2

0
0
5

L
A
N
D
E
S

B
I
O
S
C
I
E
N
C
E
.

D
O

N
O
T

D
I
S
T
R
I
B
U
T
E
.
[Human Vaccines 1:3, 95-101; May/June 2005]; 2005 Landes Bioscience
Tamar Ben-Yedidia
Ruth Arnon*
Department of Immunology; the Weizmann Institute of Science; Rehovot Israel
*Correspondence to: Ruth Arnon; Email: ruth.arnon@weizmann.ac.il
Received 01/17/05; Accepted 05/15/05
Previously published online as a Human Vaccines E-publication:
http://www.landesbioscience.com/journals/vaccines/abstract.php?id=1851
KEY WORDS
influenza, vaccine, peptide, epitope, flagella,
recombinant, human
ABBREVIATIONS
APC antigen presenting cell
CTL cytotoxic T lymphocytes
HA hemagglutinin
HLA human Leukocyte antigen
NA neuraminidase
NP nucleoprotein
PBMC peripheral blood mononuclear cells
SCID severe combined immunodeficiency
TLR toll like receptor
Review
Towards an Epitope-Based Human Vaccine for Influenza
ABSTRACT
The conventional, currently available vaccines against influenza virus, though quite
successful, suffer from a few shortcomings; one major limitation is their restriction to the
specific strains that are included in the vaccine. We review herewith some of the more
recently developed influenza vaccines and further describe our own results on the design
of epitope-based vaccine for human use. In this vaccine, a combination of B- and T-cell
epitopes are individually expressed within an immunogenic moleculesalmonella flagellin
and the resultant recombinant flagella serve both as a carrier and as an adjuvant. The
mixture of recombinant flagella expressing the appropriate epitopes was administered to
young and aged mice as well as to human/mouse chimera model in which human PBMC
are functioning within the mice body. Intranasal immunization in all these animal models
led to effective protection against challenge infection with different strains of influenza
virus.
BACKGROUNDAVAILABLE INFLUENZA VACCINES, LIMITATIONS
AND THE NEED FOR ALTERNATIVE APPROACHES
During the past decade there has been a growing awareness of the substantial medical
and economical costs associated with influenza epidemics. Prophylactic vaccination is the
major factor in combating this hazard.
The currently employed influenza vaccines consist of the trivalent (inactivated or atten-
uated) virus, based on appropriate yearly strain prediction. In the case of the attenuated
live vaccines, the virus is cold adapted, e.g., attenuated by serial passage at low temperature,
resulting in a live virus that can not cause significant illness in human.
1
In addition to the
whole virus trivalent vaccine, subunit vaccines that are based on viral envelope proteins
(HA and NA) are also approved for human use. It has been shown that such vaccines are
highly immunogenic and well tolerated in children, young adults, and among the elderly.
2
The two vaccines (subunit and attenuated) were similarly efficacious in preventing culture-
positive influenza illness.
3
Some safety concerns related to influenza vaccination include
the eliciting of immune responses not induced by natural infection, that may lead to
autoimmunity.
4
However, these do not seem to be equivocal as commented by Haber et
al.,
5
and do not present serious issue.
Egg-grown inactivated influenza vaccine makes up the bulk of the human vaccines
currently used. This vaccine is generally well tolerated, but because egg is the medium for
viral culture, this vaccine is contra indicated in people with egg allergy. Although in most
influenza seasons the method of production works well, it still has inherent limitations,
including the long (78 months) production cycle
6
and the inability to grow specific
strains (such as the highly pathogenic H5 and H7) in eggs. Of particular concern is the
problematic mass preparation of such vaccine for potential pandemic or violent strains;
these limitations were confronted in the 2004 season when due to manufacturing problems,
nearly half of the influenza shots supply (4648 million doses) was wiped out and a sudden
shortage of vaccine was encountered. Some of these drawbacks were addressed by new
approaches for vaccination as described in the following.
An example for such a new approach, aiming at accelerating the response to pandemic
influenza is the use of plasmid-based reverse genetic systems to construct influenza virions
as vaccines. Viable viruses can be generated from individually cloned cDNA copies of each
of the eight viral RNA segments; reassortment can be prospectively defined and directed,
and the extra amino acids at the HA cleavage site (which are associated with high virulence)
can be removed to allow rapid generation of a vaccine seed strain in eggs. Plasmids encoding
www.landesbioscience.com Human Vaccines 95

the internal genes of the base vaccine are
already available.
7
The expected resulting
vaccines would include the outer
membrane proteins hemagglutinin
(HA) and neuraminidase (NA) from
strains that are expected to infect in the
following year, where the polypeptides
are purified from detergent-extracted,
inactivated virions. Furthermore, this
approach allows the inclusion of addi-
tional proteins such as the Matrix and
Nucleoprotein of influenza, that are not
normally targeted in the inactivated
virus vaccines, thus enabling the
immune system to elicit a specific
response against them as well. Targeting
these conserved proteins provides also
the theoretical advantage of increasing
the breadth of protection against
serotypically diverse strains.
DNA immunization strategies which
are based mainly on the influenza nucle-
oprotein (NP)
8
have several potential
advantages: One of them is their ability
to induce a broad-spectrum immuni-
ty,
9,10
this is in comparison to the highly
specific antigenicity of the HA and NA
glycoproteins based vaccines. The
advantage of this DNA vaccines
approach is that it can also be employed
to induce immunity against the haz-
ardous influenza strains such as H5N1
11
since their manufacturing process does
not involve culturing of the harmful virus. However, the genetic
vaccination approach has not progressed rapidly towards licensure,
due to several problems including concerns of potential DNA
integration to the chromosomes, which are not too serious, but
mainly because the lack of potency as manifested by the limited
duration and extent of immunity in humans and non-human
primates.
12
Alteration of various parameters including the vector,
the dose of DNA, inclusion of CpG-ODN motifs or fusion with
specific antigenic epitopes, are expected to improve the efficacy of
these DNA vaccines.
