Immunology Letters 64 (1998) 9 – 15
Effect of pre-existing carrier immunity on the efficacy of synthetic
influenza vaccine
T. Ben-Yedidia *, R. Arnon
Department of Immunology, The Weizmann Institute of Science, Reho6ot 76100, Israel
Accepted 27 July 1998
Abstract
In our previous studies on the development of synthetic peptide-based vaccines, we have evaluated flagellin from a Salmonella
typhi vaccine strain as a carrier molecule for synthetic peptides derived from the influenza virus. The results indicated that the use
of recombinant flagella, expressing defined influenza epitopes, is adequate for induction of protection against a challenge infection.
It is of importance to show that previous exposure to the carrier does not induce suppression of the response to such vaccine. In
the present study we demonstrate that the protective effect is not impaired by pre-immunization with either the carrier flagellin
molecule alone or with the intact salmonella. The immune response was manifested both by the level of antibodies produced and
by a proliferative cellular response, as well as by an efficient protection of the mice from a sub-lethal challenge infection. Since
prior exposure to the carrier did not result in immune suppression, we conclude that Salmonella flagellin is a suitable carrier for
synthetic peptide based vaccines. © 1998 Elsevier Science B.V. All rights reserved.
Keywords: Carrier; Epitope; Influenza; Suppression; Vaccine
1. Introduction
In the design of peptide-based vaccines, one of the
major considerations is to overcome their low immunogenicity. The common approaches to this issue are the
coupling of the peptides to an immunogenic carrier
molecule, such conjugate eliciting an effective anti-peptide immune response and the use of an appropriate
adjuvant. The critical issue concerning adjuvants is the
hurdle of their approval for human use. On the other
hand, a problem that can be associated with the use of
carriers is the carrier suppression effect reported in
several instances [1– 5]. In these cases, subjects that had
been previously vaccinated or exposed to the carrier
molecule failed to react with the target epitope which
was presented on the same carrier molecule. This has
been observed when a malaria peptide bound to
* Corresponding author. Tel.: +972 8934 2471; fax: +972 8946
9712; e-mail: [email protected]
Tetanus toxoid was injected in mice that had already
been vaccinated with Tetanus toxoid alone [6]. Similar
carrier suppressive effect has been observed in other
systems [7–9]. It should be noted, however, that other
studies, similarly designed, report on an enhanced response towards the conjugated peptide after prior immunization with the carrier [3,10–12]. The mechanism
leading to the suppression is a combination of suppressor T-cells, that inhibit only the antibody response to
the hapten together with a deficiency at the B-cells level
[13].
In the present study, we investigated whether flagellin
as a carrier shows any carrier suppression effect on the
response to a peptide based vaccine against influenza.
Using recombinant methods, three epitopes derived
from influenza proteins were expressed within the flagellin molecule of Salmonella typhi vaccine strain. Each
epitope was expressed separately and the mixture of the
three chimeric flagella (cleaved from the bacteria) was
administered to mice intranasally. It was previously
0165-2478/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved.
PII S0165-2478(98)00073-X
10
T. Ben-Yedidia, R. Arnon / Immunology Letters 64 (1998) 9–15
shown that a three immunization series with this mixture protected mice against infection with several influenza strains. This protection was long lasting (7
months) and was effective even against a lethal dose
challenge. Mice that were immunized with the recombinant flagella and then challenged with a lethal dose of
the virus, lost less weight and none of them died from
the infection. Hence, this vaccine was defined as protective [14,15]. If the same approach using flagellin as a
carrier is to be considered for a human vaccine, it is
important to evaluate the carrier suppression effect of
native flagellin, since a suppression might appear as a
result of prior vaccination against salmonella or due to
former infection with the bacteria. To explore this issue,
we employed the mouse model. Prior to vaccination,
the animals were immunized once, intranasally, with
the native carrier (flagella) that does not express any
foreign epitope. Then, we examined the ability of the
chimeric vaccine to elicit specific humoral and cellular
immune responses and to confer protection was examined. We wish to report that the immune response as
well as the protective effect of the vaccine were not
suppressed. The same results were obtained when the
whole inactive salmonella was administered orally 1
month before vaccination or when young mice were
vaccinated with the native flagella and then immunized
with the recombinant protein at 18 months of age.
2. Materials and methods
2.1. Synthetic peptides and nucleotides
Two peptides corresponding to influenza NP epitopes, namely NP55-69 (RLIQNSLTIERMVLS) and
NP147-158 (TYQRTRALVRTG) as well as one peptide from the H3 subtype haemagglutinin, HA91-108
(SKAFSNCYPYDVPDYASL), were synthesized as reported previously [14].
