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
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