13
Another point that should be considered in regard to anti
influenza vaccines is its route of administration; the above vaccines
are given parenterally although the infection agent enters the host via
mucosal surfaces. Such a delivery route of a non-replicating antigen
induces relatively strong systemic response but only a limited local
mucosal immunity. In 2003, the CAIV-T (Flumist

) attenuated
vaccine was approved for human use; it is a trivalent live, cold-adapted
and temperature sensitive viruses, consisting of influenza A and B
vaccine strains. The exact strains are updated each year to antigenically
match the antigens recommended by national health authorities.
CAIV-T has a significant advantage in the convenience of adminis-
tration, intranasal spray which is preferable over a parenteral injection
by a syringe.
14
However, as regards its efficacy, a comparative study
of various commercial vaccines against influenza revealed that the
recommended live aerosol vaccines (CAIV-T) is less efficacious than
the recommended inactivated parenteral vaccines in healthy adults,
and amongst the elderly. In the latter, local IgA and systemic IgG
anti HA response are low and relatively short-lived following live
attenuated compared with inactivated vaccine. In general, the efficacy
of the current vaccines in reducing incidence of clinical influenza
and number of working days lost is limited. Among the aged popu-
lation it is even inferior and the level of protection is not sufficient.
15,16
A general limitation is due to the fact that current influenza virus
vaccines aim at inducing a strong antibody response to the viral
glycoproteins, particularly to the HA and NA, as such antibodies are
well known to be protective against infection. However, influenza
type A virus has a high tendency for changing the determinants
recognized by these protective antibodies, which necessitates repetitive
vaccination with updated vaccine strains that reflect these antigenic
changes. The available vaccines do not provide a satisfactory solution
of long-term immunity (most probably T-cell induced) or protection
against new emerging strains; they also fail to confer protection to
populations with a compromised immune system such as the elderly
and infants. In addition, the limited efficacy of current vaccines leads
to low levels of vaccination in the healthy population and eventually
the mortality attributable to influenza continues to increase.
17
Another major issue is that periodically, the human influenza
viruses are transmitted to other hosts, including mammals, raising a
concern that such a reassortment virus has the potential of a pandemic
strain that can cause infections and deaths. Since 1997, when the
avian H5N1 influenza virus was transmitted to humans and killed
six of 18 infected persons, there have been multiple transmissions of
avian influenza viruses to mammals leading to seven pandemic
events.
18,19
In most cases, the HA of human influenza strains differ
Towards an Epitope-Based Human Vaccine for Influenza
Figure 1. (A) Protection of mice from viral challenge following immunization with the mixture of flagella
expressing influenza epitopes. Young and old mice were immunized with a mixture of flagella expressing
the three murine epitopes, whereas in the human/mouse radiation chimera model, the four human epitopes
mixture (tetra vaccine) was employed. (I) Non-immunized challenged group infected with a sub-lethal
dose of H3N2 virus; () mice challenged with virus following immunization with recombinant flagella mixture.
In the human/mouse chimera, an additional control group comprised of mice that were not transplanted
with the human cells but were vaccinated with the tetra vaccine and subsequently challenged with the virus
(I) Virus titer was expressed as Log EID50 units. (B) Representative lung sections of young (left) and aged
(right) mice. Lungs sections of the aged mice shows a naturally occurring slight chronic inflammatory that
can be observed. The samples were fixed and stained with Hematoxylin, Eosin and Light green. Top: histology
of the lung of a nave mouse; middle: the lungs of non-immunized infected mice demonstrating a massive
inflammatory process. Bottom: the lung section of immunized mouse after challenge; it is comparable to the
normal lung with a limited lymphocytes cuffing.
A B
96 Human Vaccines 2005; Vol. 1 Issue 3

www.landesbioscience.com Human Vaccines 97
from the HA of avian influenza strains by its specificity to the receptor
that is expressed on the epithelial cells of the host, leading to restriction
of viral influenza strains to specific hosts. However, some avian
strains have receptor binding characteristics that are typical to human
and to avian strains, thereby increasing the potential for human
infection.
20
In view of these cumulative problematic issues, the need for alter-
natives is acute and hence, new approaches for influenza vaccines,
advanced delivery systems and adjuvants are being developed contin-
ually, aiming at improving the current vaccines and focusing on the
following aspects: (1) Avoid the hurdles associated with current
manufacturing procedures that are based on new strains every year.
(2) Safety consideration associated with mass production of live
vaccines against hazardous strains. (3) The route of immunization.
(4) The potential to confer protection against multiple strains of the
virus.
One of these approaches, which is based on conserved influenza
epitopes and applied in our laboratory, is detailed in the following.
EPITOPE-BASED VACCINES
The main benefit of immunization with an epitope-based vaccine
is the ability to immunize with a minimal structure, consisting of a
well-defined antigen which can be thoroughly characterized with
respect to its antigenicity and immunogenicity. Comprising the
adequate epitopes, such vaccine will stimulate an effective specific
immune response, while avoiding potential undesirable effects. For
example, antigenic regions that may activate suppressor mechanisms
or a response against self-antigens can be eliminated from the vaccine
preparation, thus providing a safer vaccine. However, the use of small
molecules such as peptides may create a problem of low immuno-
genicity, as compared to the multi-epitopes protein antigens, or an
entire pathogen, that is used for immunization in the conventional
vaccines. Hence, when applying the strategy of peptide-based vaccines,
it is important to take into consideration the specificity of antigen
processing, the MHC restriction of the T cell response and the
presence of both B cell and T cell epitopes.
Ideally, peptide-based vaccines should contain both B-cell epitopes
that are important for the induction of neutralizing antibodies and
T-cell epitopes that will serve to induce a protective T helper and a
CTL response. The epitopes should be specific for the pathogen and
not induce cross-reactions with self-antigens. They should also provide
a long-lasting immunity without resort to frequent booster immuniza-
tions. Since the immunogenicity of peptides as such is low, they
should be adequately presented and usually formulated within an
appropriate carrier or adjuvant. The first generation of peptide vaccines
that had been evaluated, included various B- and T-cell epitopes,
sometimes in clusters, synthesized either in tandem repeats or as
multi-antigen peptides (MAPs).
