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Protein Crystallography in Vaccine Research and Development

Authors:

Abstract

The use of protein X-ray crystallography for structure-based design of small-molecule drugs is well-documented and includes several notable success stories. However, it is less well-known that structural biology has emerged as a major tool for the design of novel vaccine antigens. Here, we review the important contributions that protein crystallography has made so far to vaccine research and development. We discuss several examples of the crystallographic characterization of vaccine antigen structures, alone or in complexes with ligands or receptors. We cover the critical role of high-resolution epitope mapping by reviewing structures of complexes between antigens and their cognate neutralizing, or protective, antibody fragments. Most importantly, we provide recent examples where structural insights obtained via protein crystallography have been used to design novel optimized vaccine antigens. This review aims to illustrate the value of protein crystallography in the emerging discipline of structural vaccinology and its impact on the rational design of vaccines.
Int. J. Mol. Sci. 2015, 16, 13106-13140; doi:10.3390/ijms160613106
International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
Review
Protein Crystallography in Vaccine Research and Development
Enrico Malito 1, Andrea Carfi 2 and Matthew J. Bottomley 1,*
1 Protein Biochemistry Department, Novartis Vaccines & Diagnostics s.r.l. (a GSK Company),
Via Fiorentina 1, 53100 Siena, Italy; E-Mail: enrico.x.malito@gsk.com
2 Protein Biochemistry Department, GSK Vaccines, Cambridge, MA 02139, USA;
E-Mail: andrea.x.carfi@gsk.com
* Author to whom correspondence should be addressed; E-Mail: matthew.j.bottomley@gsk.com;
Tel.: +39-0577-243-244; Fax: +39-0577-539-314.
Academic Editor: Charles A. Collyer
Received: 31 March 2015 / Accepted: 1 June 2015 / Published: 9 June 2015
Abstract: The use of protein X-ray crystallography for structure-based design of
small-molecule drugs is well-documented and includes several notable success stories.
However, it is less well-known that structural biology has emerged as a major tool for the
design of novel vaccine antigens. Here, we review the important contributions that protein
crystallography has made so far to vaccine research and development. We discuss several
examples of the crystallographic characterization of vaccine antigen structures, alone or in
complexes with ligands or receptors. We cover the critical role of high-resolution epitope
mapping by reviewing structures of complexes between antigens and their cognate
neutralizing, or protective, antibody fragments. Most importantly, we provide recent
examples where structural insights obtained via protein crystallography have been used to
design novel optimized vaccine antigens. This review aims to illustrate the value of protein
crystallography in the emerging discipline of structural vaccinology and its impact on the
rational design of vaccines.
Keywords: crystallization; protein engineering; paratope; meningitis; respiratory syncytial
virus (RSV); Staphylococcus aureus; human immunodeficiency virus (HIV)
OPEN ACCESS
Int. J. Mol. Sci. 2015, 16 13107
1. Introduction
Vaccination first began in the 18th Century when Edward Jenner protected humans from smallpox
by administering material from humans infected with cowpox. In the 19th Century, Pasteur, Koch,
Ramon and Mérieux pioneered the development of live-attenuated and killed vaccines, and inactivated
toxins, to protect against rabies, cholera, plague and typhoid. Additional major vaccine developments
in the 20th Century provided protection against diphtheria, tetanus, pertussis, polio, several types of
meningococcus and pneumococcus, haemophilus influenzae B, hepatitis and influenza. Collectively,
these vaccines have eliminated most of the life-threatening childhood diseases that previously caused
millions of deaths and severe morbidity, thus rendering vaccination one of the most effective medical
interventions in history [1,2]. In the 21st Century, vaccination continues to play a highly significant
and expanding role in the control and elimination of disease. Nevertheless, many important
disease-causing infections are not yet preventable by vaccination, including, respiratory syncytial virus
(RSV), human immunodeficiency virus (HIV), groups A and B streptococcus (GAS, GBS), malaria,
tuberculosis and ebola. Moreover, certain vulnerable population groups generally tend to be poorly
served by vaccination. Therefore, further research and development of novel vaccines is required to
address a plethora of currently unmet, globally-significant medical needs [3].
One of the most important advances in vaccine research over the last 10–15 years was the advent of
whole-genome sequencing technology. Genomics drove the development of the “reverse vaccinology”
approach, which overcame challenges that had not been resolved via conventional vaccinology [4].
Indeed, it was the whole-genome sequencing of Neisseria meningitidis serogroup B that enabled the
development of reverse vaccinology for the identification of recombinant antigens for a protein-based
vaccine against serogroup B meningococcus [5,6]. Since then, it has become routine to obtain the
amino acid sequence of all possible proteins that a pathogen might encode in its genome, which greatly
potentiates the early stages of vaccine discovery. However, while all antigen sequences can be readily
obtained, this information does not necessarily translate into recombinant antigens with ideal attributes
for vaccine development, nor do the sequences necessarily provide insights into antigen structures or
functions. Therefore, empirical studies are required in order to optimize the recombinant proteins for
development and to provide the degree of antigen characterization desirable prior to embarking on
clinical studies—these are the stages where protein crystallography can play a crucial role.
Over the last five years, several examples have been presented where antigen structure
determination by X-ray crystallography not only provided a highly-detailed level of antigen
characterization but, more importantly, also enabled the design of better antigens. Improvements have
encompassed structural modifications that stabilize a desirable conformation of the antigen, or that
remove undesirable biological properties such as pore-forming toxin function or catalytic activity, or
that modify the surface in order to display preferred epitopes. Indeed, the high sequence variability of
antigens on a pathogen surface represents a major hurdle to vaccine design in many cases. To fully
understand the antigenic manifestation of such sequence variability, we require insights into the
structure, dynamics and conformational variability that the antigen may possess. Structural information
can therefore help to identify solutions to these various obstacles, thus facilitating vaccine development.
This review aims to provide a concise survey of several recent advances in vaccine research and
development that have been driven by insights obtained from protein crystallography. We present
Int. J. Mol. Sci. 2015, 16 13108
several examples, from both bacterial and viral pathogens, which illustrate how high-resolution
structural information can be combined with protein engineering to generate antigens that are safe,
immunogenic, broadly-protective, stable, and easy to develop. We also conclude with an outlook of
how we expect the field to evolve in the near future.
2. Protein Crystallography for Antigen Characterization and Epitope Mapping
One of the major contributions of protein crystallography in vaccine research is the structural
characterization of antigens either alone or in complexes with the antigen-binding antibody fragments
(Fabs) of neutralizing, or “protective”, monoclonal antibodies (mAbs). The following sections provide
an overview of some recent advances and highlights in this field.
2.1. Antigen Characterization by X-ray Crystallography
2.1.1. NadA—A Surface-Exposed Meningococcal Adhesin and Vaccine Antigen
It is worthwhile to introduce the pathogen Neisseria meningitidis, since research towards a
broadly-protective serogroup B meningococcal vaccine has provided interesting examples discussed
herein. N. meningitidis is a human-specific bacterium that causes severe sepsis and meningococcal
meningitis, resulting in death or devastating long-term sequelae, and is responsible for about 50% of
bacterial meningitis worldwide, an estimated 1.2 million annual cases [7]. The meningococcal
serogroups A, B, C, W and Y are the most common, causing most of the disease, predominantly in
infants, young children, and adolescents. Due to the very rapid onset and development of disease,
mortality rates among infected individuals are as high as 10%, and sequelae are found in 11%–19% of
survivors, despite the availability of antibiotic therapies. Glyco-conjugate vaccines protecting against
serogroups A, C, W and Y have shown great efficacy [8], yet development of a conjugate vaccine
against serogroup B meningococcus was hampered due to similarity of the B polysaccharide to the
“self” neuraminic acid present on human fetal tissues [9]. Consequently, serogroup B meningococcus
is responsible for up to 90% of cases of meningitis in Europe and 30%–50% of cases in the United
States. However, the first recombinant protein-based meningococcal vaccine, Bexsero®, was approved
in Europe in 2013 [10]. Bexsero® has subsequently been approved in over 35 countries worldwide, and
two meningococcal vaccines, Trumenba® [11] and Bexsero®, have been approved for use in the United
States in late 2014 and early 2015.
Bexsero® is a multi-component vaccine composed of an outer membrane vesicle component plus
three main recombinant meningococcal proteins: the Neisserial heparin binding antigen (NHBA), the
factor H binding protein (fHbp) and the Neisseria adhesin A (NadA), as reviewed previously [12].
Here we briefly describe the structural characterization of NadA, which was not straightforward and
therefore also serves to illustrate a number of enabling technologies which may be widely relevant to
protein crystallographers in this field.
NadA is a surface-exposed protein belonging to the trimeric autotransporter adhesin (TAA) family.
All TAAs are obligate homotrimeric proteins and share a common molecular architecture made of a
conserved C-terminal integral membrane β-barrel, which anchors the proteins to the outer membrane,
and an N-terminal “passenger” domain that is responsible for adhesion [13]. The passenger domain
Int. J. Mol. Sci. 2015, 16 13109
has a modular architecture and is typically made of a central α-helical domain (the stalk) that forms
coiled-coil structures and a distinct N-terminal domain (the head) that is mainly responsible for
binding to host cellular receptors.
NadA is known to mediate adhesion to and invasion of epithelial cells [14,15] and to induce
bactericidal antibodies in immunized humans [16]. Over 40 different NadA protein sequences have
been identified and classified into two main groups, containing six variants overall [17]. The Bexsero®
vaccine contains NadA variant 3, which shares approximately 90% sequence identity with variants 1
and 2, against which it induces cross-protective antibodies. In contrast, the variants 4, 5 and 6 from
group II display only 45%–50% sequence identity with variants from group I, and the two groups are
not cross-protective.
Structural studies of NadA were initiated in order to understand the molecular basis for these
immunological differences. However, sequence analyses of NadA predicted a long and potentially
flexible molecule with a high proportion of α-helical elements likely to form coiled-coils [15], the
structure determination of which is notoriously difficult [18]. The NadA variant 3 vaccine construct,
which includes the predicted head and the entire stalk, was indeed recalcitrant to crystallization,
presumably due to its great length, intrinsic flexibility, and relatively low thermal stability. The latter is
typically correlated with poor outcomes in crystallization trials, whereas higher melting-points tend to
increase the probability of crystallization [19]. Protein engineering approaches were employed to find
expression constructs of NadA fragments that might crystallize more easily [20]. First, differential
scanning calorimetry (DSC) was used to screen multiple C-terminal truncation mutants of NadA
variant 3 designed with progressively shorter stalks. Next, the design of truncated constructs was
translated onto other NadA sequence variants (4 and 5), to explore whether slight differences in amino
acid composition could further affect crystallization propensity, an approach previously defined as
sequence homolog screening [21,22]. The most thermostable construct obtained by these strategies
(NadA variant 5, spanning residues 24–220) crystallized readily and reproducibly. Subsequently,
crystals optimized by use of additives diffracted at 2.0 Å resolution [20]. Several attempts to solve the
structure by molecular replacement, using these initial native data, were unsuccessful. However, the
previous observation that other TAAs with Asn residue substitutions at position d of the coiled-coil
heptad repeat were able to coordinate ions in the buried core of the hydrophobic coiled-coil [23]
inspired attempts to soak halides into crystals of NadA for successful experimental phasing by single
anomalous dispersion (SAD).
