Protein engineering strategies for the development of viral vaccines
Jayne F. Koellhoffer, Chelsea D. Higgins, Jonathan R. Lai⇑
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, United States
a r t i c l e i n f o
Received 24 September 2013
Revised 12 October 2013
Accepted 14 October 2013
Available online 21 October 2013
Edited by Wilhelm Just
a b s t r a c t
Vaccines that elicit a protective broadly neutralizing antibody (bNAb) response and monoclonal
antibody therapies are critical for the treatment and prevention of viral infections. However, isola-
tion of protective neutralizing antibodies has been challenging for some viruses, notably those with
high antigenic diversity or those that do not elicit a bNAb response in the course of natural infection.
Here, we discuss recent work that employs protein engineering strategies to design immunogens
that elicit bNAbs or engineer novel bNAbs. We highlight the use of rational, computational, and
combinatorial strategies and assess the potential of these approaches for the development of new
vaccines and immunotherapeutics.
? ? 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
The introduction of viral vaccines during the 20th century has
led to a significant decrease in viral disease burden worldwide
. Most viral vaccines are thought to work by inducing the pro-
duction of antibodies that block infection or reduce viral load,
thereby providing host protection or blunting infection such that
cellular immunity can be effective [2,3]. Antibodies can partici-
pate in host defense in several ways, including opsonization,
the coating of viruses to enhance uptake by phagocytic cells,
or activation of the complement family of proteins that can di-
rectly destroy pathogens or enhance phagocytic uptake. Here,
we will focus on neutralizing antibodies, which bind the virus
and prevent infection. Neutralizing antibodies are protective
against many viruses in both animals and humans [4–11]; there-
fore there has been much interest in their identification and
characterization for potential use as immunotherapeutic agents,
or to serve as templates for immunogen design. Neutralizing
antibodies have historically been identified by immunization of
animals with viral components, or from B-cell repertoires of
human vaccinees or survivors [11–17]. In recent years, an
increasing amount of structural information about neutralizing
antibodies – and their mechanisms of activity – has shifted focus
toward structure-based design of immunogens to elicit such
antibodies and of the antibodies themselves [18–34].
Neutralizing antibodies are thought to abrogate viral infectivity
by three major mechanisms (Fig. 1): (i) by blocking virus
attachment to host cells; (ii) by inhibiting viral uncoating or
conformational changes in viral envelope glycoproteins needed
for cell entry; or (iii) by inducing the formation of non-infectious
viral aggregates that cannot enter cells. In the case of enveloped
viruses, those surrounded by a lipid bilayer, the primary neutral-
ization targets are the virus envelope glycoproteins that are
responsible for mediating membrane fusion between the viral
and host cell membranes, a critical step for infection . During
the course of natural infection or vaccination, neutralizing antibod-
ies against many viruses, such as polio, mumps, and measles, are
elicited in both humans and animals. However, induction of effec-
tive neutralizing antibodies is rare or does not occur against some
viruses, notably those with high antigenic diversity such as the hu-
man immunodeficiency virus-1 (HIV-1), hepatitis C virus, and
influenza virus. Not surprisingly, this antigenic variation is
reflected in the diverse sequences of the virus envelope glycopro-
teins among strains or clades, and thus antibodies that do not bind
conserved epitopes have a narrow spectrum of activity.
Various strategies have been employed to develop vaccines
that elicit neutralizing antibodies for these high diversity viruses.
In vaccination trials, the use of adjuvants to enhance the quality
of antibody response to vaccination , nucleic-acid based
methods for the delivery of antigen [37–40], and the administra-
tion of more than one type of vaccine to boost immunogenicity
[41–43] have been attempted. However, effective vaccines for
these viruses remain elusive. A major hurdle appears to be that
the immunodominant antibody responses are directed against
the most variable parts of the envelope glycoproteins, and there-
fore most neutralizing antibodies are narrowly strain-specific. An
0014-5793/$36.00 ? 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
E-mail address: email@example.com (J.R. Lai).
FEBS Letters 588 (2014) 298–307
journal homepage: www.FEBSLetters.org
effective vaccine should be able to elicit ‘‘broadly neutralizing’’
antibodies (bNAbs) that engage conserved, less variable domains
and can therefore protect across a spectrum of genetic isolates.
Likewise, immunotherapeutics for these viruses should be direc-
ted at conserved viral epitopes or infection pathways. In this
review, we highlight recent work that utilizes novel protein
engineering strategies for the development of effective vaccines
and immunotherapeutics against highly variable viruses and
viruses for which a bNAb response does not arise during the
course of natural infection.