2.2. Preparation of recombinant flagellin
The construction of the recombinant bacteria was
performed as described by Newton et al. [16]. The
synthetic oligonucleotides were inserted into the plasmid pLS408, which was kindly provided by Professor
B.A. Stocker and eventually transformed into
Salmonella dublin SL5928 as described elsewhere [17].
The transformed S. dublin were selected for ampicillin
resistance and motility under the light microscope. Selected clones were grown overnight in LB medium
containing ampicillin and the flagellin was purified by
acidic cleavage, according to the technique described by
Ibrahim et al. [18].
2.3. Mice
Young (2–4 months) and old (18 months) BALB/c
mice were obtained from Harlan Laboratories (Rehovot, Israel) at the age of 6 weeks. They were maintained under specific pathogen-free conditions, on
Purina chow and autoclaved water ad libitum, at constant room temperature and humidity.
2.4. Influenza 6irus
Influenza strain A/Texas/1/77 (H3N2 subtype) virus
was grown in the allantoic cavity of 11-day-old embryonated hen eggs (Hafetz Hayim Hatchery, Israel). It
was used as infectious allantoic fluid and for coating
ELISA plates. Using sucrose gradient, the virus was
further purified and used for induction of cell proliferation responses. Virus growth and purification, as well as
its titration (by the hemagglutination assay) were described by Barret et al. [19]. Titers were expressed as
hemagglutination units (HAU).
2.5. Salmonella typhi
The bacteria, S. dublin 5928 was grown at 37oC in LB
medium which contained ampicillin until it reached the
desired density (1 O.D600 = 109 bacteria/ml). The culture was centrifuged, resuspended in PBS and inactivated by exposure to irradiation from a gamma beam
150-A 60Co source (produced by the Atomic Energy of
Canada, Kanata, Ontario) with F.S.D of 75 cm and a
dose rate of 2.5 Mrad.
2.6. Immunization and infection procedures
Mice were immunized intranasally (i.n.) with the
native or hybrid flagellins, (75 mg per animal in 50 ml
PBS were administered to the nostrils of mice lightly
anesthetized with ether). The mice were boosted twice,
at 3-week intervals. Immunization with 0.4× 109 inactive salmonella was given once i.p. in 0.2 ml PBS,
followed by i.n. immunization with the hybrid flagellin
mixture. Infection of mice was performed by inoculating i.n. infectious allantoic fluid containing 10 − 4 HAU
virus per mouse under light ether anesthesia, 1 month
after the last booster.
2.7. Enzyme linked immuno-sorbent assay (ELISA)
The antigens were absorbed to ELISA plates (Nunc
Immuno Plate Maxisorp F96) in carbonate buffer pH
9.6. Allantoic fluid with A/Texas/1/77 virus (100 HAU/
ml) was added in 100 ml/well buffer and incubated
overnight at 37°C. Blocking was performed with PBS
containing 1% BSA. Goat anti-mouse Ig antibodies,
conjugated to horseradish peroxidase (HRP) (Jackson
T. Ben-Yedidia, R. Arnon / Immunology Letters 64 (1998) 9–15
11
Laboratories) was used as a second antibody. The
substrate consisted of ABTS (2,2%-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid)) (Sigma) 1 mg/ml in
solution of 0.2 M Citric Acid, 0.2 M Na2HPO4 and 1 ml
H2O2 added per 10 ml of the solution. The substrate
solution (100 ml/well) was added and incubated at 37°C
for 1 h. The plates are read at 414 nm.
2.8. Proliferation response of 6irus specific T-cells
Proliferation response to the virus was assayed as
previously described [20]. Briefly, the spleen was removed 5 days after viral challenge and single cell suspension was prepared. The cells were cultured in 0.2 ml
medium in the presence of inactivated purified virus.
Proliferation was evaluated 3 days later by 3Hthymidine incorporation [21].
2.9. Statistical analysis
Statistical analysis was performed using the Stat
View II program (Abacus Concepts, Berkeley, CA,
USA) on a Macintosh IICi. F-test was utilized to
calculate probability (P) values. Results are presented
as mean and S.E. of at least two repeated independent
experiments, including eight to ten animals per each
group. The error bars refer to cumulative data from all
the mice in all the repeated experiments.
