PCPP-Formulated H5N1 Influenza Vaccine Displays Improved
Stability and Dose-Sparing Effect in Lethal Challenge Studies

ALEXANDER K. ANDRIANOV,

1

DANIEL P. DECOLLIBUS,

1

ALEXANDER MARIN,

1

ASHLEY WEBB,

2

YOLANDA GRIFFIN,

2

RICHARD J. WEBBY

2

1

Apogee Technology, Inc., Norwood, Massachusetts 02062

2

Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105

Received 4 June 2010; revised 12 August 2010; accepted 15 September 2010

Published online 19 October 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22367

ABSTRACT: The potential impact of an influenza pandemic can be mitigated through the re-
alization of a successful vaccination program. The implementation of antigen stabilization and
dose-sparing technologies is an important step in improving availability of vaccines at the time
of a pandemic outbreak. We investigated poly[di(carboxylatophenoxy)phosphazene] (PCPP) as a
potential stabilizing and immunostimulating agent for H5N1 influenza vaccine. Physicochem-
ical characterization of PCPP-formulated H5N1 influenza vaccine revealed macromolecular
complexation in the system, whereas single radial immunodiffusion assay verified antigenicity
of the formulation in vitro. PCPP-enhanced formulation displayed a fourfold increase in the
half-life at 40

◦

C compared with a nonadjuvanted vaccine. Lethal challenge studies in ferrets

demonstrated 100% protection for low-antigen dose PCPP-adjuvanted formulations (1 :g of
hemagglutinin) and at least a 10-fold antigen-sparing effect. Therefore, PCPP demonstrated an
ability to improve thermal stability of H5N1 influenza vaccine in solutions and provide for a
substantial dose-sparing effect in vivo. © 2010 Wiley-Liss, Inc. and the American Pharmacists
Association J Pharm Sci 100:1436–1443, 2011
Keywords:

vaccine adjuvants; stabilization; polymeric drug delivery systems; polyelec-

trolytes; biodegradable polymers vaccine delivery

INTRODUCTION

The emergence of highly pathogenic avian influenza
A/H5N1 virus and the increasing number of cases of
its direct transmission to humans poses a major pan-
demic threat.

1–3

It is expected that safe and effec-

tive vaccines will be the single most important public
health instrument for reducing the morbidity, mor-
tality, and economic impact of pandemic influenza.

1,4

Despite extensive experience with vaccines against
human influenza viruses, researchers face serious
challenges in developing successful vaccines against
avian influenza with pandemic potential.

3

One of the

challenges stems from the fact that, for unknown
reasons, hemagglutinin (HA) proteins of avian sub-
types of influenza A viruses are not as immunogenic
as those of human subtypes.

5

As more research is

ongoing to understand biological basis for the poor

Correspondence to: Alexander K. Andrianov (Telephone: 781-

551-9450; Fax: [phone]; E-mail: [e-mail])

Journal of Pharmaceutical Sciences, Vol. 100, 1436–1443 (2011)
© 2010 Wiley-Liss, Inc. and the American Pharmacists Association

immunogenicity of avian HA glycoproteins, a strat-
egy for enhancing immunogenicity of avian influenza
vaccine and implementing dose-sparing technologies
through the use of adjuvants appears to be of high
importance.

1,3,6

Potential challenges in pandemic vaccine manufac-

turing also lie in the uncertainties associated with
predicting the time of the potential outbreak and even
the pandemic virus itself. To maximize preparedness,
the concept of vaccine stockpiling has become a part
of a global pandemic plan, and there is a strong focus
on improving shelf life of vaccine and standardization
of assays for their monitoring.

7–9

Improvement of vac-

cine stability is also of prime importance for seasonal
strains as reduced dependence on cold-chain facili-
ties, as well as the diminished risk of vaccine losses
caused by “off-label” storage can lead to enormous an-
nual savings.

10,11

Water-soluble

polyphosphazenes,

such

as

poly[di(carboxylatophenoxy)phosphazene]

(PCPP),

have been extensively investigated in vivo as vaccine
adjuvants for seasonal influenza vaccines.