The NadA variant 5 protein exhibits an elongated structure approximately 220 Å-long, and
almost exclusively coiled-coil, which runs from the N terminus to the C terminus. The insertion along
the coiled-coil of small β-strand structures (residues N49–G84), contribute to make a broader
N-terminal region that forms the head domain (Figure 1A) and splits the coiled-coil in two regions. It
is remarkable how this sequence interruption apparently does not result in a structural perturbation of
the coiled-coil, but forms wing-like structures that protrude from the stalk and pack against the
N-terminal coiled-coil helices. Regions of flexibility or disorder were observed along the stalk, with
partial electron densities suggesting unwinding of the coiled-coil towards the C terminus, and thus
supporting the notion of flexibility as an intrinsic property of this protein.
Int. J. Mol. Sci. 2015, 16 13110
Figure 1. (A) The structure of the meningococcal antigen Neisseria adhesin A (NadA)
variant 5 (pdb 4CJD) is shown on the left, with the region experimentally determined by
X-ray crystallography labeled in red. The two main domains of NadA (head + wings and
stalk) are labeled with green and blue arrows/boxes, respectively. All other regions were
defined by homology modelling, as described previously [20]. A homology model of
NadA variant 3 is also shown, with sequence conservation among variants 1–5 depicted as
a gradient from light blue (low sequence identity) to dark blue (high sequence identity).
The modeled transmembrane anchor is shown in orange. Red dots indicate regions
that were not modeled due to lack of predicted coiled-coil periodicity or homology;
(B) A surface representation of the co-crystal structure of the staphylococcal antigen MntC
(semi-transparent light yellow surface and dark yellow cartoon) bound to Fab C1 (light and
dark grey surfaces depicting light and heavy chains, respectively) (pdb 4NNP). The
binding site of C1 on the surface of MntC (red patch) provides insights into the
mechanisms of interaction between MntC and its natural receptor MntB. The Mn
2+
-binding
site and the occluded MntB receptor binding sites on MntC are labelled. All figures were
prepared using the Pymol software (http://www.pymol.org).
Int. J. Mol. Sci. 2015, 16 13111
Elucidation of the structure of NadA variant 5 allowed building of a homology model of the vaccine
antigen NadA variant 3 (approximately 50% sequence identity overall), for which high-resolution
experimental structural information is not yet available. The homology model was used to visualize
sequence and structural differences among the variants, and for epitope mapping of a protective
bactericidal mAb (Figure 1A). Interestingly, a surface-plot of the sequence conservation revealed that
the largest solvent-exposed patches of highly-conserved NadA residues occur in the head domain,
which is also known to be functionally involved in cell adhesion [14,24]. These studies may therefore
provide a platform for the design of an optimized “head-only” antigen to be presented in multiple
copies on a scaffold. Such an antigen might be able to focus the immune response towards the largest
region of potentially cross-reactive epitopes, and ultimately generate a broader immune response.
The structural studies of NadA summarized above represent an example of a rational, biophysically
driven approach to determine the 3D structure of a vaccine antigen, which in turn provides useful
information to further elucidate both the molecular mechanism of its biological function, and its
immunological properties as a vaccine antigen.
2.1.2. Staphylococcal Solute Binding Protein Antigens
Many bacterial pathogens employ solute-binding proteins (SBPs) in nutrient uptake across
membranes, as they recognize and deliver substrates to ATP-binding cassette (ABC) importers [25].
In many pathogens, metal ion transport across the membrane is regulated by ABC importers coupled to
their SBP partners, and since metals are critical for many biological processes, the inhibition of their
acquisition represents an attractive mechanism for developing antibacterial strategies. Similarly, the
abundance, variety and surface-exposure of SBPs suggests that they may play key physiological roles
and be important virulence factors, thus also becoming targets for vaccine development.
A vaccine discovery project recently identified an SBP protein, FhuD2, as one of five conserved
antigens that play important roles in the virulence and pathogenicity of Staphylococcus aureus [26].
FhuD2 is a lipoprotein involved in iron uptake and in early stages of invasive S. aureus infection [27].
FhuD2 regulates uptake of hydroxamate iron (III) siderophores, which are organic chelators with a
very high affinity and specificity for ferric iron. S. aureus FhuD2 mediates ligand import through the
ABC transporter complex FhuCBG. As part of the characterization and validation of this candidate
vaccine antigen, a structural and functional study of FhuD2 was performed, revealing an overall fold
similar to known class III SBPs (a bilobate bean-like shape), and iron-loaded ferrichrome bound in a
central cleft between the two lobes [28]. Crystallization was initially enabled by stabilization of the
protein upon binding to ferrichrome, while in a subsequent study the protein was also crystallized in
the apo-form, taking advantage of surface-entropy reducing mutations to promote crystallization [29].
Previously, immunization of mice with FhuD2 was shown to generate protective immunity against
diverse clinical isolates of S. aureus [27]. To explore whether a more thermostable FhuD2 would
induce a more potent antibody response, as might be hypothesized based on the assumption of a better
presentation of well-ordered epitopes, mice were immunized with two forms of FhuD2 bound to its
stabilizing ligands ferrichrome or coprogen. However, similar degrees of protective immunity were
observed when compared to apo-FhuD2, thus validating the unbound form as an effective antigen
without the need for additional ligand-mediated stabilization [28].
Int. J. Mol. Sci. 2015, 16 13112
Another pair of bacterial SBPs that are known immunogens and/or potential vaccine candidates are
the manganese transport proteins MntC (S. aureus) and its orthologue SitA (S. pseudintermedius).
MntC is one of two protein antigens in a four-component protein plus polysaccharide vaccine (named
SA4Ag) [30] designed to protect broadly against S. aureus and currently in early clinical development.
As part of the S. aureus MntABC importer system, MntC chelates Mn(II) from the host environment
and presents it to the integral membrane transporter, MntB. The crystal structures of MntC and SitA
were both determined recently and found to be highly similar, as were their metal-binding properties in
solution [31,32]. Using a mouse model, active immunizations with MntC were shown to be effective at
reducing the bacterial load associated with infections by S. aureus and S. epidermidis, suggesting that
it has the potential to provide protection across multiple staphylococcal species, still to be confirmed in
human clinical trials [33]. To learn more about the function of MntC, its crystal structure was
determined in complex with an antibody fragment (Fab), obtained from a phage-display library, that
blocks Mn2+ import (Figure 1B) [34]. Structure-guided mutations of MntC residues in the region
recognized by Fab C1 induced hypersensitivity of S. aureus to reactive oxygen species, mimicking an
mntC null mutant, thus suggesting that the Fab C1 binding site on MntC overlaps with the MntB
interaction site. Since a suitable form of the integral membrane-bound MntB protein to use in binding
experiments with the MntC proteins was not available, this study showed how the co-crystal structure
with a functionally-characterized Fab can be an important tool to indirectly demonstrate the molecular
bases of antigen inhibition, in addition to providing important information on the potential
development of this antibody as a therapeutic.
In summary, the examples of NadA and MntC provided here illustrate how the availability of
antigen structures can aid the interpretation of previous results, guide further investigation, and provide
informative starting points for structural vaccinology studies.
2.2. Protein Crystallography and High-Resolution B-Cell Epitope Mapping
Almost all current vaccines act via functional antibodies that block infection, bacteremia or
viremia [35]. At the first point of host–pathogen interaction, functional antibodies bind to their target
epitopes on the pathogen surface proteins (or to epitopes on secreted proteins) and thus initiate the
acquired immune defense mechanisms. Epitope mapping is therefore an essential activity in the field
of vaccine research, in order to understand the host response to infection or immunization, and has been
boosted enormously in recent years by major developments in our abilities to isolate and expand human
B cells. The developments enabled the identification of large repertoires of antigen-specific immunoglobulin
gene sequences, and thus the easy production of recombinant human antibodies [36–38]. Firstly,
information about such interactions can improve the chances of being able to select and rationally-design
antigen molecules that elicit the desired neutralizing or protective response upon immunization.
Secondly, epitope mapping data can indicate which parts of a surface-bound antigen are actually
exposed and accessible on the surface of the pathogen, which in turn may provide insights into the
functional regions of the protein. Ultimately, an epitope is the full collection of atoms making direct
contacts with the antibody, with typical intermolecular distances of up to ~4 Å. Here, we focus on
B-cell epitopes, which underlie antigen–antibody interactions and are the crucial molecular determinants
of the antibody-mediated immune response.
Int. J. Mol. Sci. 2015, 16 13113
Many different techniques have been developed to perform epitope mapping, displaying various
advantages and limitations, as described previously [39]. For example, although fast and simple,
peptide- or fragment-based approaches tend to identify only linear peptide epitopes, thus only
partially revealing the immunogenic properties of larger conformational epitopes within their natural
three-dimensional contexts. Such approaches have some value, but are unlikely to identify all
conformational epitopes recognized by a mAb of interest. In contrast, the crystal structure of an
antigen-Fab complex can provide the complete atomic description of an epitope–paratope interface.
However, since the success rate of protein crystallization is notoriously unpredictable, efforts to
develop additional techniques for conformational epitope mapping have been extensively explored. As
a recent development, the more readily-obtained electron microscopy (EM) or hydrogen-deuterium
exchange mass spectrometry (HDX-MS) epitope mapping data has been combined with structural data
of the antigen, or a structurally-similar homolog, obtained by more lengthy protein crystallographic
structural studies, thus forming the basis of a powerful “hybrid method” approach.
Indeed, both EM and HDX-MS have emerged as increasingly powerful tools for mapping
conformational epitopes under native conditions, i.e., using full-length folded proteins, not linear
peptides or fragments [39]. For example, HDX-MS was used to map the protective epitope of a mAb
targeting the meningococcal factor H binding protein (fHbp), a Bexsero® antigen, and the results were
in close agreement with the crystal structure of the same complex [40] (Figure 2A,B). Analysis of the
Fab 12C1/fHbp complex structure in silico and subsequent sequence and structure-guided site-directed
mutagenesis studies revealed that the variant 1-specific conformational epitope targeted by 12C1 is not
dependent on just one or two key residues, but rather is determined by a large discontinuous
conformational epitope, which was optimally identified only by protein crystallography. Interestingly,
the Fab binding site on the surface of fHbp overlapped significantly with the binding site of human
factor H revealed by a previous co-crystal structure [41], and this competition for overlapping surfaces
may well contribute to the strong bactericidal efficiency of this mAb [42].