2. Viral antigen design to elicit broadly neutralizing antibodies
One promising strategy for the generation of bNAbs by vacci-
nation is ‘‘reverse engineering,’’ where structural information
gleaned from the binding of bNAbs raised in the course of natu-
ral infection is used to guide immunogen design [3,44]. In the-
ory, translation of this antibody binding information into an
immunogen designed to display specific, critical epitopes should
allow production of antibodies with similar broad neutralization
capacity in vivo, provided that the immunological evolution
pathway of the bNAb can be induced by vaccination. Thoughtful
modification of the immunogen to reflect the specific, three-
dimensional antibody-binding site is required (Fig. 2). Since the
goal of reverse engineering is to develop a peptide or protein
scaffold that mimics the natural epitope, most strategies have
utilized rational, combinatorial, or computational methods. Here
we discuss several recent examples in which these methods
were used to develop and evaluate immunogens.
2.1. Conformational mimicry of linear epitopes from HIV-1 gp41 and
HIV-1, a lentivirus, enters host cells by fusing its lipid bilayer
with the host cell plasma membrane. This fusion is facilitated by
the viral envelope glycoprotein, Env, which consists of a surface
subunit, gp120, and a transmembrane subunit, gp41 . Infection
is initiated by gp120 binding to CD4 and a co-receptor on host
cells, triggering large-scale conformational changes in gp41 that
eventually lead to membrane fusion. Antibodies directed against
Env have the potential to be neutralizing, but the generation of
bNAbs has proven to be extremely challenging. This is likely
because of the hypervariability encoded in the Env gene, the exten-
sive glycosylation of the surface of the Env protein, and structural
heterogeneity associated with gp120 that is critical for its function
as the triggering molecule for membrane fusion. During the course
of chronic infection by HIV-1, ?10% of patients develop bNAbs,
suggesting that a vaccine approach to prevent HIV-1 infection is
possible [12,45,46]. A number of HIV-1 bNAbs target linear epi-
topes in the V3 region of gp120 or the membrane-proximal exter-
nal region (MPER) of gp41. Structures of these bNAbs bound to
peptide epitopes have demonstrated that these segments contain
well-defined secondary structure when bound to the bNAbs. It is
therefore hypothesized that immunogens designed to elicit anti-
bodies that bind these segments in such conformations would be
critical for a successful vaccination strategy.
Immunogens based on the V3 loop have been designed and
have so far met with some limited success. Antibody 447-52D
wasisolated viahybridomamethods fromasubtypeB
Fig. 1. Mechanisms by which neutralizing antibodies block viral infection. Neutralizing antibodies are thought to abrogate viral infectivity by blocking virus attachment to
host cells, inhibiting viral uncoating, blocking conformational changes in viral envelope glycoproteins needed for membrane fusion or prematurely triggering the fusion
machinery, or by inducing the formation of non-infectious viral aggregates that cannot enter cells.
J.F. Koellhoffer et al./FEBS Letters 588 (2014) 298–307
HIV-1-infected individual and found to bind to the tip of the V3
loop in a b-hairpin conformation . Chakraborty et al. designed
an immunogen to mimic the tip region by inserting the epitope of
447-52D into thioredoxin, a small and stable Escherichia coli pro-
tein, and using newly introduced disulfide bonds to lock the epi-
tope in the desired conformation . This construct was able to
generate a 447-52D-like response upon immunization in guinea-
pigs. Although a fairly high antibody concentration was elicited
by this immunization strategy (50–400 lg/mL serum), the serum
was not able to effectively neutralize many primary viral isolates,
perhaps because of the low accessibility of the V3 loop on many
of these isolates .
Mor et al. synthesized a library of V3-based peptides in which
they varied the position of disulfide bonds within the peptide
. The group found that V3-peptides containing a single disul-
fide bond, regardless of position, retained flexibility and did not
form an ideal b-hairpin turn. However, installation of a second
disulfide bond led to a significant improvement in peptide rigidity
and many of these disulfide bond-containing peptides exhibited
higher affinity to 447-52D than corresponding linear V3 peptides
. The constrained V3 peptides were linked to an 18-residue
segment of the gp120 C4 region, known to induce a helper T-cell
response, and were shown to elicit a 30-fold stronger HIV-1
neutralizing response in rabbits as compared to analogous linear
V3 peptides or gp120 constructs displaying the V3 loop . These
studies suggest that carefully designed proteins that mimic natural
HIV-1 bNAb binding sites have potential to elicit neutralizing
Two of the most potent bNAbs known to target HIV-1, 2F5 and
4E10, bind linear epitopes on the MPER of gp41. The MPER is a
highly conserved, tryptophan-rich region that is believed to play
a crucial role in HIV-1 membrane fusion [48,49]. The 2F5 and
4E10 epitopes neighbor one another and appear to require binding
to only a few crucial residues within their respective epitopes .