12–14

In

1436

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ROLE OF PCPP-FORMULATED H5N1 INFLUENZA VACCINE IN LETHAL CHALLENGE STUDIES

1437

Figure 1.

Molecular formula of poly[di(carboxylatophe-

noxy)phosphazene].

these studies, PCPP has demonstrated the ability to
significantly increase hemagglutination inhibition
and serum antigen-specific immunoglobulin G titers,
as well as displayed a substantial dose-sparing effect.
PCPP-formulated vaccines have been also tested
in clinical trials and reported to be immunogenic
and well tolerated with no vaccine-related, serious
adverse events.

14,15

From the physicochemical and

formulation standpoint, PCPP (Fig. 1) is a well-
defined compound produced by a controlled synthetic
process and capable of forming water-soluble com-
plexes with antigens.

16,17

However, to this date, there

have been no reports on the effect of this adjuvant on
H5N1 influenza vaccines.

In the present paper, we investigated PCPP-

formulated H5N1 influenza vaccines in regard to their
physicochemical behavior in solution. We assessed
in vitro potency using a single radial immunodiffu-
sion (SRID) assay. Furthermore, we studied the effect
of PCPP on thermal stability of H5N1 antigen in solu-
tion and the resistance of such formulation to drying.
Finally, we evaluated the in vivo activity of PCPP-
formulated H5N1 vaccine in lethal challenge studies
in ferrets.

EXPERIMENTAL

Materials

PCPP (Sigma–Aldrich, St. Louis, Missouri) was pu-
rified by multiple precipitations using sodium chlo-
ride to produce polymer with weight-average molec-
ular weight of 855,000 g/mol and polydispersity
parameter of 2.5, as determined by gel perme-
ation chromatography using poly(acrylic acid) stan-

dards, which is typically in a good correlation with
light scattering data.

18

The following reagents were

obtained through the NIH Biodefense and Emerg-
ing Infections Research Resources Repository, NIAID,
NIH: monovalent influenza subvirion vaccine, rgA/
Vietnam/1203/2004 (H5N1), NR-4143; polyclonal
anti-influenza virus H5 HA, A/Vietnam/1203/04
(H5N1) (antiserum, goat), NR-2705. A37 agarose
(molecular biology grade, IBI Scientific, Peosta, Iowa);
N-lauroylsarcosine

sodium

salt,

polyoxyethylene

(20) sorbitan monolaurate (Tween) (TCI America,
Portland, Oregon); Coomassie Brillant Blue R-250
(J.T. Baker, Phillipsburg, New Jersey); Dulbecco’s
phosphate buffered saline (DPBS) without magne-
sium and calcium (Thermo Scientific, Logan, Utah);
glacial acetic acid, methanol, 10

× PBS concentrate

(EMD Chemicals, Gibbstown, New Jersey); acetoni-
trile (Fisher Scientific, Pittsburgh, Pennsylvania);
and sodium azide (VWR International, West Chester,
Pennsylvania) were used as received.

Methods

Physicochemical Characterization

Size-exclusion chromatography (SEC) of the for-
mulation was performed using a Hitachi high
performance

liquid

chromatography

(HPLC)

system

with

an

L-2450

diode

array

detector

(Hitachi

LaChrom

Elite

System,

Hitachi,

San

Jose, California) equipped with an ultrahydrogel
linear size exclusion column (Waters Corporation,
Milford, Massachusetts) at 25

◦

C using 0.1

× PBS

with 10% acetonitrile as a mobile phase with a flow
rate of 0.75 mL/min. Samples were prepared by
mixing vaccine formulation with solution of PCPP
in PBS to obtain a final HA concentration of 0.015
mg/mL (0.076 :M) and PCPP concentration of 0.006
mg/mL (0.007 :M). The solution was gently vortexed
immediately after mixing and incubated at room
temperature for at least 1 h prior to analysis. Peak
area and retention time of the peak attributed to
PCPP (λ

max

= 235 nm) was monitored.