Another example of epitope mapping by hybrid methods combining HDX-MS and protein
crystallography was recently reported, describing a bactericidal mAb (33E8) that targets NadA variant 3,
and which could not be crystallized (see above) [20]. The Fab 33E8/NadA interaction was mapped by
HDX-MS and combined with the homology model of NadA variant 3, built using the crystal structure
of variant 5. This study revealed the possible basis for the lack of binding of mAb 33E8 to the second
group of NadA variants 4, 5 and 6, since the conservation of the sequences between the two groups
within this epitope was relatively low [20] (Figure 1A). Similarly, a recent hybrid method approach
using negative stain electron microscopy (NS-EM) and particle reconstruction was performed to map
the binding site of a neutralizing mAb on the human cytomegalovirus (HCMV) glycoprotein gH/gL or
gH/gL/gO complexes, information which, when coupled with HDX-MS data, and a homology model
built using the crystal structure of herpes simplex virus (HSV) glycoprotein gH/gL complex [43],
provided insights into the key determinants of the conformational epitope and the 3D architecture of
the antibody/antigen complexes [44]. Collectively, these studies indicate that increased efforts to
structurally characterize antigen–antibody interfaces, by protein crystallography alone or via hybrid
methods, are required to fully understand the antigen recognition by the immune system and can
provide insights regarding the mechanism of action of protective or neutralizing mAbs.
Int. J. Mol. Sci. 2015, 16 13114
Figure 2. (A) The crystal structure of the complex fHbp-Fab 12C1 (pdb 2YPV) is depicted
with green/blue surface for N and C termini of fHbp, and light/dark gray for light and
heavy chains of Fab 12C1. The epitope and paratope surfaces are colored in red and
yellow, respectively; (B) Surface representations of fHbp (colored as in panel A), allowing
comparison of the Fab 12C1 epitope (red patch) as revealed by HDX-MS (top) and X-ray
crystallography (bottom). For clarity, the surface of fHbp only is shown, after re-orientation
(~90° about the Y-axis) of the view in A; (C) Surface locations of fHbp residues (red
patches, labeled) which when mutated to Alanine inhibit human fH binding. The entire
interface of the interaction with fH on the surface of fHbp is outlined with a black line, as
revealed previously [41].
Despite various efforts to develop alternatives, protein crystallography remains one of the most
powerful techniques allowing fine mapping of epitope–paratope interfaces. A co-crystal structure
provides a visually immediate and highly comprehensive definition of the interface. To date there are
over 100 non-similar antibody-antigen (i.e., Fab-protein) complex structures deposited in the protein
data bank (PDB), providing a wealth of information about molecular recognition by the immune
system [45]. Thus, epitope mapping has become one of the most widespread and important
applications of protein crystallography in the field of vaccine research.
Nevertheless, protein crystallography has several practical limitations and cannot always provide
epitope mapping information within short timelines. For instance: (i) the generation of crystals
typically requires relatively large amounts of sample; and (ii) even when sufficient sample is available,
there is no guarantee that the antigen–antibody complex will actually produce high-quality crystals.
Despite this, the use of Fabs, single-chain variable fragments (scFvs) or single-domain antibodies is an
emerging tool to “chaperone” the crystallization of recalcitrant proteins, and therefore it can be
anticipated that the probability-of-success when crystallizing antigen-antibody complexes may in fact
be higher than that when crystallizing antigens alone, largely due to the solubilizing and/or stabilizing
effect of the antibody component and the generation of new regions that can provide crystal packing
interfaces [46]. For example, on a “local” scale, complex formation with an antibody might stabilize
flexible surface-exposed loops in one of several relevant low-energy conformations, thus overcoming
Int. J. Mol. Sci. 2015, 16 13115
flexibility that might otherwise inhibit productive crystallization. Or alternatively, on a “whole
molecule” scale, complex formation might stabilize a large conformationally-heterogeneous protein
in a particular state otherwise difficult or impossible to crystallize. An additional benefit is that
co-crystallization enables use of the Fab as the molecular replacement search model to provide phasing
information during structure determination. The increasing ability to produce high-quality recombinant
Fabs for co-crystallization studies will facilitate this approach [36–38]—providing more, high-quality
“shots on goal” than possible when using only a few Fabs derived from standard hybridoma techniques.
Summarizing, given the value of protein crystallography in the vaccine field, we anticipate a
strong expansion of epitope mapping applications in the immediate future, likely to include an
increasing proportion of antibody fragments derived directly from individually-cloned human B-cell
sequences. Moreover, crystallographic epitope mapping can also potentiate the optimization of
therapeutic antibodies and indicate the most appropriate antibody–antigen pairs for diagnostic
applications or in vitro potency assays designed to monitor the stability of the most relevant
components of a vaccine formulation.
3. Structure-Based Antigen Design
The ultimate goal of using protein crystallography in vaccine research is to enable the design of
novel antigens with enhanced characteristics. This section reviews several notable examples of
structure-based antigen designs, some of which introduce and demonstrate promising new approaches,
and some of which have now progressed from pre-clinical into clinical trials.
3.1. Optimizing the Factor H Binding Protein Antigen of Serogroup B Meningococcus
Meningococcal factor H binding protein (fHbp) binds to human factor H (hfH) and down-regulates
complement activation, thus evading complement-mediated killing and promoting bacterial
survival [47,48]. Slightly different forms of fHbp are included in both of the recently-licensed
serogroup B meningococcal vaccines [10,11]. To date, there are over 800 distinct fHbp amino acid
sequences known [49] and they can be classified into three main variant groups, which exhibit
90%–100% sequence identity within the groups, but as little as 63%–85% sequence identity across the
groups. The fHbp sequence variation is presumably an immune evasion mechanism that appears
responsible for the lack of cross-variant protection afforded by wild-type molecules [47,50–52]. Of
these antigens, the most well-characterized is fHbp variant 1, for which several structures have been
determined [53–55]. Crystal structures of representatives from each of the variant groups 1, 2 and 3
have been determined, alone or in complex with hfH [41,54,56] or with a bactericidal mAb specific for
fHbp variant 1 [40]. The recently-approved anti-meningococcal vaccines are expected to save many
lives and much suffering by preventing invasive meningococcal disease, and yet these current
first-generation vaccines are unable to guarantee protection against all possible strains, largely due to
the high sequence variability exhibited by MenB surface antigens, especially fHbp. Therefore,
structure-based design efforts have targeted two major issues: (i) how to engineer an improved fHbp
molecule that combines the entire immunogenic repertoire of all the 3 variant groups into a single
broadly-protective antigen; and (ii) how to engineer a factor H nonbinding form of the antigen.
Int. J. Mol. Sci. 2015, 16 13116
3.1.1. Overcoming Sequence Variability
Structural studies of fHbp variant 1, combined with basic epitope mapping data, suggested that
amino acids contributing to the immunogenicity of variant 1 or variants 2 and 3 were located in
non-overlapping regions. This intriguing observation of variant-specific epitope patches led to the
development of a “chimeric” antigen displaying an immunogenic subset of variant 2 or 3 residues on
the variant 1 backbone [57]. In short, using the variant 1 fHbp as a scaffold, patches of residues from
variants 2 and 3 were grafted onto the protein surface (replacing variant 1 residues). Each patch
encompassed approximately 900–2000 Å2 of surface area, and approximately 50 different proteins
were designed and tested, in order to fully explore the strategy. The approach was successful in
generating an antigen able to elicit more broadly cross-protective antibody responses in pre-clinical
studies in mice [57], as reviewed previously [58,59]. Notably, crystal structure determination of the
most effective engineered chimeric fHbp protein, which contained over 20 simultaneous surface-exposed
point mutations, confirmed that the surface had been successfully manipulated to display an epitope
bearing the characteristics of the variants 2 and 3 groups, but without affecting the overall fold of the
protein, thus leaving the vast majority of variant 1 epitopes undisturbed.
3.1.2. Elimination of Undesirable Function
The structure of the fHbp:fH complex provided insights on the potential interference of fH binding
by fHbp with immune recognition or antibody binding when used as a vaccine antigen [41]. Since the
affinity for fH is very high and the site of interaction between fHbp and fH quite extensive, the concern
arose that a functional fHbp, able to bind fH, could have a reduced number of epitopes available for
recognition by antibodies, as these would be obscured by the bound fH. Thus, the hypothesis that the
structure of the complex could be used to design an engineered fH nonbinding antigen was advanced,
considering that this would make a superior antigen, with higher immunogenicity as the resultant
antibody responses would be directed also at epitopes in or near the now exposed fH binding site,
resulting in greater complement-mediated serum bactericidal activity (Figure 2C).
First, two double mutants of fHbp were designed (residues R341A/H337A and E283A/E304A, the
latter subsequently renumbered to E218A/E239A [60]) and studied by surface plasmon resonance
(SPR), revealing the expected loss-of-function [41]. Later, it was shown that the mutant E218A/E239A
was less immunogenic than wild-type fHbp, as it elicited up to 20-fold lower serum bactericidal titers
than those elicited by a wild-type fHbp [60]. Subsequent studies characterized the binding and
immunogenicity of other fH nonbinding mutants [56,60,61]. Structure-based design was also
performed by Granoff and co-workers to remove a charged hydrogen-bond with fH mediated by a
surface-exposed fHbp arginine [61]. This Arg-41-Ser mutation resulted in no detectable fH binding
and a nearly twenty-fold higher protective antibody response in a mouse model [62]. This design was
also supported by previous knowledge of fHbp epitopes that elicit bactericidal antibodies, allowing
confident prediction that the Arg-41-Ser substitution would have no effect on serum bactericidal
antibody responses to the mutant fHbp antigen, as subsequently confirmed [61]. The study of key fHbp
amino acids necessary for high affinity fH interactions was also extended to other fHbp variant
families, revealing how different variants engage fH in distinct ways, though all using the same
Int. J. Mol. Sci. 2015, 16 13117
molecular region overall [56]. In addition, in this same study, the crystal structure of the double mutant
E218A/E239A was determined, showing that the only detectable change compared to WT was the loss
of the side chains of E283 and E304, thus the overall fold was conserved [56]. More recently, Tang
and co-workers showed how two nonfunctional v3 fHbps retain their immunogenicity, and although
these mutants (T286A and E313A) possess a marked reduction in affinity for fH, their folding was
apparently not affected by the mutations [63]. Although some creative transgenic mouse models that
approximate the expression of human fH have shown the benefits of these nonbinding fHbps [56,61], it
is clear that rationally-designed fHbp antigens will need to be tested and compared in human clinical
trials, in order to estimate the impacts and potential benefits of the loss of fH-binding ability.
3.2. Nonbinding Mutants of Transferrin Binding Protein B
The concept of engineering non-functional forms of an antigen that do not bind to the natural ligand
has also been recently applied for a second bacterial antigen. Mammalian host transferrin (Tf) is used
as an iron source by several Gram-negative bacterial pathogens. The surface-exposed Tf binding
protein B (TbpB) mediates interaction with Tf [64,65], and in pathogenic Neisseria TbpB is thought to
orchestrate the “piracy” of the iron cargo from human Tf [66]. Consequently, TbpB is a potential
antigen for human or animal vaccines. However, data suggested that upon immunization the formation
of the TbpB/Tf complex might mask important epitopes and thereby inhibit generation of the optimal
immune response against TbpB. Therefore, TbpB point-mutants with strongly reduced ability to bind
Tf were designed based on the crystal structures of TbpB/Tf complexes, combined with insights from
homology modelling [67]. In pre-clinical tests in a pig model, a mutant Haemophilus parasuis TbpB
was shown to induce enhanced immune responses and provide superior protection. These studies
further indicated that structure-based strategies can be a powerful way to design “nonbinding” antigens
with improved pre-clinical performance.