Both antibodies have been shown to interact with the virion lipid
membrane in addition to binding to gp41, suggesting that the
structure of membrane-anchored MPER is crucial for binding by
these mAbs . Because of the breadth and potency of neutraliza-
tion exhibited by these antibodies, strategies aimed at eliciting a
2F5- or 4E10-like response are the subject of many efforts for
development of an effective anti-HIV vaccine. Both 2F5 and 4E10
were isolated well over a decade ago [48,51,52] and efforts to
HIV Envelope Spike
Identify bNAb epitope by structural
or biochemical studies
Graft onto acceptor scaffold or
optimize antigen characteristics
Confirm binding to bNAb
Use as an immunogen to
isolate new bNAbs
Fig. 2. Immunogen design by reverse engineering. Structural or biochemical studies are used to define the epitope of a bNAb. For both linear and discontinuous epitopes,
residues involved in the antibody–antigen interaction can be grafted onto a stable scaffold in the appropriate conformation, or the original antigen can be modified to
optimize recognition or other properties. Following confirmation of antibody binding, the engineered immunogen can then be used to elicit a broadly neutralizing antibody
response in animal models. In this way, additional bNabs may be identified and the potential use of the immunogen in vaccine development may be assessed. Here, we have
depicted the reverse engineering process using two HIV-1 bNAbs, b12, which recognizes a discontinuous epitope on gp120, and 2F5, which recognizes a continuous linear
epitope on gp41. PDB files used in figure: b12: PDB ID 2NYZ, 3RPT, eRU8; 2F5: PDB ID ITJI, 3LEV; MAb 11f10: PDB ID 3LEX [19,32].
J.F. Koellhoffer et al./FEBS Letters 588 (2014) 298–307
mimic their epitopes with designed immunogens have been ongo-
ing since then. Recently, several novel bNAbs have been isolated
against the MPER. One example is mAb 10E8, isolated from an
HIV-infected donor by Huang et al. . 10E8 is one of the most
potent and broadly neutralizing anti-HIV antibodies yet identified.
It was shown to bind the MPER in a conformation similar to 4E10,
but has a novel binding epitope . The presence of 10E8 and
other MPER-binding antibodies in natural infection suggests that
an appropriately designed immunogen would elicit similar
In 2010, Ofek et al. used computational methods to construct
an epitope scaffold using the 2F5 epitope . The 2F5 epitope
is conformationally flexible when not bound by the antibody,
therefore posing a particular challenge for epitope design. Upon
2F5 binding, the MPER epitope adopts a kinked, extended struc-
ture and recognition of this specific structure is postulated to be
a requirement for neutralizing activity. Ofek et al. therefore
strove to mimic this structure in their computationally designed
immunogen. The group first searched the protein data bank
(PDB) for ‘‘acceptor proteins’’ that could be used as scaffolds,
with segments that contained backbone structural similarity to
the 2F5-bound gp41 epitope. The identified proteins were re-de-
signed using RosettaDesign to introduce mutations such that the
2F5 MPER epitope side chains would be included in these scaf-
folds . These constructs were used in vaccination trials using
mice. Although some antibodies with similar binding modes to
2F5 were identified, the vaccine trials failed to produce neutral-
izing sera. However, crystal structures of the resulting antibodies
in complex with the HIV MPER demonstrated that the segment
corresponding to the 2F5 epitope adopted the desired kinked,
extended structure . Correia et al. performed a similar study
using the linear epitope of 4E10 . Appropriate scaffold pro-
teins were again identified from the PDB and optimized using
RosettaDesign. The resulting protein-4E10 epitope constructs
were found to bind with higher affinity (in some cases 100-fold
higher) to 4E10 than compared to the MPER peptide epitope
alone . These epitope-scaffolds were used in immunization
trials with rabbits, and were shown to induce antibodies that
were non-neutralizing but displayed high structural similarity
to 4E10 . As discussed above, it is known that both 2F5
and 4E10 require interaction with the virion lipid membrane
for binding . Therefore, this approach may require some
modifications to incorporate membrane-like components to elicit
2F5- or 4E10-like antibodies. Nonetheless, these studies demon-
strate that appropriate engineering of immunogens to contain
segments that mimic conformational features of linear epitopes
can be used to generate structure-specific antibodies against
Azoitei et al. have developed a computational and experimental
methodology to incorporate both the backbone conformation and
the side chains of functional motifs onto appropriate protein scaf-
folds [18,19]. In the examples above, protein grafting involved
transplantation of the protein side chains onto an ‘‘acceptor pro-
tein’’ scaffold that already contained native segments in which
the backbone conformation matched that of the linear epitope in
the bNAb-bound conformation. However, such an approach is lim-
ited in that an acceptor protein with a segment that matches the
epitope conformation of the epitope region must be identified. In
their work, Azoitei et al. developed a method to incorporate both
side chains and backbones of an epitope into a scaffold and impos-
ing the desired epitope conformation by protein design . The
authors found that epitope backbone grafting resulted in scaffolds
that bound 2F5 with up to 30-fold higher affinity than the corre-