Ultraviolet-visible light spectroscopy was con-

ducted using a Hitachi U-2810 Spectrophotomer
(Hitachi, San Jose, California) at 25

◦

C in a 1 cm

quartz cuvette (NSG Precision Cells Inc., Farming-
dale, New York). Samples were prepared at a con-
centration of 0.005 mg HA/mL (0.025 :M) and 0.006
mg/mL (0.007 :M) of PCPP. The solutions were gen-
tly vortexed prior to the analysis. Optical density was
recorded during wavelength scans from 400 to 200 nm
in 1 nm steps. All spectra are the result of average of
two repeated scans for each sample.

Single-Radial Immunodiffusion Assay

The SRID technique utilized to determine in-
fluenza HA concentrations was used as described

DOI 10.1002/jps

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 4, APRIL 2011

1438

ANDRIANOV ET AL.

previously.

19

A37 agarose was melted (1.5% in DPBS,

pH 7.4) and allowed to cool to 56

◦

C prior to the addi-

tion of 5 :L antiserum per milliliter of gel and sodium
azide at a final concentration of 0.1% (w/v) to prevent
bacterial growth. Eleven milliliter aliquot of the com-
plete gel was then transferred to warm 10 cm glass
Petri dishes (VWR International, West Chester, Penn-
sylvania) and allowed to cool to room temperature and
set. Wells were then cut into the gels using a 3 mm
diameter biopsy punch (Miltex, York, Pennsylvania)
and loaded with 10 :L of sample per well. Once the
sample solution had diffused out of the wells (

∼15

min), 10 :L of 5% (w/v) N-lauroylsarcosine sodium
salt solution in DPBS was added. Gels were then cov-
ered and placed in a moist chamber at room temper-
ature to allow diffusion for 24 h. After washing for 30
min with two changes of DPBS, the gels were pressed
dry for 30 min, as described previously,

19

and air dried

to remove residual moisture. Then the gels were sub-
merged in a staining solution containing 0.03% (w/v)
Coomassie Brilliant Blue R-250 in 29% (v/v) methanol
and 12% (v/v) acetic acid for a total time of 20 min be-
fore destaining with the same solution, less the dye,
until precipitin zones were clearly visible. Images of
the gels and calibration grid were then taken on a
Nikon D90 digital camera equipped with an AF-S
Nikkor 18–105 mm 3.5–5.6 G ED lens (Nikon Cor-
poration, Tokyo, Japan) and analyzed on Motic Im-
ages Advanced 3.2 software (Motik, Xiamen, China).
Image analysis consisted of manually outlining the
precipitin ring with an n-sided polygon (n > 30) and
using the calibrated software to determine its area in
mm

2

.

To test the effect of PCPP on the assay of HA by

SRID, subvirion vaccine was diluted to 0.025 mg HA/
mL (0.127 :M HA) with solutions of varying PCPP
concentration in PBS. Final concentrations of PCPP
in the solutions ranged from 0.0 to 0.217 mg/mL
(0.0–0.254 :M). Solutions were gently vortexed im-
mediately after mixing and incubated at room tem-
perature for at least 1 h prior to addition to gels.

Thermal Stability Studies

Samples were prepared by diluting H5N1 subunit
vaccine to an HA concentration of 0.025 mg/mL; PCPP
and Tween were added to some solutions at concen-
trations of 1.0 and 0.025 mg/mL, respectively. Pre-
liminary studies demonstrated that protein stabiliz-
ing effect of PCPP was practically unchanged (within
15%) in the range of concentration of 0.2–1.0 mg/mL
of PCPP (data not shown). Solutions were incubated
at 4

◦

C for 64 h prior to incubation at 40

◦

C. Sample so-

lutions were analyzed by SRID along with calibration
samples made from dilutions of each formulation.

Ambient Temperature Drying Studies

Ambient temperature drying studies of PCPP–H5N1
vaccine formulations were conducted using titanium
microneedle arrays, each containing 50 microneedles
of approximately 600 :m in length.