3.3. Multiple Protein F-Based Strategies for a Respiratory Syncytial Virus Vaccine
RSV is the most important viral cause of severe respiratory tract disease in children worldwide [68].
RSV accounts for over 6% of deaths in infants from 1 to 12 months old and is thus a leading viral
cause of childhood death [69] and also affects elderly and immunocompromised adults [70]. Although
there is a therapeutic humanized monoclonal antibody (palivizumab, named Synagis®) licensed by
the FDA for prophylactic use in children at high risk, and which reduces the incidence of severe
disease [71], there is currently no RSV vaccine available, despite over 40 years of targeted research.
However, there are now several clinical trials of candidate RSV vaccines ongoing, and there is eager
anticipation that these efforts will deliver a much-needed vaccine in the next five to ten years [72,73].
The vast majority of research into RSV candidate antigens has focused on the membrane-anchored
fusion glycoprotein F, a highly-conserved target of neutralizing antibodies [74]. Although there is
encouraging clinical evidence that RSV F-specific antibodies (including palivizumab) can protect
against disease, the development of an F protein antigen as a vaccine candidate has been hampered by
several factors, including the biochemical challenge that F has an intrinsic tendency to undergo large
conformational changes, a functional requirement typical of viral fusion glycoproteins for mediating
viral and cellular membrane fusion, as reviewed previously [75]. The following sections provide three
Int. J. Mol. Sci. 2015, 16 13118
distinct examples of how protein crystallography has provided structural insights that have overcome
obstacles in the research and development pathway, thus driving the design of novel F-based antigens,
each of which shows promise as a future vaccine antigen.
3.3.1. Rational Engineering of a Soluble, Stable and Homogeneous Post-Fusion F
The RSV F protein forms large (>150 kDa) trimeric structures anchored on the outer face of the
virion membrane. Electron cryotomography and negative-stain electron microscopy revealed that F
exhibits two main forms [76,77], now termed the pre-fusion and post-fusion F forms. When produced
recombinantly, pre-fusion F is only “metastable”, and readily converts to the post-fusion form which,
however, tends to aggregate via an exposed hydrophobic fusion peptide [78], thus rendering it
challenging for development as a vaccine antigen. By sequence- and structure-based modelling using
homologous F protein templates from another paramyxoviridae (parainfluenza PIV5) F protein
structure, a novel non-aggregating and highly-stable form of RSV F was designed via removal of the
fusion peptide, the transmembrane region and the cytoplasmic domain. The crystal structure of this
substantially complete form of post-fusion F was determined [79], and revealed a stable trimer which
displayed the key neutralizing antibody binding site of palivizumab and motavizumab (an affinity-enhanced
derivative) [80]. The presence of these protective epitopes, and the mAb 101F epitope, which had been
previously defined by co-crystal structures of the motavizumab and 101F Fabs in complex with target
RSV epitope peptides [81,82], were confirmed independently in a second similar crystal structure
determination of post-fusion F [83]. Together, these two structures revealed the molecular basis for the
unexpectedly high immunogenicity of the post-fusion F antigen. The engineered F antigen molecule
was readily prepared in a homogeneous, stable and reproducible format and was found to elicit high
titers of neutralizing antibodies in mice or cotton rat animal models [79]. Clinical trials are ongoing
using post-fusion versions of the F antigen.
3.3.2. An Antibody-Dependent Approach to Design and Engineer Pre-Fusion F
Significant efforts have also been made to harness the vaccine potential of the more elusive
“metastable” pre-fusion F antigen. Conceptually, the pre-fusion F conformation would be a better
vaccine target as it exposes all the functional sites and neutralizing epitopes present on virion F.
Although engineered post-fusion F can elicit high titers of neutralizing antibodies in animal
models [79], a subsequent report demonstrated that antibodies specific for the pre-fusion F form
account for most of the neutralizing activity of human sera from seropositive subjects [84]. It thus
appeared that some critical neutralizing mAb binding sites were absent in the post-fusion F form and
consequently attempts to design a stable pre-fusion F antigen intensified. Important “turning points”
that ultimately enabled informed antigen design were the discoveries of a few new anti-F neutralizing
mAbs (mouse 5C4, human D25 and AM22) with the unique property of not recognizing a stabilized
post-fusion F form. These mAbs were used for structural studies to trap the F molecule in its
pre-fusion state. Crucially, after co-expression and co-purification, the crystal structure of Fab D25
bound to RSV F in the pre-fusion conformation was determined [85]. Although the structure revealed
that the palivizumab and motavizumab epitopes were well exposed in pre-fusion F, there was a
dramatic overall change in conformation (Figure 3). Analysis of the epitope–paratope interface in this
Int. J. Mol. Sci. 2015, 16 13119
complex explained why D25 does not bind to post-fusion F and thus the crystal structure provided
mechanistic insights, suggesting that D25 neutralizes RSV by restraining F in the pre-fusion state. The
epitope recognized by D25, site Ø, which is also the target of 5C4 and AM22, is on the most exposed
apex of F, which may underlie the higher effectiveness of neutralizing antibodies against this region,
despite having a binding affinity similar to that of motavizumab. These structural studies led to
the proposal that F antigens stabilized in the pre-fusion conformation may further improve the
immunogenicity of this molecule. Indeed, stabilization of the trimer by addition of a trimerization tag
(a foldon) replacing the transmembrane region, structure-based insertion of hydrophobic packing
mutations and judicious insertion of a novel disulfide bond, forms the basis of a leading pre-fusion F
candidate antigen (Figure 3). Of note, similarly to the iterative approach of structure-based design used
for the development of high-affinity drugs, the authors developed a method to screen hundreds of
structure-guided mutations to identify those resulting in protein stabilization and favorable expression
levels. Most importantly, in mouse and nonhuman primate animal models, a stabilized pre-fusion F
molecule elicited RSV-specific neutralizing titers significantly greater than those elicited by a post-fusion
F protein and well above the protective threshold [86].
Figure 3. (A) Stabilized respiratory syncytial virus (RSV) F pre-fusion (pdb 4MMV) is
shown as light and dark surfaces for two chains, and as yellow cartoon for the third chain
of the trimer. Sites that were mutated to stabilize the pre-fusion configuration are colored
in blue, green, and pink, for the S190F-V207L pair (Cav1), the S155C-S290C double
mutant (DS), and the D486H-E487Q-F488W-D489H mutant (TriC), respectively [86].
Known epitope surfaces for Fabs D25, and for palivizumab and motavizumab, are colored
in cyan and green, respectively. A zoomed view of the region of DS and Cav1 mutants
(central box) provides details of the cavity-filling mutation S190F and of the introduction
of the disulfide bridge C155-C290; (B) Post-fusion RSV F (pdb 3RKI) [79] is shown as
surface, color-coded as in panel A.
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3.3.3. Epitope-Focused Vaccine Design to Target a Neutralizing Epitope on the F Antigen
As introduced above, development of an RSV vaccine based on the wild-type F protein has been
hampered by the large conformational changes it undergoes, and its relatively poor behavior in
solution. Consequently, alternative approaches to F-based antigen design were sought, based on the
concept of using selected scaffold proteins to display structured peptide fragments representing
neutralizing epitopes of F. This goal was greatly facilitated by determination of the crystal structure of
the RSV-neutralizing motavizumab Fab in complex with its peptidic epitope from the F protein,
wherein motavizumab was observed bound to its 24 residue peptide target which adopted a
helix-turn-helix conformation [82]. However, initial structure-based design efforts to generate RSV
epitope-scaffold immunogens were only partially successful, insofar as they induced structure-specific
anti-F antibodies but without RSV-neutralizing activity [87]. Subsequently, a major proof-of-principle
that epitope-scaffold immunization can re-elicit neutralizing antibodies against a pre-defined target
epitope was obtained by developing new computational methods to design or optimize novel scaffolds
tailored for the motavizumab epitope structure [88]. Briefly, a novel scaffold with robust biophysical,
structural and antigenic properties was designed to faithfully display the motavizumab helix-turn-helix
epitope. Achievement of the design objective was confirmed by crystal structure determination of an
epitope-scaffold alone or in complex with the motavizumab Fab, which revealed a high degree of epitope
mimicry. Further, several epitope-scaffolds were recognized by sera obtained from RSV-seropositive
humans, confirming that a clinically-relevant epitope conformation was presented. Finally, immunization
of rhesus macaques with three slightly different motavizumab epitope-scaffolds alone, or one
epitope-scaffold construct mounted in multiple copies on a hepatitis B core antigen-derived virus-like
particle (VLP), were sufficient to induce F-binding antibodies in all animals. Notably, in at least half of
the epitope-scaffold VLP immunized animals, the elicited RSV-neutralizing activity was comparable
to the neutralization titers induced by natural human infection.
3.4. Human Immunodeficiency Virus (HIV)—The Ultimate Challenge?
HIV affects more than 30 million people worldwide, killing ~2 million people per year and it
remains a major global public health threat [89]. There is currently no cure for HIV infection, though it
may be controlled with effective antiretroviral treatment. However, in many countries, the cost of
antiretroviral therapy is prohibitive. Prevention of HIV transmission is certainly the long term global
solution for the HIV pandemic and vaccination is likely the most sustainable mechanism to achieve
this goal.
The envelope glycoprotein (Env) is the only target for neutralizing antibodies of HIV-1. Env is
responsible for fusion between the viral and cell surface membranes and allows entry of HIV into the
target host cell. Env is produced as the gp160 precursor which is cleaved by furin to generate a
heterodimer composed of gp120 and gp41, three copies of which form the trimeric Env spike [90].
HIV is highly successful in thwarting the immune response, in part due to its high mutation rate that
results in high sequence diversity of the spike, thus hindering the development of potently and broadly
neutralizing antibodies.
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Over the last ~30 years, HIV vaccine design programs have attempted various strategies to generate
humoral or cellular immunity, or, more recently, both. The largest proof of the feasibility of a vaccine
preventing HIV infection came in the RV144 Phase 3 trial in Thailand, which used a recombinant
canarypox prime expressing gp120_TM and a bivalent gp120 (subtype B, MN and subtype A/E, A244)
protein boost [91]. This study showed modest efficacy over three years of follow-up. Subsequent
intensive evaluations have shown that V1V2 (variable regions 1 and 2) antibodies may be associated
with less risk of subsequent HIV infection [92]. These findings energized the field and suggested that
HIV vaccination may work and new human trials are now being conducted. A comprehensive survey
of the vast body of literature describing the search for HIV vaccine antigens is beyond the scope of this
review; the interested reader is invited to consult other recent and more extensive HIV-dedicated
resources (as excellent examples, see [93,94]). Nevertheless, to serve our particular objective in
highlighting the roles of protein crystallography in vaccine research, the following sections describe a
number of innovative approaches that have been developed in the past several years aiming to generate
an effective HIV vaccine antigen. Indeed, the development of a protective HIV vaccine remains one of
the greatest challenges facing vaccinologists today.
