SKARLAS ET AL.
Table 5. Animal immunizations with SOC4-conjugates
Immunized mice
Died
Survived
Ac–SOC4{Ac2, Aoa2}–NH2 (9)
6
5
5
6
4
4
6
3
6
6
6
0
1
1
0
2
2
0
3
0
0
0
Ac–SOC4{Ac2, [T1(CH N–O)]2}–NH2 (10)
Ac–SOC4{Ac2, [T2(CH N–O)]2}–NH2 (11)
Ac–SOC4{Ac2, [T3(CH N–O)]2}–NH2 (12)
Ac–SOC4{Ac2, [T4(CH N–O)]2}–NH2 (13)
Ac–SOC4{Ac2, [T5(CH N–O)]2}–NH2 (14)
Ac–SOC4{Ac2, [T6(CH N–O)]2}–NH2 (15)
Ac–SOC4{Ac2, [T7(CH N–O)]2}–NH2 (16)
Ac–SOC4{Ac2, [T8(CH N–O)]2}–NH2 (17)
Adjuvant
Control
Figure 4. CD spectra of the carrier Ac–SOC4[(Ac)2,(Aoa)2]–NH2 (9) and the
Ac–SOC4{Ac2, [T7(CH N–O)]2}–NH2 conjugate (16) in 50% TFE/H2O.
virus vaccines primarily include only the killed vaccines that are
inactivated. Subunit vaccines can be used in a variety of formats,
but typically they all are targeted to the HA gene [23].
temperature controller. Spectra were obtained using a quartz cell
of 1 mm path length and the concentration of the tested com-
pounds was 100 µM or 50 µM. Experiments were performed in
TFE/H2O mixtures (0, 50 and 100%). Spectra were obtained with
a 1 nm bandwidth, a scan speed of 50 nm/min and a response of
1 s. The signal-to-noise ratio was improved by accumulating three
scans. All CD spectra are reported in terms of ellipticity units per
mole ([ꢀ] in deg cm2 dmol−1). The percentage helical content
was estimated on the basis of the [ꢀ]222 nm values, in different
environments, as described by Chen et al. [19] (Figure 4).
Both antibodies and activated T lymphocytes were produced
in response to viral infection. There are reports indicating that
influenzavirus-specificIgGresponseisCD4+T-celldependentand
others describing CD4+ T-cell independent antibody responses.
CTL response is considered to be directed against the M and NP
proteins, while other investigations pointed out that CTL response
in addition to strong antibody formation is more effective in
preventing the disease [21,24–26].
Aiming at contributing to the development of a vaccine
that remains the first choice for prophylactic intervention, a
reconstituted model of HA, mimicking its antigenic properties
was designed, synthesized and tested in mice for the induction
of protective immunity. Four HTL (T1, T3, T7 and T8) and four CTL
(T2, T4, T5 and T6) epitopes were coupled in two copies each to
the tetrameric SOC4, (Lys-Aib-Gly)4, carrier. The SOC4-conjugates
were synthesized in three steps: (i) solid phase synthesis of
the SOC4 carrier bearing two amino-oxy-acetyl (NH2 –OCH2CO)
groups on the first and third Lys-NεH2 residues, (ii) solid phase
synthesis of each epitope and creation of an aldehyde group and
(iii) chemoselectiveligationofthealdehyde-epitopestothecarrier
through the formation of an oxime bond. All final products were
obtained in sufficient yields and high purity as confirmed by HPLC
and ESI-MS (Tables 3 and 4).
The CD spectra of the amino-oxy-acetyl SOC4 carrier,
Ac–SOC4[(Ac)2,(Aoa)2]–NH2 (9), in TFE/H2O (50/50 v/v) and 100%
TFE exhibitedapositivebandat192 nmandtwonegativebandsat
205 and 222 nm typical of helical structure. The helical content of
the carrier, estimated on the basis of the [ꢀ]222 value, was found to
be 15% at 50% TFE/H2O [19]. These helical features, in agreement
with our previous studies, were conserved even after the attach-
ment of the T-cell epitopes to the carrier, as for example in the
case of Ac–SOC4{Ac2, [T7(CH N–O)]2}–NH2 (16) confirming the
persistence of the carrier-helical conformation in its conjugated
forms (Figure 4). One may assume that the coupled T-cell epitopes
of the HA protein will conserve their initial topology, which is
critical for expressing their immunogenic properties.
Biological Assays
The animal experiments were performed in the laboratories of
the co-authors Droebner and Planz at the Friedrich-Loeffler-
Institut (FLI), Tuebingen, Germany. The FLI is an unique ‘high-
safety’ BSL3 building where experiments with wild-type H5N1
influenza virus are allowed to be performed. The experiments
were performed with an original field isolate from a mallard that is
highly pathogenic to mice without adaptation to the mammalian
host. The carrier (9) and the SOC4-conjugates (10–17) were
dissolved in ddH2O. Immunizations were performed according
to Papamattheou et al. [20]. Groups of six female bagg albino
(BALB)/c mice (at an age of 6–8 weeks) were immunized
subcutaneously (s.c.) with 50 µg of each SOC4-conjugate dissolved
in ddH2O and emulsified in complete Freund’s adjuvant (CFA)
on day 0. Two boosters of half-doses of each SOC4-conjugate
in incomplete Freund’s adjuvant (IFA) followed at days 25 and
50. Twenty-five days past the third boost, the animals were
infected intranasally with 2 × 105 pfu (100× MLD50) H5N1
(A/mallard/Bavaria/1/2006, MB1) influenza virus. The survival rates
were determined after a 14-day period [21]. The experiments were
performed twice.
Results and Discussion
Although avian influenza has been extensively studied in the past
30 years, our knowledge of the immune response to this pathogen
remains rather limited. Vaccination is the most efficient method
for preventing influenza and its severe complications. However,
antigenic drift has a major impact on the vaccine effectiveness [22]
and the development of a universal influenza vaccine is still at a
preclinical or clinical phase. Current avian influenza vaccines are
divided into whole viral vaccines and subunit vaccines. The whole
Immunization experiments (Table 5) indicated 50% survival
of mice when immunized with the SOC4-conjugate bearing
two copies of T7 (16) and 30% survival when immunized with
conjugates bearing two copies of T4 (13) and T5 (14) (Figure 5).
Previous biological studies [21], performed in the same laboratory
under comparable conditions, showed 60% survival of mice. It is
c
wileyonlinelibrary.com/journal/jpepsci Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2011; 17: 226–232
Skarlas, Theodore