20–22

Using the

X-Y-Z micropositioning system, the coating procedure
was performed by submerging the microneedles into
the wells in the coating reservoir and then immedi-
ately removing them, followed by a drying step in
which the arrays were purged with anhydrous nitro-
gen gas at ambient temperature. The formulation was
fed to a 50 microwell reservoir, using a Genie Plus
syringe pump (Kent Scientific, Torrington, Connecti-
cut). A stereo zoom microscope (STZ-45-BS-FR) with
a digital camera (Caltex Scientific, Irvine, California)
was used to monitor the process.

Stability of antigen in the ambient temperature

drying process was studied by dissolving microneedle
formulations and comparing their antigenicity with
the antigenicity of the coating solution. Specifically,
samples were rinsed with 1 mL of PBS to dissolve the
coating formulation, and the resulting solution was
analyzed by SRID assay.

In Vivo Challenge Studies

Three groups of four 4-month-old ferrets shown to
be seronegative for H5N1, and circulating human
H1N1 and H3N2 influenza viruses were purchased
from Triple F Farms (Sayre, Pennsylvania). The ex-
perimental groups were as follows: (1) 10 :g HA in-
tramuscularly (i.m.), (2) 1 :g HA i.m., and (3) 1 :g
HA adjuvanted with 66 :g PCPP i.m. The dose of
antigen was selected on the basis of previous studies
of PCPP formulations with influenza antigens.

12–14

Nine weeks after vaccination, all animals were anes-
thetized with inhaled isoflurane and challenged with
1

× 10

6

egg infectious dose 50 (EID

50

) of A/Vietnam/

1203/2004 (H5N1) in 1 mL of PBS. Animals were
monitored daily for disease signs and were eutha-
nized if severe clinical signs were present. All ani-
mal studies were approved by the St. Jude Children’s
Research Hospital Institutional Animal Care and Use
Committee.

RESULTS AND DISCUSSION

Macromolecular Interactions in PCPP–Antigen
Formulations

Physicochemical characterization of PCPP-adjuvan-
ted

H5N1

formulations

can

provide

valuable

clues for understanding their functional properties,
including biological activity. In particular, molecular
interactions between polyphosphazene adjuvant and
vaccine antigen appear to be of prime importance
as ability of PCPP to form noncovalent complexes
with protein molecules was previously linked to its

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ROLE OF PCPP-FORMULATED H5N1 INFLUENZA VACCINE IN LETHAL CHALLENGE STUDIES

1439

biological performance.

17

Thus, it was important

to

establish

whether

such

interactions,

which

are typically defined as polymer–protein complex
formation,

23

take place in the PCPP–H5N1 antigen

system.

Macromolecular interactions in PCPP–H5N1 for-

mulation were studied using SEC by monitoring the
PCPP peak, which provided high UV absorbance,
short retention time, and minimal overlap with peaks
associated with vaccine formulation (Fig. 2a). As seen
from Figure 2a, polyphosphazene peak underwent re-
markable changes upon addition of the antigen for-
mulation as manifested in both shift toward higher
retention time and reduction of the peak area. The
first indicates a significant molecular size reduction
and, in light of the previous findings on the com-
paction of PCPP conformation upon reactions with
proteins,

17

suggests macromolecular interactions in

the system. Figure 2b shows that this effect was ob-
served in a broad range of PCPP/antigen ratios and
was most pronounced at the high antigen content,
which is also in an agreement with previously re-

Figure 2.

(a) Size-exclusion chromatography profiles of

PCPP (dotted line), antigen (dashed line), and PCPP–anti-
gen mixture (solid line) in aqueous solution (PBS, pH 7.4,
0.015 mg/mL of HA, 0.006 mg/mL of PCPP, 235 nm, only
a portion of chromatogram is shown related to molecules
with a molecular weight in excess of 1000 g/mol); (b) A shift
in the peak retention time ( RT) of PCPP chromatogram
upon addition of antigen as a function of PCPP:antigen
ratios.

ported conformational changes of the complex.