3.4.1. Scaffold-Based and Multi-Copy Approaches in the Design of HIV Antigens
Some of the earliest breakthroughs in HIV structural biology were achieved over 15 years ago by
co-crystallization of gp120 sub-domains with deletions in the variable regions together with soluble CD4
(the host receptor) and Fab fragments [95]. Several different structurally- and computationally-based
HIV antigen design projects have followed, mainly focused on gp120 and portions of gp41, and some
promising results have been obtained. For example, scaffolds displaying known HIV gp120 and gp41
epitopes in a more stable fashion than in their native context have been generated. Namely, starting
from antibody/epitope co-crystal structures, Schief and co-workers described computational methods
for the design of optimized epitope scaffolds that showed high conformational stability and were
therefore good candidates for the presentation of known structural epitopes to the immune system [96].
In a similar study, the same group performed the grafting of a discontinuous gp120 epitope onto
a scaffold protein unrelated to gp120, with the appropriate retention of structural and antigenic
properties [97]. Further, Kwong and co-workers grafted neutralizing epitopes of HIV-1 gp41 onto a
protein backbone scaffold more stable than gp41 itself [98]. Continued efforts to identify and
characterize antibodies able to broadly cross-neutralize many strains of HIV are likely to further fuel
this epitope-based approach to vaccine design.
In addition to the structure-based computational design of conformationally-correct epitope
scaffolds, different groups have used the structures of gp120 bound to the CD4-receptor as the starting
point to design mutations that lock full-length or truncated forms of gp120 in the CD4-bound
conformation [99,100]. When the stabilized molecules were used to immunize small animals, they
were found to elicit a higher proportion of antibodies targeting the conserved CD4 and co-receptor
binding sites than the wild-type antigen. Recently, an elegant approach to target the germline B-cell
precursor of an affinity-matured broadly-neutralizing antibody has been developed [101]. The authors of
this study designed an HIV gp120 outer-domain immunogen that bound to VRC01-class broadly
neutralizing antibodies and their germline precursors. Of note, when presented on a lumazine synthase
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self-assembling nanoparticle, this immunogen was able to activate germline and mature VRC01-class
B-cells [101]. This approach may be particularly useful for the elicitation of rare antibodies targeting
specific neutralizing epitopes.
3.4.2. Structure Determinations of the HIV Envelope Glycoprotein (Env) Trimer
Despite being a great focus of attention, Env has been highly resistant to structural characterization,
in particular via crystallization, mainly due to its heterogeneous metastable nature, conformational
heterogeneity and extensive glycosylation. Over the last 12–18 months, major advances have been
achieved, using both X-ray crystallography and cryo-electron microscopy (cryo-EM) to structurally
characterize the Env trimer and Env–antibody or Env–receptor interactions. The crystallographic
studies first required production of a soluble, stable, cleaved form of the Env trimer, which was
achieved by inserting a covalent disulfide bridge between gp120 and gp41, coupled with a point
mutation to generate a more stable gp41 trimer (Ile to Pro, in the N-terminal heptad repeat).
Ultimately, several protocol adjustments were combined to obtain a well-behaving form of Env,
structurally and antigenically similar to the native form, and suitable for crystallization. The crystal
structure of an HIV-1 Env trimer was first determined at ~4.7 Å resolution [102], and subsequently at
~3.5 Å resolution [103], revealing a stem-and-head spike structure, with gp41 in the pre-fusion
conformation and the trimer apex stabilized by inter-protomer interactions of the gp120 V1, V2 and
V3 variable loops. The mature closed-state pre-fusion structure, the target of most neutralizing
antibodies, was shown to be covered with N-linked glycosylations (25–30 per gp120-gp41 protomer)
and rich in sequence-variable regions, major hallmarks of immune evasion strategies [103]. A very
similar Env structure was simultaneously determined by cryo-EM at 5.8 Å resolution [104]; both the
X-ray and EM structures revealed complexes with broadly-neutralizing antibody Fab fragments, and
thus provided insights into protective epitopes on Env, which are potential sites of vulnerability to
antibodies and ideal platforms for continued structure-based design of candidate vaccine antigens.
Early efforts have been made to convert this sort of engineered Env construct into next-generation
antigens [105]. However, it remains to be seen whether these most recent structural insights will
ultimately lead to the design and development of an effective vaccine antigen for clinical trials.
4. Enabling Technology for Protein Crystallography in Vaccine Research
During the last ~80 years, fueled by technological advances crystallography has made dramatic
progress and a revolutionary expansion, becoming integral to modern biology, medicine, and drug
discovery [106,107]. The determination of macromolecular structures using X-ray diffraction involves
many technologies such as molecular biology, bioinformatics, and more generally physical sciences.
Progress in recombinant DNA technology, crystallization methods, synchrotrons, computing, phasing
and refinement algorithms, drove the strong expansion of crystallography, which resulted in more than
100,000 structures of biological macromolecules and macromolecular assemblies being deposited in
the Protein Data Bank as of 2014 [108]. Although structure determination by protein crystallography is
still not a high-throughput discipline, several recent advances have increased throughput and the
probability-of-success of crystal structure determination, often stimulated by the observations that have
emerged from large-scale structural genomics initiatives over the last two decades [109].
Int. J. Mol. Sci. 2015, 16 13123
4.1. A Short Introduction to X-ray Crystallography of Proteins
The birth of protein crystallography can be traced back to the observation by Bernal and Crowfoot
of the first X-ray diffraction pattern from crystals of pepsin, which revealed that proteins had an
ordered three-dimensional structure [110]. The first protein structure to be determined was that of
myoglobin [111,112], followed soon after by those of haemoglobin and lysozyme, the first structure of
an enzyme, in 1960 [113] and 1962 [114,115]. While a comprehensive and recent overview of the
pipeline of protein crystallography and of its developments can be found elsewhere [116], here we
provide a concise summary of the main steps and technical challenges of the method.
In order to solve the three-dimensional structure of a protein by X-ray crystallography, the first
prerequisite is to crystallize the macromolecule of interest. Crystals are made of billions of the same
molecules in an ordered array, and this arrangement allows the magnification of the diffraction signal
that is essential to overcome the weak, and not measurable, diffraction from a single molecule.
The crystalline state confers to each molecule the same scattering properties. However, since proteins
and nucleic acids do not naturally arrange into a regular and periodic manner as is typical of crystals,
crystallization is often the rate-limiting step in protein crystallography. Several strategies for
overcoming the intrinsic difficulties of the crystallization of biological macromolecules have been
devised (see below). Once highly-ordered and well-diffracting crystals have been obtained, data
collection experiments are performed, where the diffraction pattern of the crystals is recorded by
placing them into an X-ray beam. When X-rays strike the crystal, the atoms it contains will produce
scattered X-ray waves, and the energy (or amplitudes) and positions of these scattered waves are
recorded during data collection. In theory, by summing all the scattered waves the structure of the
macromolecule can be solved. However, the origin of each wave must first be determined, which
corresponds to observing the time of arrival of the X-ray peaks scattered in different orientations. This
information is not directly accessible or experimentally measurable, and this is known as the phase
problem, which is another major obstacle in the structure determination by X-ray crystallography.
Different computational and experimental methods to solve the phase problem are available today, and
their development is discussed below. Once phases have been assigned to each scattered wave, their
summation in three dimensions generates the electron density distribution of the molecule of the
crystal. This process is performed by use of the Fourier transformation, which requires (1) the structure
factor amplitudes measured during data collection; and (2) their relative phase angle. The initial
electron density obtained upon solving the structure is subsequently used to trace or fit models of
the crystallized molecule, thus providing the first picture of the structure of the protein. The
coordinates of the model structure are subsequently subjected to refinement, to improve their overall
quality by tweaking calculated model parameters such as atom positions and displacement (also called
temperature or B-factors), until they best describe the experimentally observed data. Refinement will
determine the final quality of a crystal structure, which also depends on the resolution of the X-ray
diffraction data. A typical crystallographic dataset can enable structure determination in a resolution
range from high (~1 Å) to low (~3.5 Å), providing sufficient detail to observe the positions of all
(non-Hydrogen) backbone and side chain atoms, or the shape of the molecule and the secondary
structure elements, respectively. There is currently no other technique that can routinely deliver such
highly-detailed and precise atomic-level information, and such data has over the last 50 years provided
Int. J. Mol. Sci. 2015, 16 13124
innumerable structural and functional insights, enabled many protein engineering efforts, as well as the
design of small-molecule inhibitors now available as pharmaceutical agents to treat a variety of
medical conditions.
4.2. Advances in Protein Crystallization
The production of high-quality crystals for X-ray crystallography is famously a major bottleneck in
structure determination. Although there is no apparent correlation between crystallization propensity
and protein structure, nor “magic bullets” for the production of good crystals, a variety of methods to
aid crystallization exists and these have been extensively reviewed [117–119]. Structural genomics
initiatives have been a major driving force for the automation and progress of large-scale expression,
purification of recombinant proteins, and protein crystallization methods [120–123]. A list of advances
that facilitate protein crystallization can be attempted by dividing these into two groups, based on how
time- and labor-intensive they are. Group 1 includes those that involve or require cloning strategies
and were enabled or facilitated mainly by the development of high-throughput recombinant methods
and new cloning vectors; Group 2 includes more general and simple rescue tools or strategies to obtain
better crystals or to improve existing sub-optimal crystals.
4.2.1. Group 1
i. High-throughput domain-hunting strategies to search for optimal expression constructs
for the bacterial production of difficult target proteins [124,125];
ii. Mutations to stabilize the target protein [126,127];
iii. Surface entropy reduction (SER): mutation of surface residues to create patches of low
entropy that can preferentially mediate crystal contacts [128];
iv. Sequence homolog screenings: sequence variability between homolog proteins, if
localized on the surface, can favor better packing and crystallization [21];
v. Fusion proteins: highly crystallizable proteins (i.e., T4 lysozyme) covalently fused
to disordered regions of the target protein, well-known to aid crystallization of
GPCRs [129,130].
4.2.2. Group 2
i. Binding partners (or chaperone-assisted crystallography): Fab fragments, single-domain
antibodies, synthetic antibodies, and more general substrates, may reduce conformational
freedom of the target protein and thus enhance propensity to form the ordered lattice
required for crystallization [118,131,132];
ii. In situ proteolysis: the addition of trace amounts of proteases in crystallization trials
can help to enzymatically eliminate flexible or disordered regions that might hinder
crystallization [133];
iii. Seeding: separating crystal nucleation and growth, use of seeds from microcrystalline
material or precipitate to streak in a freshly prepared protein solution [134,135];
Int. J. Mol. Sci. 2015, 16 13125
iv. Reductive methylation: targets lysine residues, modifying their primary free amines to
tertiary amines, and thus likely decreasing disorder on the protein surface [136,137].
