destabilization. Only
upon the addition of a denaturant to a destabilized mutant variant
did seven additional backbone amide sites exchange cooperatively.
Segmentwise analysis of the HX kinetics of the mutant variants further
confirmed that the observed increase in cooperativity was due to the
smoothing of the ruggedness of the free energy landscape of folding
of the protein by the chemical denaturant
Hydrogen/Deuterium Exchange Mass Spectrometry Provides Insights into the Role of Drosophila Testis-Specific Myosin VI Light Chain AndroCaM
In
Drosophila testis, myosin VI plays a special role, distinct
from its motor function, by anchoring components to the unusual actin-based
structures (cones) that are required for spermatid individualization.
For this, the two calmodulin (CaM) light-chain molecules of myosin
VI are replaced by androcam (ACaM), a related protein with 67% identity
to CaM. Although ACaM has a similar bi-lobed structure to CaM, with
two EF hand-type Ca2+ binding sites per lobe, only one
functional Ca2+ binding site operates in the amino-terminus.
To understand this light chain substitution, we used hydrogen–deuterium
exchange mass spectrometry (HDX-MS) to examine dynamic changes in
ACaM and CaM upon Ca2+ binding and interaction with the
two CaM binding motifs of myosin VI (insert2 and IQ motif). HDX-MS
reveals that binding of Ca2+ to ACaM destabilizes its N-lobe
but stabilizes the entire C-lobe, whereas for CaM, Ca2+ binding induces a pattern of alternating stabilization/destabilization
throughout. The conformation of this stable holo-C-lobe of ACaM seems
to be a “prefigured” version of the conformation adopted
by the holo-C-lobe of CaM for binding to insert2 and the IQ motif
of myosin VI. Strikingly, the interaction of holo-ACaM with either
peptide converts the holo-N-lobe to its Ca2+-free, more
stable, form. Thus, ACaM in vivo should bind the myosin VI light chain
sites in an apo-N-lobe/holo-C-lobe state that cannot fulfill the Ca2+-related functions of holo-CaM required for myosin VI motor
assembly and activity. These findings indicate that inhibition of
myosin VI motor activity is a precondition for transition to an anchoring
function
Isolation of a potently neutralizing and protective human monoclonal antibody targeting yellow fever virus
Yellow fever virus (YFV) causes sporadic outbreaks of infection in South America and sub-Saharan Africa. While live-attenuated yellow fever virus vaccines based on three substrains of 17D are considered some of the most effective vaccines in use, problems with production and distribution have created large populations of unvaccinated, vulnerable individuals in areas of endemicity. To date, specific antiviral therapeutics have not been licensed for human use against YFV or any other related flavivirus. Recent advances in monoclonal antibody (mAb) technology have allowed the identification of numerous candidate therapeutics targeting highly pathogenic viruses, including many flaviviruses. Here, we sought to identify a highly neutralizing antibody targeting the YFV envelope (E) protein as a therapeutic candidate. We used human B cell hybridoma technology to isolate mAbs from circulating memory B cells from human YFV vaccine recipients. These antibodies bound to recombinant YFV E protein and recognized at least five major antigenic sites on E. Two mAbs (designated YFV-136 and YFV-121) recognized a shared antigenic site and neutralized the YFV-17D vaccine strai
Pan-protective anti-alphavirus human antibodies target a conserved E1 protein epitope
Alphaviruses are emerging, mosquito-transmitted pathogens that cause musculoskeletal and neurological disease in humans. Although neutralizing antibodies that inhibit individual alphaviruses have been described, broadly reactive antibodies that protect against both arthritogenic and encephalitic alphaviruses have not been reported. Here, we identify DC2.112 and DC2.315, two pan-protective yet poorly neutralizing human monoclonal antibodies (mAbs) that avidly bind to viral antigen on the surface of cells infected with arthritogenic and encephalitic alphaviruses. These mAbs engage a conserved epitope in domain II of the E1 protein proximal to and within the fusion peptide. Treatment with DC2.112 or DC2.315 protectsmice against infection by both arthritogenic (chikungunya andMayaro) and encephalitic (Venezuelan, Eastern, and Western equine encephalitis) alphaviruses through multiple mechanisms, including inhibition of viral egress and monocyte-dependent Fc effector functions. These findings define a conserved epitope recognized by weakly neutralizing yet protective antibodies that could be targeted for pan-alphavirus immunotherapy and vaccine design