21,22
For example, recently such a
MAP malaria vaccine containing minimal Plasmodium falciparum
circumsporozoite protein repeat epitopes was assessed for safety and
immunogenicity in volunteers. This is the first demonstration in
humans that a peptide vaccine containing minimal T and B cell
epitopes can elicit antibody titers comparable to multiple exposures
to the whole pathogen.
23
Immunogenicity of peptides can also be augmented by the use of
macromolecular carriers to which the desired epitope is either
complexed or covalently attached.
24,25
Alternatively, the carriers could
comprise a recombinant fusion product, expressing the desired epitope
as a foreign one.
26,27
Employing this latter approach, a synthetic
oligonucleotide coding for the particular epitope is inserted into a
plasmid containing a gene of a viral or bacterial protein, thus leading
to a recombinant organism expressing this epitope. This recombinant
agent can then serve as an intact live vaccine, or be used for the
isolation of the recombinant protein which would, in turn, serve as
a synthetic recombinant vaccine. This approach has been employed
in our lab in the study of influenza vaccine, as described in the fol-
lowing. Further improvement of influenza peptide vaccine can be
achieved by local mucosal delivery of the immunogen, as well as by
employing adequate adjuvants for augmenting the immune
response. A number of novel adjuvants have been described during
the past few years, including bacterial toxins such as cholera toxin,
28
cytokines,
29
bacterial wall components
30
and others.
31
In regard to the choice of the epitopes, indicated above, one of
the major antigens of the influenza virus is the envelope glycoprotein
haemagglutinin (HA). It plays a key role in initiating viral infection
by binding to sialic acid-containing receptors on host cells and thus
mediates the subsequent viral entry to the cell and membrane
fusion.
32,33
Protection against influenza requires adequate titer of
antibodies against this protein; hence, influenza vaccines efficacy is
routinely evaluated by their ability to induce anti HA antibodies.
The receptor-binding site within the HA (HA 116261)
34
is located
in close proximity to one of the major conserved antigenic epitopes
(HA91108) of all H3 strains.
35
In earlier studies conducted in our
laboratory we have investigated this epitope; when conjugated to
Tetanus toxoid and either emulsified in Freunds adjuvant or conju-
gated to muramyl-dipeptide, it was used for parenteral immunization
of rabbits. The anti peptide antibodies in their sera were able to
inhibit the haemagglutination capacity and also neutralize the virus,
as manifested by inhibition of plaque formation in tissue culture in
vitro. Moreover, immunization of mice with this conjugate, elicited
an in-vivo anti-viral immune response against challenge infection, as
observed by the reduced viral load in their lungs.
36,37
In a comple-
mentary study, this peptide, that represents a B cell epitope, was
either expressed within a carrier protein or anchored to an outer
membrane protein proteosome vesicle and used for local (nasal)
immunization of mice. When administered without the aid of an
adjuvant, it led to a partial in vivo protection. The elevated titer of
lungs IgA specific to the peptide and to the intact virus is indicative
of a successful induction of local immunity.
38,39
In later studies, additional epitopes were included in the vaccine
in an effort to improve the level of protection that had been achieved
by immunization with the single B cell epitope HA 91108. For this
purpose, two T cell epitopes were examined: the T helper epitope
(NP 5569)
40
and a CTL epitope (NP 147158).
41
Both epitopes
are derived from the inner nucleoprotein (NP). The internal viral
proteins are known to be conserved and do not undergo frequent
antigenic variations that characterize the envelope glycoproteins.
After viral entry to the epithelial layer, they are processed, cleaved
and exposed to the immune system when they are presented by the
appropriate MHC molecule. As such, they activate cellular immune
responses as manifested by cytokines secretion, lymphocytes prolif-
eration and CTL killing. Since small peptides are poor immunogens
and in view of our previous studies with the B cell epitope HA
91108, the T cell epitopes were also anchored to proteosomes.
Intranasal administration of the B cell epitope together with the T
cell epitopes significantly improved its efficacy in clearing the virus
from the lungs of the infected mice. This is an indication for the
contribution of the cellular arm of the immune response to the overall
protective effect induced by this epitope-based immunogen. In all
Towards an Epitope-Based Human Vaccine for Influenza

those experiments, the peptides as such, that were included as
controls, induced only limited immune responses and did not lead
to protection against infection.
FLAGELLA AS A CARRIER FOR INFLUENZA EPITOPES
In an effort to further augment the immunogenic response to the
influenza epitopes, they were expressed, individually, in a macromol-
ecular carrier, the flagellin of Salmonella vaccine strain. As detailed
below, the choice of flagella is based on three considerations: (1) The
early finding that using attenuated Salmonella as a carrier induced
an immune response against several foreign antigens, including a
peptide epitope of cholera toxin.
42
This was subsequently confirmed
by us for the B cell epitope of influenza.
38
(2) The unique properties
of flagella as polymers of the protein flagellin, to express multiple
copies of any foreign epitope expressed in a recombinant construct.
(3) The capacity of flagellin to interact with Toll-like receptor
(TLR5), allowing it to enhance the immune response to any foreign
epitope that it expresses.
The immune system includes two components: the innate response,
which is a rather non-specific, first line of defense against infections,
and the adaptive immune response, which represents specific resistance
to a wide variety of antigens, which is weak at first, but develops in
the long term into strong memory responses.
43
New vaccination
strategies rely on the incorporation of effective adjuvants, which
stimulate the innate immune response and, in turn, activate the
adaptive immune response. Dendritic cells (DCs) can process and
present antigens, resulting in the activation of T- and B-cell responses
and the establishment of protective immunity.
Toll like receptors (TLRs) are expressed in immature and mature
DCs and monocytes. These are the molecules that link pathogen
recognition and the induction of adaptive immunity against it.