In addition to the developments above, a considerable amount of crystallization data has been
amassed, and this is now being used to develop statistical analyses for predictive strategies for
crystallization [138–140]. Also, progress in instrumentation for crystallization droplets imaging now
allows further developments towards automatic ranking and classification of the droplets [141].
4.3. Advances Facilitating the Determination of Crystal Structures
As for crystallization methods, technological advances and structural genomics initiatives have
largely powered progress in methods for protein structure determination and refinement, moving the
field towards automatic crystallographic structure solution tools. Astonishing progress has been
accomplished in all fields from synchrotron radiation/X-ray diffraction data collection, to phasing and
structure refinement.
Synchrotron radiation is now the main source for X-ray diffraction, and it virtually entirely replaced
sealed-tube and rotating-anode generators that were common in crystallographic laboratories until the
1990s. Compared to a rotating-anode source, the increase of the X-ray flux of a third-generation
synchrotron facility is of 20 million times [142]. The potential of synchrotron radiation application for
crystallography was first recognized in the late 1940s, and enabling technologies that allowed the
construction of synchrotrons with appropriate high energies started to develop in the 1960s. The first
use of synchrotron radiation for protein crystallography can be traced back to the 1980s. In the early
2000s, a new hybrid pixel X-ray detector (PILATUS) was introduced, which is now in standard user
operation on an increasing number of beam lines [143]. The introduction of the PILATUS, which
operates in single-photon counting mode and possesses a fast readout time and the absence of readout
noise, profoundly changed data-collection strategies [144]. Among the most recent developments of
synchrotron facilities are automation, remote user access, and industrial service provision [145–147].
More recently, the advent of free-electron lasers (FELs), which deliver extremely intense femtosecond
X-ray pulses, allowed the development of serial femtosecond crystallography (SFX) [148].
SFX promises to overcome two major limitations of protein crystallography, small crystal size and
radiation damage, and examples of applications have been published recently [149–151].
The availability of tunable X-rays from synchrotron sources in the 1990s allowed the
implementation of the multi-wavelength anomalous dispersion (MAD) phasing method [152]. At the
time, the commonly used phasing method was multiple isomorphous replacement (MIR), where
heavy-metal ions were incorporated, mostly by time- and labor-intensive soaking experiments into
native crystals [153]. Measurements of the perturbation of the diffraction pattern of heavy-metal
soaked crystals compared to native crystals were then used to obtain information on the possible values
of the phase angle, with the critical caveat that measurements had to be performed with very high
accuracy, and native and derivative crystals needed to be isomorphous [154]. The introduction of
MAD overcame these obstacles, as only one single crystal containing atoms capable of anomalous
scattering (commonly seleno-methionine labelled proteins) was needed. However, to perform a MAD
experiment required the collection of a number of X-ray wavelengths, and combined with the power of
synchrotrons, a MAD experiment potentially induced severe radiation damage that could often
Int. J. Mol. Sci. 2015, 16 13126
compromise the measurement of the anomalous signal itself [155]. Later, as more high-throughput
methods for structure determination were needed, as well as methods that would induce less or no
radiation damage, single anomalous dispersion (SAD) was developed [156,157]. Being faster and easier to
perform, phasing by SAD has been successfully adapted in high-throughput pipelines [158,159]. Other
phasing methods that have gained popularity with the advent of high-throughput crystallography projects
are those that exploit the soakings of halides in native crystals [160]. These are particularly
advantageous in cases where a protein does not bind heavy-metal atoms or cannot be prepared as a
seleno-methionine variant. Halide anions, such as bromide or iodide, have been shown to be easily
incorporated into the crystal solvent regions around protein molecules, and as such they allow
measuring an anomalous signal, thus providing phasing power [161].
Due to the increasing number of protein structures available today in the PDB, molecular
replacement (MR) is now the most widely used phasing method [162]. The method was introduced in
the 1960s; it is based on the availability of a suitable related model (the template) and consists of a
trial-and-error search where all possible orientations and positions of the template model are explored
in the unit cell of the unknown crystal target [163]. Perhaps, the most critical step for the success of
MR is the selection and modification of the template model structure, which is usually made based on
sequence homology. Progress in bioinformatics and sequence manipulation software, and the
availability of many sequences and structures in the databases allow accurate multiple alignments that
can aid in the selection of an optimal template for MR [164]. In addition, several automated software
tools that streamline the process of finding homologs and generating a suitable template, as well as
running the MR searches using different softwares such as Phaser [165], and Molrep [166], and
performing the initial refinement, are now available [167–169]. The automation of crystallographic
structure solution has also seen tremendous progress over the last decade with the specific
development of many new software tools [170,171] and user support [172].
5. Conclusions and Outlook
Here, we have reviewed how protein crystallography can play a key role in vaccine research and
development processes. Once the potentially-useful antigens of a pathogen have been identified,
structural biology can have an impact on several stages of product development. The value of
structure-based antigen design can be perceived at several stages along the pathway, for example (i) to
eliminate undesirable regions of the antigen (catalytically-active sites, or immunodominant decoy
epitopes); (ii) to stabilize the antigen in the most beneficial conformation; (iii) to guide presentation of
the most relevant antigenic epitopes, preferably with an orientation tailored to elicit a targeted immune
response; (iv) to assemble the antigen in multi-valent arrays for enhanced immunogenicity; (v) to
identify ideally-located sites for molecular conjugation, either of other protein antigens (thus creating
larger polypeptides with multiple antigen features) or of smaller molecules, to enable site-specific
labelling with antigenic oligosaccharides or immune-potentiator compounds. During later stages of
vaccine development, structure-based design can also be used to build-in biophysical or biochemical
features that enhance the productivity, stability and safety of the vaccine antigens.
Here, we initially focused on some of the contributions made by X-ray crystallography in the
characterization of protein antigens. With emerging technical advances, we anticipate that several other
Int. J. Mol. Sci. 2015, 16 13127
techniques may also play growing roles in this field in the very near future. In particular, cryo-EM has
become the preferred method to study icosahedral viruses (featuring high symmetry) [173–175] and has
already shown promise in the characterization of large antigens and membrane proteins with structures
now determined at atomic resolution [176,177]. In particular, EM is an excellent method to rapidly study
antibody–antigen interactions at moderate resolution and with low sample quantity requirements [44,178]
and can be combined with crystallographic data to provide detailed structural information. This
hybrid approach may be particularly powerful when considering the increasing ease with which
antibodies can be cloned from human B-cells and can be recombinantly produced and purified with
moderate throughput. Another area, partly introduced above, is the use of HDX-MS for the rapid
characterization of protein–protein interfaces, with obvious application to antigen–antibody complexes.
In contrast with crystallographic or cryo-EM approaches, HDX-MS also holds the intriguing possibility
that such studies might be applicable to polyclonal antibody–antigen mixtures [179]. Further, the addition
of electron transfer dissociation (ETD) technology to the HDX-MS approach is likely to enable
improvements in the resolution of structural MS-based epitope mapping studies [180].
Collectively, these discussions point to the growing intercalation of the fields of human
immunology and structural biology. We expect X-ray crystallography to continue to deliver the core
information needed for precision design of optimized antigens. However, we also eagerly anticipate
the continued development and ensuing contributions of other structural technologies and increasing
computational power, thus potentiating the tool-kit available when attempting to address the urgent
need for the development of antigen components of novel vaccines designed to control or eliminate
infectious disease.
Acknowledgments
We are grateful to Ethan Settembre and the Structural Biology team at Novartis Vaccines &
Diagnostics for useful discussions contributing to the preparation of this manuscript.
Author Contributions
Enrico Malito, Andrea Carfi and Matthew J. Bottomley contributed equally to numerous topical
discussions and to preparing the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
Abbreviations
Context-relevant definitions of abbreviations or professional terms used herein, listed in the order in
which they appear in the manuscript:
Reverse vaccinology: a genomic approach for the sequence-based computational identification of
proteins for experimental testing as candidate vaccine antigens.
mAb: a monoclonal antibody, a protein produced by the clones of a single hybrid cell obtained from
the fusion of a B cell with a tumor cell (or, more recently, after cloning the relevant gene fragments
Int. J. Mol. Sci. 2015, 16 13128
from a mammalian B cell followed by recombinant expression) and with binding targeting a specific
antigen; those discussed herein are of the IgG class, and each IgG is composed of four chains: two
identical heavy chains (each of ~50 kDa) and two identical light chains (each of ~25 kDa), such that
each mAb is ~150 kDa. There is significant flexibility in several regions of a mAb, such that they are
highly recalcitrant to crystallization.
Fab: the fragment antigen-binding of a mAb, encompassing the antigen combining site. A Fab is a
heterodimer, composed of one entire light chain (variable-light and constant-light regions, VL and CL)
and part of one heavy chain (the variable-heavy and constant-heavy-1 regions, VH and CH1). Fabs are
much less flexible than mAbs, and consequently are more likely to crystallize than mAbs.
Meningitis: a potentially-fatal inflammation of the meninges, those membranes surrounding the
brain and spinal cord, usually caused by pathogenic infections.
Glyco-conjugate: a conjugate of a polysaccharide or oligosaccharide component derived from the
cultured target pathogen and covalently linked to a carrier protein which enhances the immune
response against the saccharide (the most common carriers are inactive toxins, such as CRM197,
diphtheria toxoid, or tetanus toxoid).
Structural Vaccinology: the use of structural biology data to drive the design and/or optimization of
vaccine antigens.
Crystal packing interfaces: typically relatively small interfaces needed to mediate the protein-protein
contacts that result in assembly of the ordered array of molecules that defines a protein crystal. Such
interfaces are not necessarily physiologically-relevant, i.e., do not necessarily correspond to intermolecular
contacts observed in solution under native conditions.
Serum bactericidal activity (SBA): serum bactericidal antibodies have been accepted as the
surrogate for protection against meningococcus. In an SBA assay, bacteria are killed based on the
cumulative action of specific antibodies in the serum directed against antigens on the bacteria. The
amount of serum required for efficient bactericidal killing thus gives an indication of the functionality
of the antibodies raised by the antigen.
Foldon: a tag of approximately 27 amino acids that promotes trimerization, derived from the
trimeric C-terminal domain of the protein fibritin of the T4 bacteriophage.
Epitope mapping: the identification of the amino acid residues (or atoms) of an antigen that are
contacted by a specific antibody.
Epitope grafting: the use of genetic engineering to insert epitope residues of one antigen into the
amino sequence of a second (scaffold) antigen.
Paratope: the amino acid residues (or atoms) of an antibody that are contacted by a specific antigen.
Surface entropy reduction: the use of genetic engineering to replace amino acids (typically Arg,
Lys, Glu, Asp) exposed on a recombinant protein surface with smaller, less flexible amino acids
(typically Alanine) such that the protein surface exhibits lower entropy.