Fig. 1. Anti-influenza virus antibody response in the sera of vaccinated mice. Antibodies were assayed by ELISA. Total Ig level was
assayed in the serum 1 week after the last immunization with the
flagellin construct. (), normal mouse serum; (2), Mice immunized
with control flagellin, which does not express the influenza epitopes;
(), mice immunized with the triple epitope construct; (), mice
immunized with the carrier flagellin and subsequently with the flagellin that expresses influenza epitopes. The level of antibodies is
significantly higher (in dilutions 1:9 – 1:243) in the groups that were
immunized with the triple epitope construct (with or without pre-vaccination with the carrier) as compared to the normal mice and to the
mice immunized with the control flagellin (P B0.05).
3.1. Humoral response
ing to investigate the effect of the carrier given at the
age of 2–3 months, on vaccination against influenza at
the age of 18 months. As previously described, the mice
were bled 1 week after the last booster and the specific
anti-viral response was evaluated. These results, as presented in Fig. 2, also indicate that the pre-vaccination
BALB/c mice, 2–3 months old, were immunized once
i.n. with the native isolated flagella and 3 weeks later
they were immunized with the mixture of chimeric
flagella that express the three influenza epitopes. The
immunization was given three times i.n., at 3-week
intervals. One control group was immunized only with
the native flagella and another only with the hybrid
flagella mixture (three immunizations). Antibodies specific for the flagellin could have been detected in the
serum already 1 week after the first immunization as
determined by ELISA (data not shown) while an additional three immunizations were needed in order to find
serum antibodies against the influenza virus. The results
for antibodies specific for the virus are presented in Fig.
1. As shown, no reduction in antibody level was observed as a result of prior exposure to the native
flagella. If at all, there is a small increase.
In another experiment, the effect of exposure to the
carrier alone, in young age, was evaluated in old mice.
Since, in many cases the salmonella infection or vaccination against it occur in young age but the vaccination
against influenza is given to the elderly, it was interest-
Fig. 2. Anti-influenza virus antibody response in the sera of vaccinated aged mice. Antibodies were assayed by ELISA. Total Ig level
was assayed in serum samples diluted 1:243, 1 week after the last
immunization with the triple flagellin construct. (A) mice immunized
with the native flagellin; (B) mice immunized with the triple epitope
construct; (C) mice immunized with the carrier and 15 months later,
with the flagellin that expresses influenza epitopes. Groups B and C
are not significantly different from each other, while both of them are
higher than group A.
3. Results
12
T. Ben-Yedidia, R. Arnon / Immunology Letters 64 (1998) 9–15
dex in response to the inactive virus is 3.4, while in the
group that was immunized only with the hybrid flagellins, it is 2.9. Statistically, there is no significant
difference between the two groups. In both cases the
response was significantly higher as compared to the
control untreated group.
3.3. Protection from sub-lethal infection
Fig. 3. Anti-influenza virus antibody response in the sera of vaccinated mice after oral pre-exposure to inactivated whole salmonella.
Antibodies were assayed by ELISA. Total Ig level was assayed in
samples diluted 1:81, 1 week after the last immunization with the
triple flagellin construct. (A) mice immunized with the native flagellin;
(B) mice immunized with the triple epitope construct; (C) mice
immunized with salmonella and 3 months later, with the flagellin that
expresses influenza epitopes. There is no significant difference between groups B and C, while both of them are higher than group A.
with the carrier does not reduce the production of
antibodies and may even cause a slight enhancement.
Thus, no carrier suppression was observed either when
the flagella was given a short (Fig. 1) or long (Fig. 2)
time before the vaccination against influenza epitopes
which are presented on the same carrier.
In humans, carrier suppression effect may arise following infection with the salmonella bacterium. In this
case, the bacteria invade orally and infect the intestine.
To simulate this situation more closely in the mouse
model, the animals (2 – 3 month old) were administered
orally with the inactive bacteria and 3 weeks later
immunized i.n. with the hybrid flagella mixture. One
week after the last immunization they were bled and
antibodies specific for the virus were detected in the
samples. These data, as shown in Fig. 3, are similar to
the previous results and are not indicative of any
suppression induced by the prior exposure to the carrier.