Upon binding to their specific ligand, a signaling cascade in the anti-
gen presenting cells (APCs) is initiated and eventually leads to the
activation of NF-B and mitogen-activated protein kinases. This
triggers the maturation and activation of APCs, which include
upregulation of the major histocompatibility complex (MHC) and
costimulatory molecules and secretion of proinflammatory cytokines
and chemokines, as well as enhancing their ability to prime naive T
cells. It is well established that in the bacterial flagellum, its specific
antigen flagellin binds to host tissues and activates host inflammatory
signaling pathways via TLR5.
44
It has been reported that flagellin
acts as an effective adjuvant capable of enhancing T-cell responses in
vivo.
45
Moreover, recombinant Salmonella, expressing foreign epitopes
in its flagellin and used as live vaccines, induced specific T-cell
responses against these epitopes,
37,33,38,39
suggesting that flagellin is
indeed a suitable carrier for the development of vaccines that might
induce strong and specific immune responses against foreign antigens.
In our lab, we employed the recombinant synthetic approach, in
which the advantages of the flagellin as a carrier and adjuvant were
combined and directed towards the influenza virus by inserting viral
epitopes into the flagellum structure. As mentioned above, in this
system, oligonucleotides coding for specific peptide epitopes of anti-
gens of unrelated pathogens, are inserted at the unique EcoRV site
of pLS408 that specifies chimeric flagellins. In many instances, this
leads to the production of functional flagella when the plasmid is
placed in a flagellin-deficient delta aroA live-vaccine strain of
Salmonella dublin. The foreign epitope is then exposed at the surface
of the flagellar filaments, as shown by the immobilizing effect of
anti-epitope antibody and by immunogold electron microscopy.
This is a general methodology that can be applied for various anti-
gens; however, if the antigen incorporated into the flagellin is too
large, it interferes with the folding, transport, or assembly of the
flagella.
46
Consequently, this approach is limited to presentation of
small antigens such as peptides, and hence, the flagella provide an
ideal vehicle for the epitope-based vaccines.
Many epitopes from various pathogens including HIV,
47
Hepatitis B virus,
48
E. coli,
42
Streptococcus pyogenes
46
and Schistosoma
mansoni
49
were already expressed within this system and encouraging
results were obtained as deduced from the ability of these constructs
(either the whole bacteria or the purified, cleaved flagella) to generate
specific immune responses as well as protection against the respective
disease in an animal model. In the following, there is a detailed
description of our results with recombinant flagella expressing
influenza epitopes aiming at the design of a vaccine for human use.
EFFICACY OF RECOMBINANT FLAGELLA VACCINE
IN YOUNG AND AGED MICE
For the purpose of designing a vaccine against influenza that will
be efficient against various strains of the virus, we focused on three
conserved epitopes, a B cell epitope as well as two T cell epitopes;
they were expressed individually within the flagella system that was
described above. The B cell epitope, as mentioned above, comprises
the 18-residue peptide corresponding to the sequence HA91108,
which is common to all H3 strains,
35
and is derived from an inner,
conserved region of the protein. In earlier studies, this epitope by
itself, either conjugated to a macromolecular carrier
36
or expressed in
flagella
50
induced partial protection against infection. The amino
acids sequence of the T helper epitope that is derived from the viral
Nucleoprotein (residues 5569) is shared by the human infective
strains H1N1, H2N2, H3N2 and also by swine (H9N2) specific
viruses. The CTL epitope also resides in the conserved nucleoprotein,
between amino acid residues 147 and 158. Many influenza strains,
including H1N1, H3N2, H3N3 as well as the avian strains H5N1,
H7N3, H9N2 and others also share this sequence. As determined by
sequence query in http://www.ncbi.nlm.nih.gov/btast/.
The resultant peptide-based recombinant vaccine, comprising a
mixture of recombinant flagella, each expressing individually one
epitope, provoked efficient cross-strain protection and long-term
immunity against influenza infection in young mice.
51
It is worthy
of mention that if the B cell epitope was omitted, the combination
of the two T cell epitopes by themselves provided only partial
protection,
50
emphasizing the contribution of the humoral response
to the protection of mice. The most successful results were obtained
with the mixture of all three epiotopes, administered by intranasal
immunization of young (three months old) mice with the recombi-
nant flagella without the aid of external adjuvant. Furthermore, it
induced both humoral and cellular responses and showed protective
efficacy against viral infection by several strains of influenza, indicating
that the protection was mediated both by humoral and cellular
specific immune responses.
50
Although influenza infection in the general population is usually
associated with minor symptoms and is causing primarily an economic
burden, it can also lead to mortality in immuno-compromised
populations, which is relatively high among the elderly and among
patients with chronic diseases (cardiac, pulmonary or renal). These
populations have increased risk of hospitalization.
52
In this population
there is a decline in the activity of the immune system, the production
of naive lymphocytes by the central lymphoid organs, the thymus
Towards an Epitope-Based Human Vaccine for Influenza
98 Human Vaccines 2005; Vol. 1 Issue 3

www.landesbioscience.com Human Vaccines 99
and bone marrow, is reduced and these individuals are more suscep-
tible to various diseases and infections.
53
In addition, this group is
under particular risk of rapid spread of infection in retirement homes
where a large numbers of aged people are living in close proximity.
Considerable efforts have been invested in the development of a
vaccine that will be effective in the aged population. In our lab, the
efficacy of the above synthetic recombinant epitope-based vaccine
expressed in flagella as a carrier, was studied in aged mice. Thus, the
same mixture of flagella expressing conserved B-cell and T-cell epitopes
of influenza was evaluated for its efficacy in old mice as compared to
the young ones. In this study, C57/Bl/6j and BALB/c mice, three
months and 24 months old were immunized intranasally three times
with the recombinant flagella mixture and then infected with a
sub-lethal dose of H3N2 influenza virus (strain A/Texas/1/77). A
significant reduction (>99%) in viral load in their lungs, that was
comparable to the decrease of virus titer in the young mice, was
demonstrated in the aged mice. This was further supported by the
histology of lungs sections removed from the mice; the overall
appearance of the lungs from the vaccinated group after infection,
was comparable to the lungs of naive mice controls. In the mice that
were infected but not vaccinateda massive infection and damaged
tissues were observed,
54
as illustrated in Figure 1.