sponding side-chain only grafting construct . Therefore, back-
bone grafting may prove to be more successful for the generation
of bNAbs than side chain only grafting, although no immunization
trials have yet been performed with constructs designed using this
2.2. Computational and combinatorial redesign of HIV-1 gp120
While some bNAbs, such as 2F5 and 4E10, target linear epitopes
on viral glycoproteins, many known bNAbs target epitopes that
consist of discontinuous protein segments. Therefore, neutraliza-
tion requires recognition of a specific three-dimensional conforma-
tion of the viral antigen. This presents a challenge for immunogen
design, which must accurately recapitulate the three-dimensional
antibody binding epitope. An additional challenge is that many vir-
al glycoproteins are structurally heterogeneous; therefore, it is cru-
cial to design immunogens that mimic the structure of the epitope
that is relevant for antibody neutralization. The case of HIV-1
gp120 highlights these problems facing effective viral immunogen
design. In 2009, Chen et al. used modeling and binding experi-
ments to explore the differences between the potent HIV-1 bNAb
b12, which targets the CD4-binding site on gp120, and poorly neu-
tralizing antibodies that also target the CD4-binding site . They
found that even slight differences in the binding site, on the order
of a few angstroms, were sufficient to result in differing antibody
neutralization capabilities. For example, antibody b13, whose
angle of approach to the CD4-binding site differs from that of
b12 only by a 17? rotation of the variable region, binds a substan-
tially different conformation of gp120 than does b12 . There-
fore, one explanation for the failure of viral immunogens to be
translated into effective vaccines thus far may be that immunogen
design has not been sufficiently precise in the nature of the inter-
actions with the antibodies they elicit.
Recent attempts to overcome these challenges have turned to
advances in computational and combinatorial protein design
methods. Azoitei et al. used a combined computational and
experimental approach to graft the non-linear, discontinuous
b12 epitope into an acceptor protein scaffold . The resulting
immunogen, 2bodx_43, was observed to bind tightly to b12 but
not to other CD4 binding site-targeting or non-neutralizing anti-
bodies such as b13 . In 2010, Wu et al. used protein engi-
neering to generate a variant of gp120 that preserved the CD4
binding site, but eliminated other antigenic regions by substitut-
ing surface exposed residues not included in the CD4 binding site
with simian immunodeficiency virus homologs or other non-HIV
residue identities (the ‘‘resurfaced gp120’’ core, Fig. 3A). This de-
sign was intended to focus interactions on the CD4-binding site,
since antibodies that interact with other segments of HIV-1
gp120 would not bind the resurfaced segments. The resurfaced
gp120 core was used as bait for screening using broadly neutral-
izing serum from human patients, and a potent bNAb, VRC01,
was identified . As expected from the gp120 variant design,
VRC01 binds the CD4 binding site ; other ‘‘VRC01-like’’ anti-
bodies have been identified from HIV-infected individuals and
have been shown to broadly neutralize across HIV genetic iso-
Interestingly, all of the ‘‘VRC01-like’’ antibodies derive from the
IGHV1-2germline segment, a promising observation for the induc-
tion of similar bNAbs since antibodies originating from this germ-
line segment are estimated to be present in ?2% of the human
antibody repertoire . However, the predicted germline progen-
itors of VRC01 and VRC01-like antibodies do not show binding to
wild-type gp120. Therefore, it is unlikely that gp120 itself could
elicit VRC01-like antibodies since it is unable to engage the appro-
priate unmutated progenitors. To address this problem, Jardine
et al. have recently used a combination of computationally-guided
antigen design and in vitro screening to engineer an HIV-1 gp120
immunogen that binds to multiple VRC01-class bNAbs and their
J.F. Koellhoffer et al./FEBS Letters 588 (2014) 298–307
germline precursors (Fig. 3B) . Jardine et al. first built a
homology model of a germline precursor to VRC01 bound to
gp120; the model revealed areas of potential steric clashes be-
tween gp120 glycans and the germline antibody (Fig. 3B, inset).
The group generated a gp120 outer domain (OD) construct where
these clashes were removed. Rosetta computational protein inter-
face was then used to identify additional mutations at the CD4-
binding site that were predicted to increase the affinity for the
VRC01 germline progenitor, and libraries with these mutations
were screened using yeast display. gp120 outer domain variants
identified from this screen were subjected to further rounds of
computational design and library screening, as well as selection
for retention of binding to CD4. The final result of this iterative pro-
cess was a construct termed eOD-GT6, which was shown to bind
with nanomolar affinity to VRC01 and VRC01-like antibodies and
with lower (micromolar) affinity to germline progenitors .
eOD-GT6 was fused to a self-assembling virus-like nanoparticles
and was shown to activate germline and mature VRC01-class B
cells . The eOD-GT6 construct has not yet been evaluated in
vaccination trials; rabbits, mice and macaques lack a germline seg-
ment that bears homology to IGHV1-2. Nonetheless, eOD-GT6 may
be a promising immunogen candidate to elicit VRC01-like bNAbs.