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... Several approaches have been developed in recent years and are currently explored for epitope identification. Classical methods, such as X-ray crystallography and NMR spectroscopy, require relatively large amounts of material with high purity [13,14]. On the other hand, mass spectrometry (MS) based methods, together with proteolytic digestion, have been developed using hydrogendeuterium exchange of antigen-antibody complexes (HDX), chemical cross-linking, fast photochemical oxidation of proteins (FPOP), and alanine scanning of proteolytic peptide fragments [15][16][17][18][19][20][21][22][23][24][25]. ...
... For epitope identification, proteolytic extraction was employed with a chymotryptic digest mixture submitted to the affinity column, and non-binding peptide fragments were removed by washing (supernatant fraction). Elution by mild acidification (pH 3) and MALDI-MS provided a single peptide corresponding to the ADA heavy chain sequence (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29) [49]. The epitope was ascertained by additional fragmentation and MS analysis, and was located at the begin of the CDR1 region ( Figure 4B and Figure S6). ...
... For epitope identification, proteolytic extraction was employed with a chymotryptic digest mixture submitted to the affinity column, and nonbinding peptide fragments were removed by washing (supernatant fraction). Elution by mild acidification (pH 3) and MALDI-MS provided a single peptide corresponding to the ADA heavy chain sequence (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29) [49]. The epitope was ascertained by additional fragmentation and MS analysis, and was located at the begin of the CDR1 region ( Figures 4B and S6). ...
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Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (KD) of a protein–antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled (“conformational”) antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA–aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.
... Facilitated by the improvements in high-throughput B cell technologies, the structural insights into human mAbs have been instrumental in directing the immune response to conserved antigenic sites 72,[238][239][240][241] . Complementary to this approach, structure-based design has been employed to completely remove domains containing sites targeted by non-neutralizing antibodies or to identify possible positions for the introduction of glycosylation sites to mask such epitopes 242 . ...
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Monoclonal antibodies (mAbs) are appealing as potential therapeutics and prophylactics for viral infections owing to characteristics such as their high specificity and their ability to enhance immune responses. Furthermore, antibody engineering can be used to strengthen effector function and prolong mAb half-life, and advances in structural biology have enabled the selection and optimization of potent neutralizing mAbs through identification of vulnerable regions in viral proteins, which can also be relevant for vaccine design. The COVID-19 pandemic has stimulated extensive efforts to develop neutralizing mAbs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with several mAbs now having received authorization for emergency use, providing not just an important component of strategies to combat COVID-19 but also a boost to efforts to harness mAbs in therapeutic and preventive settings for other infectious diseases. Here, we describe advances in antibody discovery and engineering that have led to the development of mAbs for use against infections caused by viruses including SARS-CoV-2, respiratory syncytial virus (RSV), Ebola virus (EBOV), human cytomegalovirus (HCMV) and influenza. We also discuss the rationale for moving from empirical to structure-guided strategies in vaccine development, based on identifying optimal candidate antigens and vulnerable regions within them that can be targeted by antibodies to result in a strong protective immune response. Monoclonal antibodies (mAbs) are appealing as potential therapeutics and prophylactics for viral infections. This Review describes advances in antibody discovery and engineering that have led to the development of mAbs that target viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus and Ebola virus, and also considers the implications for vaccine development.
... Knowledge of sequence and 3D structure can deepen our functional understanding of a protein and can facilitate the tailored design of vaccine antigens with enhanced properties [24][25][26][52][53][54]. Upon crystal structure determination and detailed manual and computational analyses of fHbp variant1-2,3.x, ...
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Invasive meningococcal disease can cause fatal sepsis and meningitis and is a global health threat. Factor H binding protein (fHbp) is a protective antigen included in the two currently available vaccines against serogroup B meningococcus (MenB). FHbp is a remarkably variable surface-exposed meningococcal virulence factor with over 1300 different amino acid sequences identified so far. Based on this variability, fHbp has been classified into three variants, two subfamilies or nine modular groups, with low degrees of cross-protective activity. Here, we report the crystal structure of a natural fHbp cross-variant chimera, named variant1-2,3.x expressed by the MenB clinical isolate NL096, at 1.2 Å resolution, the highest resolution of any fHbp structure reported to date. We combined biochemical, site-directed mutagenesis and computational biophysics studies to deeply characterize this rare chimera. We determined the structure to be composed of two adjacent domains deriving from the three variants and determined the molecular basis of its stability, ability to bind Factor H and to adopt the canonical three-dimensional fHbp structure. These studies guided the design of loss-of-function mutations with potential for even greater immunogenicity. Moreover, this study represents a further step in the understanding of the fHbp biological and immunological evolution in nature. The chimeric variant1-2,3.x fHbp protein emerges as an intriguing cross-protective immunogen and suggests that identification of such naturally occurring hybrid proteins may result in stable and cross-protective immunogens when seeking to design and develop vaccines against highly variable pathogens.
... An example of NMR structure of the antigen and antibody complex is the variable fragment of 0.5b (HIV-1 neutralizing antibody), which is bound to the third hypervariable region (V3) of gp120 protein of HIV-1 [53]. Cryo-electron microscopy (Cryo-EM) is another method that has been used to determine macromolecular structures with a resolution that is sometimes comparable to X-ray crystallography [54]. Samples are flash frozen in cryo-EM; hence crystallization is not required. ...
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Technological revolutions in several fields have pushed the boundaries of vaccine design and provided new avenues for vaccine development. Next-generation vaccine platforms have shown promise in targeting challenging antigens, for which traditional approaches have been ineffective. With advances in protein engineering, structural biology, computational biology and immunology, the structural vaccinology approach, which uses protein structure information to develop immunogens, holds promise for future vaccine design. In this review, we highlight various vaccine development strategies, along with their advantages and limitations. We discuss the rational vaccine design approach, which focuses on structure-based vaccine design. Finally, we discuss antigen engineering using the epitope-scaffold approach, gaps in structural vaccinology, and remaining challenges in vaccine design.
... In fact, several comprehensive reviews of individual methods have been published in this century. [9][10][11][12][13][14][15][16] Table 1 lists common experimental methods for epitope mapping. There are two major classifications of epitopes primarily based on the experimental method used for their identification. ...
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This mini-review presents a critical survey of techniques used for epitope mapping on the SARS-CoV-2 Spike protein. The sequence and structures for common neutralizing and non-neutralizing epitopes on the Spike protein are described as determined by X-ray crystallography, electron microscopy and linear peptide epitope mapping, among other methods. An additional focus of this mini-review is an analytical appraisal of different deep mutational scanning workflows for conformational epitope mapping and identification of mutants on the Spike protein which escape antibody neutralization. Such a focus is necessary as a critical review of deep mutational scanning for conformational epitope mapping has not been published. A perspective is presented on the use of different epitope determination methods for development of broadly potent antibody therapies and vaccines against SARS-CoV-2.
... Undoubtedly, high-resolution epitope mapping plays a critical role in vaccine research and development projects. 20 Information on antigen-antibody interactions usually guides the rational design of antigens and antibodies that can elicit desired neutralizing or protective responses, while also providing insights into functional regions. Previously, only low-resolution (5.5 Å) information on the AM14 epitope of PreF was obtained from a crystal structure of a ternary complex between DS-Cav1 and antigen-binding fragments (Fabs) of AM14 and motavizumab. ...
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Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infections resulting in medical intervention and hospitalizations during infancy and early childhood, and vaccination against RSV remains a public health priority. The RSV F glycoprotein is a major target of neutralizing antibodies, and the prefusion stabilized form of F (DS-Cav1) is under investigation as a vaccine antigen. AM14 is a human monoclonal antibody with the exclusive capacity of binding an epitope on prefusion F (PreF), which spans two F protomers. The quality of recognizing a trimer-specific epitope makes AM14 valuable for probing PreF-based immunogen conformation and functionality during vaccine production. Currently, only a low-resolution (5.5 Å) X-ray structure is available of the PreF-AM14 complex, revealing few reliable details of the interface. Here, we perform complementary structural studies using X-ray crystallography and cryo-electron microscopy (cryo-EM) to provide improved resolution structures at 3.6 Å and 3.4 Å resolutions, respectively. Both X-ray and cryo-EM structures provide clear side-chain densities, which allow for accurate mapping of the AM14 epitope on DS-Cav1. The structures help rationalize the molecular basis for AM14 loss of binding to RSV F monoclonal antibody-resistant mutants and reveal flexibility for the side chain of a key antigenic residue on PreF. This work provides the basis for a comprehensive understanding of RSV F trimer specificity with implications in vaccine design and quality assessment of PreF-based immunogens.
... MX has also aided significantly in applications that have important outcomes for industry such as protein engineering for increasing enzyme efficiency [246], changing substrate specificity [247,248], engineering new enzymes [249], vaccine development [250], etc. Another important contribution by MX is generating the information for the understanding of the function [251] or catalytic mechanism of the macromolecules [252,253] by revealing their atomic details. ...
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Protein Crystallography or Macromolecular Crystallography (MX) started as a new discipline of science with the pioneering work on the determination of the protein crystal structures by John Kendrew in 1958 and Max Perutz in 1960. The incredible achievements in MX are attributed to the development of advanced tools, methodologies, and automation in every aspect of the structure determination process, which have reduced the time required for solving protein structures from years to a few days, as evident from the tens of thousands of crystal structures of macromolecules available in PDB. The advent of brilliant synchrotron sources, fast detectors, and novel sample delivery methods has shifted the paradigm from static structures to understanding the dynamic picture of macromolecules; further propelled by X-ray Free Electron Lasers (XFELs) that explore the femtosecond regime. The revival of the Laue diffraction has also enabled the understanding of macromolecules through time-resolved crystallography. In this review, we present some of the astonishing method-related and technological advancements that have contributed to the progress of MX. Even with the rapid evolution of several methods for structure determination, the developments in MX will keep this technique relevant and it will continue to play a pivotal role in gaining unprecedented atomic-level details as well as revealing the dynamics of biological macromolecules. With many exciting developments awaiting in the upcoming years, MX has the potential to contribute significantly to the growth of modern biology by unraveling the mechanisms of complex biological processes as well as impacting the area of drug designing.
... The power system is a part of the industrial Internet of Things [1][2], which enables the intelligent interconnection of power communications through the Internet of Things technology, and ultimately promotes the development of smart grids [3][4]. As an important part of our country's energy, the power system plays a vital role in production development and people's lives [5][6]. ...
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With the increasing demand of users and power supply quality in the power grid market, this puts forward higher requirements on the power system. However, the traditional power system has many limitations. People need to be able to upgrade on the basis of the traditional power grid to adapt to changing requirements. The purpose of this paper is to study the design of multi-mode heterogeneous fusion terminal in power system. This article is based on two wireless network technology solutions, GPRS wireless public network and LTE-230 wireless private network, a hardware development platform with STM32F107VCT6 microprocessor as the core, and a μ C/OS-III embedded real-time operating system software development platform, has designed the multi-mode heterogeneous fusion terminal of the electricity information collection system. This article deeply analyzes the demand for multi-mode heterogeneous fusion terminals of the electricity consumption information collection system, which is mainly used for the collection of electricity consumption information and the marketing and distribution automation management of power services; then it analyzes the work of the GPRS network and the LTE-230 network Principle and network system structure. This article designs the communication driver and application program, and analyzes the software architecture in detail. According to the logic processing structure of the internal data of the multi-mode heterogeneous fusion terminal, this article designs the data forwarding process in detail. In this paper, protocol conversion test, interface test and communication test are carried out on multi-mode heterogeneous fusion terminal. The test results show that when the system buffer K is 2 or 4, that is, if the average size of each message in the system is 100 Bytes, and the buffer is set to 800 Bytes, the system loss rate will approach zero.