TOWARDS A HUMAN INFLUENZA VACCINE
This approach for vaccination against influenza, namely the
expression of immunogenic peptides from conserved regions of the
virus within the immunogenic flagella was further investigated, with
the purpose of constructing a vaccine for human use. As manifested
by the studies with the murine vaccine, the cellular responses con-
tribute to the efficacy of the vaccine; hence, a primary step in adapting
this approach for humans was to define the appropriate T cell epitopes
for the vaccine. The protein processing and presentation of epitopes
on cell surface by MHC molecules is the basis for induction of
specific cellular responses. Different peptides are defined as human
or murine epitopes due to the different processing of antigens by the
mouse and by the human cells and also due to the distinct specificities
of the MHC class I in both species.
55
In addition, since the cellular
immune response in humans is restricted to specific HLAs, any epi-
tope-based vaccine must take into consideration the need to induce
response in a broad range population. This can be overcome by the
use of vaccines comprising several peptides, which would be effective
in different populations, as well as by selection of those epitopes
restricted to the HLAs which are most frequent in the population of
interest. Furthermore, this approach allows the induction of all arms
of the immune response. It is also possible to design a multi-epitope
vaccine that will protect against more than one infecting pathogen
and/or several viral strains in one vaccine preparation.
In the case of our influenza vaccine study, the previously
described B-cell epitope from the HA surface antigen (HA 91108)
was included also in the human vaccine, but the T-cell epitopes that
are MHC restricted were replaced with epitopes that are restricted to
the most prevalent human HLAs. An additional consideration, as in
the case of the murine vaccine, was the selection of epitopes that are
conserved among the different strains of influenza virus in order to
allow a cross strain protection effect. According to these criteria,
three T cell epitopes were selected: one is a universal T helper epitope
HA307319 that binds a wide range of class II HLA molecules
including DR1, DR2, DR4, DR5, DR7, DR9 and others.
56-58
The
other two epitopes, NP335350 and NP380393, were from the
inner protein of the virus, the nucleoprotein, that is conserved among
various strains and is also known as an inducer of the cellular immune
response.
59
The sequence of the T-cell epitopes, their HLA specificity
and a list of the strains that encompass them are presented in Table 1.
The designed vaccine comprised a mixture of the recombinant
flagella constructs expressing individually the above four epitopes,
and was denoted tetra vaccine.
For the evaluation of this candidate influenza vaccine, a model of
human/mouse radiation chimera was employed. The observation of
Mosier et al.
60
that human PBMC can be adoptively transferred
intraperitonealy into the SCID mouse and that the engrafted cells
survive for an extended period of time producing high levels of
human Ig, has offered many new possibilities in clinical immunology
research. An alternative approach to generate human/mouse
chimeras in lethally irradiated mice radio protected with SCID bone
marrow was described by Lubin et al.
61
In this model, a marked
human humoral
62
as well as cellular (CTL) responses
63
could be
generated by immunization with either foreign antigens, or with
allogeneic cells.
61,64
This latter model was employed in our studies.
Human/mouse chimera were immunized intranasally with the
tetra vaccine described above, expressing the influenza epitopes
HA9118, HA307319, NP335350 and NP380393. Following
a single administration, the mice were challenged with a sub-lethal
dose of influenza virus with different HA and NA specificity, namely:
H1N1, H2N2 and H3N2. Human Ig specific to the virus were
detected in the serum and in the lungs of the chimera mice following
immunization and challenge, indicating of successful activation of the
transplanted cells. No antibodies were detected in the non-transplanted
immunized mice. Lung virus titers were measured by injecting serial
Towards an Epitope-Based Human Vaccine for Influenza
Table 1 Characteristics of the T-cell epitopes included in the tetra vaccine
Epitope Sequence HLA restriction Influenza strain Reference
(for HLA restriction)
NP 335-350 SAAFEDLRVLSFIRG A2, A3, Aw68, B37 DQw5 H1N1, H2N2, H3N2, H5N2, 66-69
H5N1, H6N1, H9N2,
NP 380-393 ELRSRYWAIRTRSG B8, B27 H1N1, H1N2, H2N2, H5N1, 69, 70
H5N2, H6N1, H6N2, H6N9,
H7N7, H9N2, H11N1, H14N5
HA 307-319 PKYVKQNTLKLAT DR1, DR2, DR4, DR5, DR7, H1N1, H3N1, H3N2, H3N8, 56-58, 71-73
DR9, DR51, DR52A DQ6, Dw1,
Dw13, Dw14, Dw15,
The list of influenza strains was determined according to sequence query in http://www.ncbi.nlm.nih.gov/blast/.

dilutions of the lungs homogenates of the individual mice to embry-
onated hen eggs and determination of the haemagglutenation activ-
ity. Regarding the level of infection, the results demonstrated that in
the lungs of the immunized chimera a significant clearance of the
virus (ca 100 fold reduction) had occurred, as shown in Figure 1. This
was shown after challenge with different influenza strains.
Furthermore, in a subsequent experiment, the immunization with
the tetra vaccine protected the mice from a lethal dose of the virus:
20 days after the lethal dose infection with A/Texas/1/77 (H3N2) all
the vaccinated chimeric mice survived, while none of the mice in the
control group that were vaccinated but not transplanted with the
human PBMC survived. In an additional control group, human
PBMC-transplanted mice that were not immunized, all the mice lost
weight following challenge infection and half of them died. This
partial protection is probably due to existing memory immunity
against the virus in the donors cells. The transplanted mice that were
immunized with the recombinant flagella also lost weight as a result
of the infection, but they recovered rapidly and as mentioned, 100%
survival was monitored in this group.