The capacity of the vaccine to reduce virus burden in
the lungs after sub-lethal challenge was examined in
mice that were either pre-exposed to the carrier before
vaccinating with the hybrid flagella or not. The challenge infection with a sub-lethal dose of influenza A/
Texas/77 strain, was given 1 month after the last
immunization and 5 days later, the lungs were removed
in order to measure the virus titer. The results presented
in Table 1 show the reduction of influenza virus titers in
the lungs of the mice as a result of the vaccination. The
virus titer was compared in mice that were vaccinated
with flagellin that expresses influenza epitopes (with or
without pre-vaccination with the native flagellin) to
those vaccinated only with the control flagellin. The
mean level of reduction in viral titer by the immunization with the mixture of three epitopes was in 2.239
0.09 orders of magnitude, while the reduction of virus
titer in the group that was pre-vaccinated with the
native flagellin was 1.749 0.15. Statistically, there is no
difference between the two groups in the ability of the
vaccine to effect virus burden in the lungs of infected
mice. In both cases, the reduction in the virus titer was
\ 95%.
Similar results were obtained in old mice that had
been immunized with the native flagellin at young age
3.2. Cellular response
The cellular immune response was evaluated by measuring the in vitro proliferation induced by inactivated
virus in splenocytes obtained from the vaccinated mice,
with and without pre-immunization with the carrier.
This response was measured after three immunizations
according to the regular schedule, followed by a sublethal challenge infection. Fig. 4 depicts the results. All
mice that were vaccinated with the hybrid flagellin
construct showed a significant proliferative response to
the inactive virus in-vitro. In the mice that were previously vaccinated with the flagellin, the stimulation in-
Fig. 4. Proliferation of splenocytes following in-vitro stimulation with
inactive influenza virus. (A) untreated, normal mice; (B) immunized
with flagellin that expresses influenza epitopes; (C) immunized with
the carrier and then with the hybrid flagellin. White square with black
diagonal lines, unstimulated in vitro; Square with black and white
lines, in vitro stimulated with 500 HAU influenza virus per well. The
proliferation of groups B and C is significantly (PB 0.05) higher than
the proliferation observed in group A, whereas the differences between groups B and C is not significant.
T. Ben-Yedidia, R. Arnon / Immunology Letters 64 (1998) 9–15
13
Table 1
Lung virus titers in mice infected with sub-lethal dose of influenza
Age at challenge
Native flagella
Hybrid flagella
Native and hybrid flagella
D
Young
6.23 90.33
6.23 90.33
4.009 0.24
—
—
4.49 9 0.48
2.23 90.09
1.74 9 0.15
Aged
5.50 90.50
5.50 90.50
1.099 0.25
—
—
0.78 90.30
4.41 90.55
4.72 90.58
Both young and aged mice were immunized with the native flagella and then with the chimeric one. One month after the last immunization, they
were infected with sub-lethal dose of A/Texas/1/77. In the young, the chimeric flagella was given 3 weeks after the immunization with the carrier
while in the aged, immunization with the chimeric flagella took place 15 months after immunization with the carrier. Evaluation of virus titers
took place 5 days after the infection, the titers are expressed as mean values of log EID50. In the right column the value of virus titer in the mice
immunized with the hybrid flagella was subtracted from the log EID50 value of the mice immunized only with the control flagellin. In both age
groups, the amount of virus in the lungs of the mice that were immunized with the hybrid flagella is significantly reduced as compared to the
groups that were immunized with the native flagella, (PB0.05).
and then immunized with influenza epitopes expressing
flagella, followed by a viral challenge at an old age (18
months). The vaccine capacity to reduce virus burden
was not disturbed by the early immunization with the
flagellin. The efficacy of the vaccine is apparently better
in the aged mice, probably due to memory responses
induced by the vaccine. These results give further support to the main issue dealt with in this study, namely,
that exposure of the animals to the vaccinating carrier,
in this case Salmonella flagellin, does not reduce the
efficacy of future vaccination with epitopes presented
on the same carrier.
In another experiment, the effect of prior oral exposure to the whole bacteria was evaluated. The mice,
2-months-old, were administered orally with irradiated
S. typhi 5928 and 3 weeks later they were immunized
i.n. with the hybrid flagella originating from the same
strain of salmonella expressing influenza epitopes. One
month after the last immunization, the mice were chal-
Fig. 5. Protection of BALB/c mice immunized i.n. with native flagellin as control (A); or triple construct (B); or pre-immunized orally
with inactive salmonella and then, immunized i.n. with the triple
vaccine (C); one month following the last immunization the mice
were challenged i.n. with live virus. Virus titer in the lungs, 5 days
after challenge, is presented as mean values of log EID50. The
protection level is not significantly effected by the pre-immunization
with the inactive bacteria.
lenged and then the virus titer in their lungs was
evaluated as shown in Fig. 5. There is a small difference
between the two groups in the ability of the vaccination
to reduce virus burden, but it is statistically not significant.