Furthermore, the concept of targeting of immunogens to engage
both early and fully matured forms of antibodies along the immu-
nological evolution pathway merits further evaluation.
2.3. Identification of conserved epitopes in influenza hemagglutinin
Influenza is an RNA virus that causes respiratory tract infection
in mammals and some bird species . Influenza is an enveloped
virus containing two coat proteins: hemagglutinin (HA), which is
responsible for host cell receptor binding and membrane fusion,
and neuraminidase (NA), which cleaves sialic acid residues to re-
lease newly budding viral particles from the host cell [35,58]. As
is the case with HIV-1 Env, a high degree of antigenic diversity is
tolerated in both HA and NA, thus explaining influenza’s ability
to evade host immune responses and cause repeated infections
in a single host. Highly infectious, pandemic influenza outbreaks
occur approximately every 10–12 years, and are the result of large
changes in or recombination of HA and/or NA, termed antigenic
shift. These highly contagious and lethal outbreaks are cause for
great concern worldwide. In particular, the H5N1 influenza resur-
gence in 2004 has led to concerns over its pandemic potential
and highlighted the need for a vaccine that is effective against mul-
tiple H5N1 genetic isolates .
In 2011, Giles and Ross utilized a novel computational antigen
design technique termed COBRA (computationally optimized
broadly reactive antigen) to generate a synthetic HA protein capa-
ble of eliciting a broadly neutralizing antibody response against
H5N1 avian influenza . H5N1 viruses are divided among 10
distinct clades, which are geographically diverse and classified
according to phylogentic distance among HA genes. One approach
to address the vast sequence diversity in circulating H5N1 isolates
has been to generate consensus-based viral proteins for use as
immunogens, where a population of H5N1 sequences are aligned
and the most common residue at each position within the viral
protein is selected. This strategy has been met with some success,
with consensus-based H5N1 HA immunogens eliciting broad anti-
body responses in mice, ferrets and macaques [59–61]. However,
Giles and Ross raise concerns over the bias inherent in consen-
sus-based methods, which rely on the available input sequences
and are therefore subject to sampling bias. The authors point out
that the majority of H5N1 HA sequences from human isolates arise
from clade 2 and therefore there are concerns that these sequences
do not accurately reflect the true diversity of vial sequences circu-
lating in avian population. The COBRA method was used to
overcome these concerns by using multiple rounds of consensus
Fig. 3. Computational and combinatorial redesign of HIV-1 gp120 analogs. (A) Structure of the gp120 core showing the CD4 binding site in yellow and residues that were
modified in the ‘‘resurfaced gp120’’ in red . Residues for modification were chosen to eliminate other antigenic regions on gp120 and thereby focus the immune response
to this molecule on the CD4 binding site. This modified gp120 molecule was used as bait for screening using broadly neutralizing serum from human patients, resulting in the
identification of VRC01, a potent bNAb. (gp120 PDB ID: 2NXY). (B) Schematic of gp120 engineering scheme by Jardine et al. in order to engage VRC01 germline precursors
. Inset shows native gp120 (green) and the engineered molecule, eOD-GT6 (pink) with binding to the VRC01 germline antibody (cyan). (eOD-GT6 and the germline
precursor were taken from PDB ID 4JPK; gp120 from PDB ID 2NXY.) Potential steric clashes between gp120 glycans at positions N276 and N463 and the germline precursor
were predicted; these glycans are drawn in on the inset figure in red sticks. Both Asn residues were mutated to Asp in eOD-GT6, thereby removing these clashes.
J.F. Koellhoffer et al./FEBS Letters 588 (2014) 298–307
The COBRA method was performed as follows: 129 unique HA
sequences representing clade 2 H5N1 viruses were grouped into
phylogenetic subclades and further divided into individual out-
break groups based on the time and geographic location of isola-
tion. Consensus HA sequences were generated for each outbreak
group and then consensus sequences were generated for each
subclade based on the results for the outbreak groups. These subc-
lade consensus sequences were again aligned to generate a final
consensus sequence, termed ‘‘clade 2 COBRA HA’’ . Phyloge-
netic comparison of this final consensus sequence with all human
isolates of H5N1 HA sequences demonstrated that clade 2 COBRA
HA retained a clade 2-like sequence but did not fall specifically
within any subclade classification. Indeed, clade 2 COBRA HA rep-
resents a unique HA sequence that has not been previously isolated
. VLPs displaying clade 2 COBRA HA on the surface were shown
to bind to the HA receptor sialic acid; mice, ferrets, and cynomol-
gus macaques vaccinated with clade 2 COBRA HA VLPs demon-
strated protective levels of anti-HA antibodies to a series of viral
isolates representing each subclade of H5N1 clade 2 [25,26]. Inter-
estingly, the clade 2 COBRA HA VLPs were found to elicit
higher-titer antibodies to a panel of H5N1 HA proteins than a mix-
ture of VLP vaccines expressing representative HA molecules from
clade 2.1, 2.2, and 2.3 . This result suggests that the COBRA-
derived sequence elicits a more efficient antibody response than
an immunogen designed from a single genetic isolate.