65
These results emphasize the
high efficacy of the tetra vaccine in the humanized mice and its
potential for the development of a vaccine for human use. A start-up
company has been established aiming at the development of such
vaccine and its evaluation in clinical trials.
CONCLUDING REMARKS
As emerges from this review article, effective intervention means
for influenza prevention are available and approved for healthy adults
and children, where the level of protection afforded by the different
current influenza vaccines varies from moderate to high, with no
serious adverse events. However, the efficacy demonstrated in the
various clinical trials with these vaccines depends on the degree of
match between the vaccinating and infecting strain of the virus.
Hence, development of new influenza vaccines is needed in order to
overcome the limitations. The main emphasis in this paper is on our
own study that focuses on the design of an epitope-based vaccine
against influenza.
As delineated, a vaccine comprising a B cell epitope as well as
three T cell epitopes recognized by the most prevalent HLA molecules
in the population was evaluated using the human/mouse chimera
animal model. All four epitopes stem from highly conserved regions
of the hemagglutinin and nucleoprotein, and, they were expressed
individually in a novel vehicleflagellawhich serves as both carrier
and adjuvant. The resulting tetra vaccine comprising a mixture of
the four flagella expressing these epitopes was administered
intranasally to the humanized mice. The successful results
obtained indicate that this route of administration is appropriate for
the stimulation of local immune response in the nasal cavity and
lungs, which are the primary site of influenza infection.
Furthermore, the concomitant induction of effective local immune
response in the lungs is demonstrated by the presence of specific,
broad-spectrum anti-influenza antibodies in the lung homogenates,
reactive with different influenza strains that encompass various
hemagglutinin and neuraminidase glycoproteins. Most significantly,
the efficacy of this vaccine was further evidenced by the high level of
protection against challenge infection with several influenza strains,
as well as against lethal dose challenge. These results pave the way for
the development of a multi-valent universal influenza vaccine.
References
1. Jin H, Lu B, Zhou H, Ma C, Zhao J, Yang CF, Kemble G, Greenberg H. Multiple amino
acid residues confer temperature sensitivity to human influenza virus vaccine strains
(FluMist) derived from cold-adapted A/Ann Arbor/6/60. Virology 2003; 306:18-24.
2. Mischler R, Metcalfe I. Inflexal a trivalent virosome subunit influenza vaccine: Production.
Vaccine 2002; 20:B17-23.
3. Beyer W, Palache A, De Jong J, Osterhaus A. Cold-adapted live influenza vaccine versus
inactivated vaccine: Systemic vaccine reactions, local and systemic antibody response, and
vaccine efficacy. A meta-analysis. Vaccine 2002; 20:1340-53.
4. Geiger M, Geiger D, Zahalsky A. Influenza vaccination and Guillain Barre syndrome.
Clinical Immunology 2003; 107:116-21.
5. Haber P, DeStefano F. Letter to the editor. 2003; 109:359.
6. Wood J. Developing vaccines against pandemic influenza. Philos Trans R Soc Lond B Biol
Sci 2001; 356:1953-60.
7. Webby R, Webster R. Are we ready for pandemic influenza? Science 2003; 302:1519-1522.
8. Fu TM, Guan L, Friedman A, Schofield TL, Ulmer JB, Liu MA, Donnelly JJ. Dose depen-
dence of CTL precursor frequency induced by a DNA vaccine and correlation with pro-
tective immunity against influenza virus challenge. J Immunol 1999; 162:4163-70.
9. Ulmer JB, Donnelly JJ, Parker SE, Rhodes GH, Felgner PL, Dwarki VJ, Gromkowski SH,
Deck RR, DeWitt CM, Friedman A, et al. Heterologous protection against influenza by
injection of DNA encoding a viral protein. Science 1993; 259:1745-9.
10. Michell J, Green T, Bright R, Ross T. Induction of heterosubtypic immunity to influenza
A virus using a DNA vaccine expressing hemagglutinin-C3d fusion proteins. Vaccine 2003;
21:902-14.
11. Epstein SL, Tumpey TM, Misplon JA, Lo CY, Cooper LA, Subbarao K, Renshaw M,
Sambhara S, Katz JM. DNA vaccine expressing conserved influenza virus proteins protec-
tive against H5N1 challenge infection in mice. Emerg Infect Dis 2002; 8:796-801.
12. Kem G, Greenberg H. Novel generations of influenza vaccines. Vaccine 2003; 21:1789-95.
13. Bardiya N, Bae J. Influenza vaccines: Recent advances in production technologies. Appl
Microbiol Biotechnol 2005; 67:299-305.
14. Belshe R. Current status of live attenuated influenza virus vaccine in the US. Virus Res
2004; 103:177-85.
15. Demicheli V, Rivetti D, Deeks J, Jefferson T. Vaccines for preventing influenza in healthy
adults. Cochrane Database Syst Rev 2004; 3:CD001269.
16. Liddle B, Jennings R. Influenza vaccination in old age. Age Ageing 2001; 30:385-389.
17. Glezen W. Control of influenza. Tex Heart Inst J 2004; 31:39-41.
18. Stephenson I, Nicholson K, Wood J, Zambon M, Katz J. Confronting the avian influenza
threat: Vaccine development for a potential pandemic. Lancet Infect Dis 2004; 4:499-509.
19. Subbarao K, Klimov A, Katz J, Regnery H, Lim W, Hall H, Perdue M, Swayne D, Bender
C, Huang J, Hemphill M, Rowe T, Shaw M, Xu X, Fukuda K, Cox N. Characterization of
an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness.
Science 1998; 279:393-6.
20. Saito T, Lim W, Suzuki T, Suzuki Y, Kida H, Nishimura SI, Tashiro M. Characterization of
a human H9N2 influenza virus isolated in Hong Kong. Vaccine 2001; 20:125-33.