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C-Reactive protein (CRP) is an important marker for in vitro diagnosis (IVD) of inflammation. However, CRP immunoturbidimetric kits from different manufacturers exhibit inconsistency in evaluation, making clinical diagnosis challenging. The use of immunological methods in diagnosis means that the differences in epitopes across kits may directly lead to inconsistent results. Therefore, to provide consistent results, it is essential to perform epitope mapping of different kits. The composition of antibodies in a single kit is typically complex, with a combination of polyclonal antibodies or monoclonal antibodies. Here, we show an epitope screening strategy for complex antibodies in a kit based on hydrogen-deuterium exchange mass spectrometry (HDX-MS). We applied this workflow to successfully map the epitopes for three kits from three different manufacturers and compared their quantitative results. We obtained different quantitative results using kits from different manufacturers upon epitope mapping, confirming the correlation between the quantitative results and the epitopes. Thus, we have established a workflow based on HDX-MS to screen epitopes in IVD kits. This work helps determine the quantitative accuracy of a kit based on structural information, can guide the design and production of IVD reagents, and further improves the accuracy of IVD.
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Essential oils from black cumin seeds (Nigella sativa) have largely been used in the manufacturing of nutraceuticals and functional food products due to the presence of a wide variety of bioactive compounds. However, their applications in the pharmaceutical sector have recently attracted interest and started blooming. The present research elucidates the in silico and in vitro efficacies of active leads from essential oil of N sativa against the human pathogenic bacterium Staphylococcus aureus. Biofilm development has become an inevitable situation in the health care sector. Lowering the efficacies of antimicrobial drugs is one of the vital ramifications that resulted in the emergence of multidrug resistance. Clumping factor B (clfB) of S aureus plays a key role in the human immune functions during pathogenesis. Through STRING analysis, the interacting protein partners of clfB were found to regulate biofilm pathway. Therefore, eight ligands from essential oil are docked with the critical clfB protein, which revealed p‐cymene, thymoquinone and carvacrol as the robust ligands with highest binding affinity. Therefore, antibiofilm potential of N sativa essential oil at in vitro states was evaluated against S aureus. Further, real time PCR analysis showed that the expression of clfB and intercellular adhesion gene (icaA and icaD) was significantly altered upon treatment with essential oil. Altogether, the findings confirmed the antibiofilm efficacy of N sativa essential oil against S aureus. Hence, the essential oil from N. sativa was envisaged to be promising candidate to treat S aureus biofilm mediated infection.
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The successful approach to solving crystal structures of coiled-coil proteins with the program AMPLE is discussed.
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Since the publication of our first book 'Vaccine Design: Innovative Approaches and Novel Strategies' in 2011, the field of vaccinology has advanced significantly. This has prompted the need for this new volume, which aims to distil the most important new findings to provide a timely overview of the field. As before the book has been divided into two main parts. The first explores in considerable depth the key innovations that we think are dramatically changing the field; both for preclinical as well as clinical vaccine research fields. Some of the topics covered include: applications of deep sequencing, cellular screens to interrogate the human T and B cell repertoires, microbial comparative genomics, quantitative proteomics, structural biology, novel strategies for vaccine administration, T-cell inducing vaccines, etc. The second part focuses on diseases for which current medical treatment is not sufficiently effective and that could be either prevented or treated by vaccination. The examples that we have used comprise very different diseases including infectious diseases (e.g. Malaria, Tuberculosis, HIV, and Staphylococcus aureus) as well as cancer. We believe that these will be the vaccines of the future, the 'vaccines for 2020'. This book is essential reading for everyone working in vaccine R&D in academia, biotechnology companies, and the pharmaceutical industry and a recommended volume for all Microbiology libraries.
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Objective: To determine the safety and efficacy of prophylaxis with palivizumab in reducing the incidence of hospitalization because of respiratory syncytial virus (RSV) infection in high-risk infants. Methods: A randomized, double-blind, placebo-controlled trial was conducted at 139 centers in the United States, the United Kingdom, and Canada. During the 1996-1997 RSV season, 1,502 children with prematurity (35 weeks) or bronchopulmonary dysplasia (BPD) were randomly assigned to receive five injections of either palivizumab ( 15 ing/kg) or an equivalent volume of placebo by intramuscular injection every 30 days. The primary endpoint was hospitalization with confirmed RSV infection. Children were observed for 150 days (30 days from the last injection). Those with hospitalization as a result of RSV infection were evaluated for total number of days in the hospital, total days with increased supplemental oxygen, total days with moderate or severe lower respiratory tract illness, and incidence and total days of intensive care and mechanical ventilation. The incidence of hospitalization for respiratory illness not caused by RSV and the incidence of otitis media were also evaluated. The placebo and palivizumab groups were balanced at entry for demographics and RSV risk factors. Ninety-nine percent of children in both groups completed the protocol and about 93% received all five scheduled injections. Results: Palivizumab prophylaxis resulted in a 55% reduction in hospitalization as a result of RSV (10.6% placebo vs 4.8% palivizumab). Children with prematurity but without BPD had a 78% reduction in RSV hospitalization (8.1% vs 1.8%); children with BPD had a 39% reduction (12.8% vs 7.9%). When gender, entry age, entry weight, BPD, and gestational age were included in a logistic regression model, the effect of prophylaxis with palivizumab remained statistically significant. The palivizumab group had proportionally fewer total RSV hospital days, fewer RSV hospital days with increased oxygen, fewer RSV hospital days with a moderate to severe lower respiratory tract illness, and a lower incidence of intensive care unit admission. Palivizumab was safe and well tolerated. No significant differences were observed in reported adverse events between the two groups. Few children (0.3%) discontinued injections for related adverse events. Reactions at the site of injection were uncommon (1.8% placebo vs 2.7% palivizumab); the most frequent reaction was mild and transient erythema. Mild or moderate elevations of aspartatc aminotransferase occurred in 1.6% of placebo recipients and 3.6% of palivizumab recipients; for alanine aminotransferase, these percentages were 2.0% and 2.3%), respectively. Hepatic and renal adverse events related to the study drug were similar in the two groups. Conclusions: Monthly intramus-cular administration of palivizumab is safe and effective for prevention of serious RSV illness in premature children and those with BPD.
Article
Objective. To determine the safety and efficacy of prophylaxis with palivizumab in reducing the incidence of hospitalization because of respiratory syncytial virus (RSV) infection in high-risk infants. Methods. A randomized, double-blind, placebo-controlled trial was conducted at 139 centers in the United States, the United Kingdom, and Canada. During the 1996 to 1997 RSV season, 1502 children with prematurity (less than or equal to 35 weeks) or bronchopulmonary dysplasia (BPD) were randomized to receive 5 injections of either palivizumab (15 mg/kg) or an equivalent volume of placebo by intramuscular injection every 30 days. The primary endpoint was hospitalization with confirmed RSV infection. Children were followed for 150 days (30 days from the last injection). Those with hospitalization as a result of RSV infection were evaluated for total number of days in the hospital, total days with increased supplemental oxygen, total days with moderate or severe lower respiratory tract illness, and incidence and total days of intensive care and mechanical ventilation. The incidence of hospitalization for respiratory illness not caused by RSV and the incidence of otitis media were also evaluated. The placebo and palivizumab groups were balanced at entry for demographics and RSV risk factors. Ninety-nine percent of children in both groups completed the protocol and similar to 93% received all five scheduled injections. Results. Palivizumab prophylaxis resulted in a 55% reduction in hospitalization as a result of RSV (10.6% placebo vs 4.8% palivizumab). Children with prematurity but without BPD had a 78% reduction in RSV hospitalization (8.1% vs 1.8%); children with BPD had a 39% reduction (12.8% vs 7.9%). When gender, entry age, entry weight, BPD, and gestational age were included in a logistic regression model, the effect of prophylaxis with palivizumab remained statistically significant. The palivizumab group had proportionally fewer total RSV hospital days, fewer RSV hospital days with increased oxygen, fewer RSV hospital days with a moderate/severe lower respiratory tract illness, and a lower incidence of intensive care unit admission. Palivizumab was safe and well tolerated. No significant differences were observed in reported adverse events between the two groups. Few children discontinued injections for related adverse events (0.3%). Reactions at the site of injection were uncommon (1.8% placebo vs 2.7% palivizumab); the most frequent reaction was mild and transient erythema. Mild or moderate elevations of aspartate aminotransferase occurred in 1.6% of placebo recipients and 3.6% of palivizumab recipients; for alanine aminotransferase these percentages were 2.0% and 2.3%, respectively. Hepatic and renal adverse events related to the study drug were similar in the two groups. Conclusions. Monthly intramuscular administration of palivizumab is safe and effective for prevention of serious RSV illness in premature children and those with BFD.
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The natural human antibody response is a rich source of highly specific, neutralizing and self-tolerant therapeutic reagents. Recent advances have been made in isolating and characterizing monoclonal antibodies that are generated in response to natural infection or vaccination. Studies of the human antibody response have led to the discovery of crucial epitopes that could serve as new targets in vaccine design and in the creation of potentially powerful immunotherapies. With a focus on influenza virus and HIV, herein we summarize the technological tools used to identify and characterize human monoclonal antibodies and describe how these tools might be used to fight infectious diseases.
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Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the entire genome sequence of a virulent serogroup B strain (MC58) was used to identify vaccine candidates. A total of 350 candidate antigens were expressed in Escherichia coli, purified, and used to immunize mice. The sera allowed the identification of proteins that are surface exposed, that are conserved in sequence across a range of strains, and that induce a bactericidal antibody response, a property known to correlate with vaccine efficacy in humans.
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Neisseria meningitidis is a human pathogen, which, in spite of antibiotic therapy, is still a major cause of mortality due to sepsis and meningitis. Here we describe NadA, a novel surface antigen of N. meningitidis that is present in 52 out of 53 strains of hypervirulent lineages electrophoretic types (ET) ET37, ET5, and cluster A4. The gene is absent in the hypervirulent lineage III, in N. gonorrhoeae and in the commensal species N. lactamica and N. cinerea. The guanine/cytosine content, lower than the chromosome, suggests acquisition by horizontal gene transfer and subsequent limited evolution to generate three well-conserved alleles. NadA has a predicted molecular structure strikingly similar to a novel class of adhesins (YadA and UspA2), forms high molecular weight oligomers, and binds to epithelial cells in vitro supporting the hypothesis that NadA is important for host cell interaction. NadA induces strong bactericidal antibodies and is protective in the infant rat model suggesting that this protein may represent a novel antigen for a vaccine able to control meningococcal disease caused by three hypervirulent lineages.