3. Anti-viral monoclonal antibodies
As an alternative to vaccination strategies, where immunogens
are designed to elicit a neutralizing antibody response, monoclonal
antibodies (mAbs) can be administered directly for therapeutic
intervention. mAbs have been used clinically to treat a variety of
diseases since the 1980s . The first patient to be treated with
a mAb in the United States was in 1980 for non-Hodgkins lym-
phoma . Because of the ability of mAbs to precisely target spe-
cific cell proteins and receptors, mAb
therapeutics quickly accelerated. Despite the clear advantages that
mAbs offered over traditional chemotherapeutic regimes in terms
of specific cell targeting, early therapeutic trials were limited by
the immunogenicity of murine antibody scaffolds upon repeated
exposure, resulting in shorter mAb half-life, lack of antibody effec-
tor functions, and human anti-mouse antibody (HAMA) responses
causing fever, chills, rash, nausea, headaches, and rarely anaphy-
laxis [62–64]. These limitations have been overcome by the devel-
antibodies. Currently, there are 30 mAbs approved by the FDA for
clinical use in the United States. Twenty-one of these are fully
humanized, six are human-murine chimeras, and three are murine
. One of these antibodies, Palivizumab, is a fully humanized
mAb approved for prophylaxis against respiratory syncytial virus,
demonstrating that mAb therapy can be effective against viral tar-
gets [16,66]. Palivizumab remains the only therapy to significantly
reduce RSV hospitalization rates in high risk infants (from 10.6% to
4.8%) and to reduce subsequent morbidity/mortality . How-
ever, it is worth noting that the cost-effectiveness of Palivizumab
has been questioned, as the antibody is administered in monthly
injections for all five months of the RSV season. On average, seven-
teen children must be treated in this manner to prevent one RSV-
related hospital admission, and fifty-nine must be treated to pre-
vent one ICU admission .
Traditionally, mAbs against viral targets have been isolated
from immunized/infected animals or humans. The study of
antibody-antigen complexes, mainly by X-ray crystallography,
has allowed for antibody engineering (primarily within the com-
plementarity determining regions, CDRs) to enhance the potency
therapy for cancer
and fully humanized
of these naturally occurring antibodies. Computer-aided analysis
has allowed for antibody engineering even in the absence of
high-resolution structural data. Through engineering based on
structural examination or computational methods, rational muta-
tions can be introduced into mAbs in order to improve affinity or
convey other desirable properties, such as cross-reactivity or solu-
bility, thus facilitating the identification of neutralizing antibodies
against therapeutically challenging viruses. Here we highlight
some recent examples of the engineering of anti-viral mAbs. We
end with a discussion of synthetic antibody engineering and the
potential for this technique to be applied to viral therapeutics.
3.1. Rational engineering of a potent VRC01-like mAb
Diskin et al. used structural information to increase the potency
of a VRC01-like mAb against HIV-1 . The group started with
NIH45-46, a clonal variant of VRC01 that was isolated from the
same donor patient but has enhanced neutralization abilities.
Structurally, the two mAbs are highly similar, containing 85% se-
quence identity in the heavy chain variable (VH) domains and
96% sequence identity in light chain variable (VL) domains. How-
ever, NIH45-46 includes a four-residue insertion within the third
heavy chain complementarity determining region (CDR-H3) rela-
tive to VRC01; these residues were observed to contribute to bind-
ing of the antibody to the CD4 binding site on gp120. Diskin et al.
carefully examined the crystal structures of both VRC01 and
NIH45-46 bound to gp120. They noted that a key interaction be-
tween CD4 residue 43 (a phenylalanine) and a hydrophobic pocket
on gp120 was not mimicked by either antibody, and hypothesized
that mutating residues on the mAbs to interact with this hydro-
phobic pocket would increase mAb potency and breadth (Fig. 4).
A series of NIH45-46 mutants were therefore constructed contain-
ing hydrophobic amino acid substitutions at residue 54, a Gly in
the native NIH45-46, which is in close proximity to the gp120
hydrophobic pocket. As expected, NIH45-46 containing G54W or
G54F mutations showed increased neutralization potency .
Remarkably, NIH45-46G54Wshowed a substantial increase in the
breadth of HIV-1 strains that it neutralized compared to the parent
NIH45-46, with up to 2000-fold higher neutralization abilities
against some HIV-1 strains . This work provides an elegant
example of how structure based rational design can be used to con-
struct potent anti-viral antibodies that have the potential to be
used in passive immunization or treatment of viral infection.