All these results indicate that infection with
salmonella or exposure of the immune system to the
flagellin molecules before vaccination with constructs
that include flagellin as a carrier, does not interfere with
the effective protection elicited by vaccination.
4. Discussion
Since small peptides are likely to be poor immunogens, the enhancement of their immunogenic capacity is
usually achieved by their conjugating to appropriate
carrier molecules. Hence, while developing peptide
based vaccines, primary consideration should be given
to the choice of the carrier. A particular problem that
might arise from the use of a carrier is the phenomenon
referred to as carrier suppression which is manifested as
diminished immune response towards the conjugated
peptide as a result of prior exposure to the carrier [2].
The mechanism that leads to such suppression is not
due to nonspecific suppresser phenomena, a study by
Schutze et. al. revealed that it was partially mediated by
suppressor T-cells which specifically inhibit the antihapten but not the anti-carrier antibody response but
may also be caused by a deficiency at the B-cell level
which contributes to the T-cells suppressive effect [13].
To ascertain that a carrier does not elicit such a suppression is of particular importance if an optimal ‘ideal’
carrier is identified which might be considered for more
than one peptide vaccine or when the carrier is derived
from a common pathogen that infects frequently the
target population for the vaccine. Such circumstances
may lead to reduction of the immune response to the
vaccine instead of elevating it by the conjugation to the
carrier.
14
T. Ben-Yedidia, R. Arnon / Immunology Letters 64 (1998) 9–15
The present study deals with a synthetic peptide
based vaccine against influenza employing S. flagellin as
a carrier.
A mixture of recombinant flagellin expressing three
different influenza epitopes was demonstrated to induce
efficient immune response against the virus with significant protection against challenge infection [14]. The
flagellin originates from a salmonella vaccine strain and
as such can be a candidate suitable for human vaccination. The recombinant salmonella is an aromatic compound depedent, flagellin negative live vaccine strain of
salmonella. Since there is a double mutation the risk for
emergence of revertants is reduced, moreover, the vaccine is a purified fine suspension of the flagella, which
does not include the whole bacteria. In a random
screening of human sera taken from healthy blood
donors, high levels of anti flagellin antibodies were
detected in 30% of the samples (data not shown). These
people had probably been exposed to the flagellin as a
result of prior natural infection with the salmonella. If
there is a carrier suppressive effect, these people might
fail to induce an efficient immune response towards
anti-influenza vaccine based on flagellin as a carrier.
We have therefore addressed the issue of the suppressive effect that the flagellin carrier might have on the
efficacy of the above mentioned recombinant vaccine.
The results reported in this study, indicate that a single
pre-immunization of the mice with native flagella 3
weeks before the immunization with the peptide-flagellin hybrid constructs, did not lead to a decrease in the
anti-viral responses as compared with immunized mice
that were unprimed by flagellin.
The intranasal route of administration of the carrier
that was applied in the first series of experiments was
applied in order to determine suppressive effect that
flagellin might induce if it will be used as a carrier
molecule for other vaccine preparations but this is not
the natural mean of exposure to salmonella in humans.
In order to simulate the natural infection, mice were
administered orally with whole inactive salmonella,
from which the flagellin originates and were vaccinated
with the recombinant flagella expressing the influenza
epitopes 1 month later. Their immune response against
influenza, as well as their ability to resist influenza
infection were then evaluated. As is apparent from the
results (Fig. 5), the oral route, similarly to the intranasal route of administration of the flagellin did not
interfere with the efficacy of further vaccination with
the hybrid construct in which this flagellin served as a
carrier. The humoral and cellular immune responses
eventually led to protection of the mice from live virus
infection, with no significant differences between the
groups that had been previously vaccinated with the
carrier or not.
Influenza infection is particularly risky among the
aged population, hence the efficacy of vaccination at
this age is most important. We have previously shown
that following vaccination with the hybrid flagella construct and challenge, the reduction of viral titer in the
lungs of the aged was even more efficient than in the
young mice [20].. These results were reconfirmed in the
present study. Immunization in the old seems to have a
particular advantage in elevating local specific immune
response as compared to immunization of young mice.
Although the mechanism underlying this local immunity is still unknown, the relative increase in CD8 +
cells in the lungs of the vaccinated aged mice, suggest a
role for local T-cells activity in reducing the viral titer
after vaccination and infection [20].