21. Olive C, Toth I, Jackson D. Technological advances in antigen delivery and synthetic pep-
tide vaccine developmental strategies. Mini Rev Med Chem 2001; 1:429-38.
22. Jackson D, Purcell A, Fitzmaurice C, Zeng W, Hart D. The central role played by peptides
in the immune response and the design of peptide-based vaccines against infectious diseases
and cancer. Curr Drug Targets 2002; 3:175-96.
23. Nardin EH, Oliveira GA, Calvo-Calle JM, Castro ZR, Nussenzweig RS, Schmeckpeper B,
Hall BF, Diggs C, Bodison S, Edelman R. Synthetic malaria peptide vaccine elicits high lev-
els of antibodies in vaccinees of defined HLA genotypes. J Infect Dis 2000; 182:1486-96.
24. Sciutto E, Fragoso G, Manoutcharian K, Gevorkian G, Rosas-Salgado G, Hernandez-
Gonzalez M, Herrera-Estrella L, Cabrera-Ponce J, Lopez-Casillas F, Gonzalez-Bonilla C,
Santiago-Machuca A, Ruiz-Perez F, Sanchez J, Goldbaum F, Aluja A, Larralde C. New
approaches to improve a peptide vaccine against porcine Taenia solium cysticercosis. Arch
Med Res 2002; 33:371-8.
25. Jacob C, Grossfeld S, Sela M, Arnon R. Priming immune response to cholera toxin induced
by synthetic peptides. Eur J Immunol 1986; 16:1057-62.
26. Gedvilaite A, Frommel C, Sasnauskas K, Micheel B, Ozel M, Behrsing O, Staniulis J,
Jandrig B, Scherneck S, Ulrich R. Formation of immunogenic virus-like particles by insert-
ing epitopes into surface-exposed regions of hamster polyomavirus major capsid protein.
Virology 2000; 273:21-35.
27. Liu Z, Xiao Y, Chen Y. Epitope-vaccine strategy against HIV-1: Today and tomorrow.
Immunobiology 2003; 208:423-8.
28. Lee S, Halperin S, Salloum D, MacMillan A, Morris A. Mucosal immunization with a
genetically engineered pertussis toxin S1 fragment-cholera toxin subunit b chimeric pro-
tein. Infection and Immunity 2003; 71:2272-5.
29. Murtaugh M, Foss D. Inflammatory cytokines and antigen presenting cell activation. Vet
Immunol Immunopathol 2002; 87:109-21.
30. Mittenbuhler K, vd Esche U, Heinevetter L, Bessler W, Huber M. Lipopeptides:
Adjuvanticity in conventional and genetic immunization. FEMS Immunol Med Microbiol
2003; 37:193-200.
31. Arnon R, Ben-Yedidia T. Old and new vaccine approaches. Int Immunopharmacol 2003;
3:1195-204.
Towards an Epitope-Based Human Vaccine for Influenza
100 Human Vaccines 2005; Vol. 1 Issue 3

www.landesbioscience.com Human Vaccines 101
32. Skehel J, Wiley D. Receptor binding and membrane fusion in virus entry: The influnza
hemagglutinin. Ann Rev Biochem 2000; 69:531-69.
33. Eckert D, Kim P. Mechanisms of viral membrane fusion and its inhibition. Annu Rev
Biochem 2001; 70:777-810.
34. Wilson I, Skehel J, Wiley D. Structure of the haemagglutinin membrane glycoprotein of
influenza virus at 3 A resolution. Nature 1981; 289:366-73.
35. Laver W, Air G, Dopheide T, Ward C. Amino acid sequence changes in the haemagglutinin
of A/Hong Kong (H3N2) influenza virus during the period 196877. Nature 1980;
283:454-7.
36. Muller GM, Shapira M, Arnon R. Anti-influenza response achieved by immunization with
a synthetic conjugate. Proc Natl Acad Sci USA 1982; 79:569-73.
37. Shapira M, Jolivet M, Arnon R. A synthetic vaccine against influenza with built-in adju-
vanticity. Int J Immunopharmacol 1985; 7:719-23.
38. McEwen J, Levi R, Horwitz R, Arnon R. Synthetic recombinant vaccine expressing
influenza haemagglutinin epitope in Salmonella flagellin leads to partial protection in mice.
Vaccine 1992; 10:405-11.
39. Levi R, Aboud-Pirak E, Leclerc C, Lowell G, Arnon R. Intranasal immunization of mice
against influenza with synthetic peptides anchored to proteosomes. Vaccine 1995;
13:1353-9.
40. Gao X, Liew F, Tite J. Identification and characterization of T helper epitopes in the nucle-
oprotein of influenza A virus. J Immunol 1989; 143:3007-14.
41. Taylor P, Davey J, Howland K, Rothbard J, Askonas B. Class I MHC molecules rather than
other mouse genes dictate influenza epitope recognition by cytotoxic T cells.
Immunogenetics 1987; 26:267-72.
42. Newton S, Jacob C, Stocker B. Immune response to cholera toxin epitope inserted in
Salmonella flagellin. Science 1989; 244:70-2.
43. Heine H, Lien E. Toll-like receptors and their function in innate and adaptive immunity.
Int Arch Allergy Immunol 2003; 130:180-92.
44. Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol 2003; 21:335-76.
45. Eaves-Pyles T, Murthy K, Liaudet L, Virag L, Ross G, Soriano FG, Szabo C, Salzman AL.
Flagellin, a novel mediator of Salmonella-induced epithelial activation and systemic inflam-
mation: I kappa B alpha degradation, induction of nitric oxide synthase, induction of
proinflammatory mediators, and cardiovascular dysfunction. J Immunol 2001; 166:1248-60.
46. Stocker B, Newton S. Immune responses to epitopes inserted in Salmonella flagellin. Int
Rev Immunol 1994; 11:167-78.
47. Newton S, Joys T, Anderson S, Kennedy R, Hovi M, Stocker B. Expression and immuno-
genicity of an 18-residue epitope of HIV1 gp41 inserted in the flagellar protein of a
Salmonella live vaccine. Res Microbiol 1995; 146:203-16.