3.2. Computational redesign of broad dengue virus antibodies
Recent work by Tharakaraman et al. utilizes a novel computa-
tion approach that is not reliant on crystal structure information
to increase the potency of an antibody against dengue virus
(DENV) . DENV is a flavivirus responsible for 50–100 million
human infections per year, ranging in severity from an acute febrile
illness to a fatal hemorrhagic fever or shock syndrome . DENV
consists of four serotypes (DENV1–4), which vary from one another
at the amino acid level by 25–40%. Importantly, DENV infection is
characterized by a marked antibody-dependent enhancement of
replication, whereby higher levels of viral replication and lethality
are observed in DENV survivors during a second infection with any
of the other three serotypes . Because of this phenomenon, pre-
vious vaccination trials against DENV have failed and current inter-
est focuses on antibodies that can inhibit multiple serotypes.
In their work, Tharakaraman et al. computationally redesigned
4E11, an antibody directed against the DENV viral envelope glyco-
protein (E) . 4E11 was previously identified from mice and
found have potent neutralizing ability for DENV1–3 but limited
neutralization potential against DENV4. Therefore, the group
J.F. Koellhoffer et al./FEBS Letters 588 (2014) 298–307
strove to improve the affinity of 4E11 to DENV4, while maintaining
the affinity of the antibody to DENV1–3 by relying on computa-
tional docking models. Recently, the structure of the 4E11 single
chain variable fragment (scFv) in complex with E domain III from
all four serotypes was reported , but when the Tharakaraman
et al. study was initiated, these structures were not yet available
. The group first used a multivariate logistic regression analysis
(MLR) to identify key physiochemical features that define natural
antigen–antibody interfaces, which allowed them to more accu-
rately predict the correct antibody–antigen complexes than stan-
dard docking protocols relying solely on energy minimizing
functions. Using the MLR results, models of 4E11 bound to the E
protein of DENV1–4 were generated. The models were used to de-
sign mutations that were predicted to increase antibody affinity for
DENV4 while not detrimentally affecting contacts with DENV1–3.
One mutant, 4E5A, contained five amino acid substitutions and
was found to have the greatest increase in affinity towards DENV4.
Compared to 4E11, the redesigned 4E5A displayed a 450-fold
enhancement in affinity to DENV4 and a 15-fold enhancement in
affinity to DENV2, while maintaining affinity to DENV1 and DENV3.
In vitro neutralization assays showed that 4E5A neutralized DENV4
with >75-fold increased potency, while maintaining potency
against DENV1–3. Additionally, 4E5A demonstrated potent antivi-
ral activity against all four DENV strains in a mouse model of infec-
tion, causing a significant reduction in viremia at both 1 and 5 mg/
Recent work from the Varani laboratory has also applied
computational methods to improve the neutralization capabili-
ties of an anti-DENV antibody . Here, NMR epitope mapping
was used to define the binding site of a broadly neutralizing
human mAb, DV32.6, against all four DENV serotypes. This struc-
tural information was used to filter the results of computational
docking of DV32.6 to DENV1–4. Analysis of the best docking
models allowed the group to rationally engineer DV32.6 mutants
that bound only to one serotype (thereby demonstrating an abil-
ity to increase antibody specificity) or that bound more tightly to
the eptiopes on DENV1–4, resulting in a mutant that was up to
40 times more effective than the parent DV32.6 at neutralizing
DENV . Of note, this group has used computational modeling
in the past to explore why an anti-DENV antibody failed to neu-
tralize viral infection  and to examine the binding of two
neutralizing mAbs to the influenza HA protein . The exam-
ples outlined here demonstrate how computational methods
can be applied to the study of antibody–virus interactions, and
how this information can be used to develop more effective
3.3. Synthetic antibodies targeting intermediates of Ebola virus fusion
In contrast to computational modeling, where a starting anti-
body is required, synthetic antibody engineering shows promise
for the development of novel antibodies against viral targets for
which few antibodies are available. Synthetic antibodies contain
antigen-binding sites that are constructed entirely from ratio-
nally designed, man-made diversity . A key advantage com-
engineering does not rely on previous human or animal infec-
tion/immunization. Therefore, it is possible to select antibodies
against traditionally non-immunogenic targets, or to target viral
epitope conformations that may not arise during natural infec-
tion. Synthetic antibody libraries displayed on the surface of
M13 filamentous bacteriophage (phage display) have been used
to select antibodies that bind to targets such as ubiquitin ,
histones , and hemoglobins , and to select against pre-
cise antigenic conformations . Antibodies with exquisite
specificity have been obtained from synthetic antibody libraries,
such as antibodies that can distinguish between chicken and
quail lysozyme , which differ by only four amino acids,
and those that can differentiate between two conformations of
the same enzyme (caspase) . An additional advantage is that
the initial libraries are typically constructed on human scaffolds,
therefore the resulting antibodies do not require extensive engi-
neering to ‘‘humanize’’ prior to therapeutic use .