The mice are grown under strict conditions and there
is no evidence of exposure to any pathogen. However,
this also could have been a possible explanation for the
more pronounced effect in the old in such sub-clinical
exposure to the virus. Such sub-clinical levels of influenza may induced memory responses. The results obtained in the present study demonstrate no suppressive
effect of pre-exposure to flagellin in the aged mice that
were subsequently immunized with the hybrid flagellin
expressing the influenza epitopes (Fig. 2).
The cumulative results presented in this paper suggest that if flagellin is used as a carrier for peptide
based synthetic vaccines, the carrier suppression effect
should not present a point of concern. This outcome is
significant in view of the usefulness of flagellin as a
convenient and efficient carrier that may serve also as
an adjuvant in synthetic vaccine preparations, allowing
intranasal vaccination without additional adjuvant.
References
[1] A.R. Lussow, M.T. Aguado, G.G. Del, P.H. Lambert, Immunol.
Lett. 25 (1990) 255 – 263.
[2] L.A. Herzenberg, T. Tokuhisa, K. Hayakawa, Ann. Rev. Immunol. 1 (1983) 609 – 632.
[3] D.A. Herrington, D.F. Clyde, J.R. Davis, S. Baqar, J.R. Murphy, J.F. Cortese, R.S. Bank, E. Nardin, D. DiJohn, R.S.
Nussenzweig, et al., Bull. WHO (1990), 68, pp. 33 – 37.
[4] J.M. Lieberman, D.P. Greenberg, V.K. Wong, S. Partridge, S.J.
Chang, C.Y. Chiu, J.I. Ward, J. Pediatr. 126 (1995) 198–205.
[5] T. Barington, M. Skettrup, L. Juul, C. Heilmann, Infect. Immun.
61 (1993) 432 – 438.
[6] J.D. Di, S.S. Wasserman, J.R. Torres, M.J. Cortesia, J. Murillo,
G.A. Losonsky, D.A. Herrington, D. Sturcher, M.M. Levine,
Lancet 2 (1989) 1415 – 1418.
[7] A. Kaliyaperumal, V.S. Chauhan, G.P. Talwar, R. Raghupathy,
Eur. J. Immunol. 25 (1995) 3375 – 3380.
[8] C.O. Jacob, R. Arnon, M. Sela, Mol. Immunol. 22 (1985)
1333 – 1339.
[9] F.R. Vogel, C. Leclerc, M.P. Schutze, M. Jolivet, F. Audibert,
T.W. Klein, L. Chedid, Cell. Immunol. 107 (1987) 40 –51.
[10] J.A. Chabalgoity, Villareal, B. Ramos, C.M. Khan, S.N.
Chatfield, R.D. de Hormaeche, C.E. Hormaeche, Infect. Immun.
63 (1995) 2564 – 2569.
[11] L.D. Lise, D. Mazier, M. Jolivet, F. Audibert, L. Chedid, D.
Schlesinger, Infect. Immun. 55 (1987) 2658 – 2661.
T. Ben-Yedidia, R. Arnon / Immunology Letters 64 (1998) 9–15
[12] H.M. Etlinger, D. Gillessen, H.W. Lahm, H. Matile, H.J. Schonfeld, A. Trzeciak, Science 249 (1990) 423–425.
[13] M.P. Schutze, C. Leclerc, F.R. Vogel, L. Chedid, Cell. Immunol.
104 (1987) 79 – 90.
[14] R. Levi, R. Arnon, Vaccine 14 (1996) 85–92.
[15] R. Levi, R. Arnon, in: R.M. Chanock, F. Brown, H. Ginsberg,
E. Norrby (Eds.), Vaccine, CSHL Press, New York, 1995, pp.
311 – 316.
[16] S.M.C. Newton, C.O. Jacob, B.A.D. Stocker, Science 244 (1989)
70 – 72.
.
15
[17] J. McEwen, R. Levi, R.J. Horwitz, R. Arnon, Vaccine 10 (1992)
405 – 411.
[18] G.F. Ibrahim, G.H. Fleet, M.L. Lyons, R.A. Walter, J. Clin.
Microbiol. 22 (1985) 1040 – 1044.
[19] T. Barrett, S.C. Inglis, in: W.J. Mahy (Ed.), Virology: A Practical Approach, IRL Press, Washington, D.C., 1985, pp. 119–151.
[20] T. Ben-Yedidia, L. Abel, R. Arnon, A. Globerson, Mech. Ageing Devel. 104 (1998) 11 – 23.
[21] C. Fayolle, E. Deriaud, C. Leclerc, J. Immunol. 147 (1991)
4069 – 4073.