48. Wu J, Newton S, Judd A, Stocker B, Robinson W. Expression of immunogenic epitopes of
hepatitis B surface antigen with hybrid flagellin proteins by a vaccine strain of Salmonella.
Proc Natl Acad Sci USA 1989; 86:4726-30.
49. Ben-Yedidia T, Tarrab-Hazdai R, Schechtman D, Arnon R. Intranasal administration of
synthetic recombinant peptide-based vaccine protects mice from infection by Schistosoma
mansoni. Infect Immun 1999; 67:4360-6.
50. Levi R, Arnon R. Synthetic recombinant influenza vaccine induces efficient long-term
immunity and cross-strain protection. Vaccine 1996; 14:85-92.
51. Arnon R, Levi R. Synthetic recombinant vaccine induce anti-influenza long-term immu-
nity and cross-strain protection. New York: Plenum Press, 1996.
52. Bouree P. Immunity and immunization in elderly. Pathol Biol (Paris) 2003; 51:581-5.
53. Meyer K. The role of immunity in susceptibility to respiratory infection in the aging lung.
Respir Physiol 2001; 128:23-31.
54. Ben-Yedidia T, Abel L, Arnon R, Globerson A. Efficacy of anti-influenza peptide vaccine
in aged mice. Mech Ageing Dev 1998; 104:11-23.
55. Braud V, McMichael A, Cerundolo V. Differential processing of influenza nucleoprotein in
human and mouse cells. Eur J Immunol 1998; 28:625-35.
56. Ikeda K, Katagiri M. Analysis of binding capacities between HLA class II molecules and
synthetic peptides HA307-319. Hokkaido Igaku Zasshi 1993; 68:318-24.
57. OSullivan D, Sidney J, Del Guercio M, Colon S, Sette A. Truncation analysis of several
DR binding epitopes. J Immunol 1991; 146:1240-6.
58. Rothbard J, Busch R, Bal V, Trowsdale J, Lechler R, Lamb J. Reversal of HLA restriction
by a point mutation in an antigenic peptide. Int Immunol 1989; 1:487-95.
59. Gotch F, Rothbard J, Howland K, Townsend A, McMichael A. Cytotoxic T lymphocytes
recognize a fragment of influenza virus matrix protein in association with HLA-A2. Nature
1987; 326:881-2.
60. Mosier D. Adoptive transfer of human lymphoid cells to severely immunodeficient mice:
Models for normal human immune function, autoimmunity, lymphomagenesis, and
AIDS. Adv Immunol 1991; 50:303-25.
61. Lubin I, Segall H, Marcus H, David M, Kulova L, Steinitz M, Erlich P, Gan J, Reisner Y.
Engraftment of human peripheral blood lymphocytes in normal strains of mice. Blood
1994; 83:2368-81.
62. Marcus H, David M, Canaan A, Kulova L, Lubin I, Segall H, Denes L, Erlich P, Galun E,
Gan J, et al. Human/mouse radiation chimera are capable of mounting a human primary
humoral response. Blood 1995; 86:398-406.
63. Segall H, Lubin I, Marcus H, Canaan A, Reisner Y. Generation of primary antigen-specific
human cytotoxic T lymphocytes in human/mouse radiation chimera. Blood 1996; 88:721-30.
64. Bocher WO, Marcus H, Shakarchy R, Dekel B, Shouval D, Galun E, Reisner Y. Antigen-
specific B and T cells in human/mouse radiation chimera following immunization in vivo.
Immunology 1999; 96:634-41.
65. Ben-Yedidia T, Marcus H, Reisner Y, Arnon R. Intranasal administration of peptide vac-
cine protects human/mouse radiation chimera from influenza infection. Int Immunol
1999; 11:1043-51.
66. Carreno B, Anderson R, Coligan J, Biddison W. HLA-B37 and HLA-A2.1 molecules bind
largely nonoverlapping sets of peptides. Proc Natl Acad Sci USA 1990; 87:3420-4.
67. Carreno B, Koenig S, Coligan J, Biddison W. The peptide binding specificity of HLA class
I molecules is largely allele-specific and nonoverlapping. Mol Immunol 1992; 29:1131-40.
68. Benjamin R, Madrigal J, Parham P. Peptide binding to empty HLA-B27 molecules of
viable human cells. Nature 1991; 351:74-77.
69. Carreno B, Turner R, Biddison W, Coligan J. Overlapping epitopes that are recognized by
CD8+ HLA class I-restricted and CD4+ class II-restricted cytotoxic T lymphocytes are con-
tained within an influenza nucleoprotein peptide. J Immunol 1992; 148:894-9.
70. Suhrbier A, Schmidt C, Fernan A. Prediction of an HLA B8-restricted influenza epitope by
motif. Immunology 1993; 79:171-3.
71. O'Sullivan D, Arrhenius T, Sidney J, Del Guercio MF, Albertson M, Wall M, Oseroff C,
Southwood S, Colon SM, Gaeta FC, et al. On the interaction of promiscuous antigenic
peptides with different DR alleles. Identification of common structural motifs. J Immunol
1991; 147:2663-9.
72. Barber LD, Bal V, Lamb JR, O'Hehir RE, Yendle J, Hancock RJ, Lechler RI. Contribution
of T-cell receptor-contacting and peptide-binding residues of the class II molecule HLA-
DR4 Dw10 to serologic and antigen-specific T-cell recognition. Hum Immunol 1991;
32:110-8.
73. Yamamoto K, Fukui Y, Esaki Y, Inamitsu T, Sudo T, Yamane K, Kamikawaji N, Kimura A,
Sasazuki T. Functional interaction between human histocompatibility leukocyte antigen
(HLA) class II and mouse CD4 molecule in antigen recognition by T cells in HLA-DR and
DQ transgenic mice. J Exp Med 1994; 180:165-71.
Towards an Epitope-Based Human Vaccine for Influenza