Despite the advantages offered by synthetic antibody phage dis-
play, this technique has only recently been applied to the discovery
of antibodies targeting viral epitopes. Our laboratory used this
technology to target fusion intermediates of the Ebola virus (EBOV)
envelope glycoprotein (GP) . EBOV is a highly pathologic mem-
ber of the Filoviridae family of viruses that causes severe hemor-
rhagic fever . Viral entry is mediated by GP, which consists of
three copies each of a surface subunit, GP1, and a transmembrane
subunit, GP2 [35,84]. GP1 binding to cell surface receptors initiates
uptake of the virus into the endosome. Here, host cysteine prote-
ases cleave GP removing most of GP1; this cleavage event has been
shown to be necessary for EBOV entry [85,86]. Cleavage is hypoth-
esized to be important for viral entry for two reasons: (i) cleavage
is thought to unmask the receptor binding site for Neimann Pick
C1, an endosomal cholesterol transporter which was recently
is that syntheticantibody
Fig. 4. Rational engineering of a potent VRC01-like mAb. Diskin et al. noted that neither VRC01 nor mAb NIH45-46 (blue) recapitulated a key interaction between CD4 (pink)
residue 43 (a Phe) and a hydrophobic pocket on gp120 (green) . A series of NIH45-46 mutants were constructed to contain hydrophobic amino acid substitutions at
residue 54, a Gly in NIH45-46 (shown in sticks in inset). NIH45-46 containing G54W or G54F mutations showed increased neutralization potency and breadth.
J.F. Koellhoffer et al./FEBS Letters 588 (2014) 298–307
shown to be a critical intracellular receptor for EBOV entry 
and (ii) cleavage appears to prime the GP2 subunit for large-scale
conformational changes which ultimately lead to fusion of the viral
and host endosomal membranes . However, structural changes
in GP associated with endosomal proteolytic cleavage are incom-
pletely defined, and the precise timing of cleavage within the
endosome is poorly understood. Additionally, it was unclear
whether epitopes on the proteolytically cleaved GP were available
for virus neutralization. Therefore, we strove to identify novel
mAbs capable of distinguishing between the uncleaved (GPUNCL)
and proteolytically cleaved (GPCL) forms of GP. However, there
are limited sources of natural human Ebola virus antibodies since
survivors generally have low serum antibody titers, and many re-
sponses are dominated by antibodies that bind preferentially with
a secreted, dimeric form of GP (sGP) that is not relevant to mem-
brane fusion [13,88–91].
To identify mAbs with specific recognition profiles toward
GPUNCLand GPCL, we used a synthetic antibody binding fragment
(Fab) library based on a human anti-maltose binding protein Fab
scaffold. This library, ‘‘Library F’’, contains binomial tyrosine/serine
randomization in non-structural positions of CDR-H1 and CDR-H2,
and additional variation at CDR-H3 and CDR-L3 encoding the nine
residues Tyr/Ser/Gly/Ala/Phe/Trp/His/Pro/Val in a 5:4:4:2:1:1:1:
1:1 ratio . This amino acid distribution mimics the observed
distribution found in natural CDR segments . We screened Li-
brary F against protein mimics of GPUNCLand GPCL. We identified
antibodies with distinct recognition profiles: FabCLbound prefer-
entially to GPCL(EC50= 1.7 nM), whereas FabUNCLbound specificity
(EC50= 75nM) .
GP-containing pseudotyped viruses indicated that these antibodies
inhibited GPCLor GPUNCL-mediated viral entry with specificity that
matched their recognition profiles (IC50s: 87 nM for IgGCL; 1 lM for
FabUNCL) . This work demonstrates that epitopes on GPCLare
available for neutralization by antibodies, and may lead to the
development of new tools for dissecting intermediates of EBOV en-
try. Importantly, these results demonstrate the applicability of syn-
thetic antibody engineering to the study of viral membrane fusion,
paving the way for synthetic antibody libraries to be screened
against other viral targets for which there are limited sources of
natural, human antibodies.
Rational, computational, and combinatorial methods hold great
promise for application of protein engineering principles to viral
vaccine and immunotherapeutic development. Furthermore, the
number of bNAb characterization and structural studies have been
increasing steadily over recent years, providing additional infor-
mation on which to base design. While much progress has been
made in engineering new viral immunogens and antibodies, the
challenge moving forward will be evaluation of these reagents in
appropriate animal models. We expect this future work will pro-
vide new fundamental insight into requirements for protection
from and neutralization of viruses in vivo.
J.R.L. acknowledges funding from the National Institutes of
Health (R01-AI090249). J.F.K. was supported in part by NIH Medi-
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