17

To

understand the decrease in peak area of PCPP in
the mixture (Fig. 2a), UV absorbance profiles were
recorded to eliminate a possibility of mass loss during
chromatographic separation. Analysis of PCPP spec-
tra revealed significant hypochromic effect upon ad-
dition of antigenic formulation. Both methods showed
a good correlation displaying a reduction in the opti-
cal density of approximately 20%. As UV absorbance
changes can be also construed as indicators of macro-
molecular interactions,

23,24

this provides further sup-

port for the formation of a complex between PCPP
and H5N1 antigen.

In Vitro

Potency Testing of PCPP-Adjuvanted H5N1

Formulation by SRID

SRID assay, as recommended by the European Phar-
macopoeia and the WHO,

10

has been used for sev-

eral decades to determine the antigen content (HA
potency) of all human-inactivated influenza vaccines.
SRID assay is based on the diffusion and reaction
of antigen in an agarose gel, containing specific an-
tibodies to the antigen measured, and the area of
the stained reaction (immunoprecipitation) zone

11,25

correlates with the amount of HA. As stain inten-
sity and reaction zone area are influenced by struc-
tural properties of HA, the method has been employed
to investigate the stability of various influenza vac-
cine formulations.

11

We investigated the applicabil-

ity of SRID for monitoring the antigenicity of PCPP-
adjuvanted H5N1 vaccine formulations.

The results of SRID analysis at various concen-

trations of PCPP are shown in Figure 3. Although
addition of PCPP to vaccine formulations resulted
in a slightly more intense staining of the gel inside
the reaction zone, it practically did not obscure or
interfere with the measurement of the precipitation
ring demonstrating that PCPP-adjuvanted formula-
tions were compatible with standard SRID method

Figure 3.

Effect of PCPP on single radial immunodiffu-

sion immunoprecipitation zones at 0.008 (a), 0.1 (b), and 0.5
mg/mL (c) of PCPP (0.020 mg/mL of HA).

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ANDRIANOV ET AL.

Figure 4.

Effect of PCPP on SRID zone reduction

(0.025 mg/mL of HA, n

= 5).

in all formulations employed in the present studies
with PCPP concentrations as high as 1.5 mg/mL. The
darkening effect, clearly seen in vaccine-free PCPP
solutions, appears to be more pronounced at higher
polymer concentration and suggests interactions be-
tween PCPP and Coomassie R-250 dye used in the
staining procedure.

Although PCPP formulations appear to be compat-

ible with SRID assay in a very broad concentration
range, addition of PCPP to HA protein formulations
can lead to some decrease in the reaction zone as com-
pared with their polymer-free counterparts (Fig. 4) in
the range of very low PCPP concentrations (PCPP/
HA ratio under 4:1). In all cases, the maximum re-
duction is limited to less than 10% (Fig. 4), and the
phenomenon had no practical implications on formu-
lations studied further as all of them were prepared
at a higher PCPP/HA ratio.

These results, however, can present interest as an

independent manifestation of macromolecular inter-
actions observed by physicochemical methods. It is
noteworthy that the effect of PCPP was unexpectedly
observed not at a high concentration of PCPP, which
could be reasonable to predict in case of antigen-
destabilizing effect, but rather at very low PCPP/HA
ratio, when the formation of complexes with multiple
protein ligands is anticipated. It has to be emphasized
that the basis for antigen quantification by SRID
lies in the formation of antigen–antibody complexes,
which become insoluble and thus detectable because
diffusing antigen is gradually consumed, and equimo-
lar complex is formed. Thus, it can be hypothesized

that PCPP can facilitate formation of insoluble struc-
tures in areas of higher antigen concentration playing
the role of a “cross-linker” with a resulting reduction
of the reaction zone. The phenomenon is not observed
when PCPP/HA ratio is sufficiently high to prevent
polymer binding with multiple protein molecules.

PCPP Improves Stability of H5H1 Vaccine in Solutions

As influenza vaccines are formulated in solutions,
they are subjected to physical and chemical degrada-
tion in an aqueous environment and have to be stored
at a relatively narrow temperature range, usually be-
tween 2

◦

C and 8

◦

C.

10

Improvement of their thermal

stability is an important objective as it reduces depen-
dence of their distribution and storage on expensive
“cold chains.”

10

In case of H5N1 vaccines, formula-

tions with improved stability will also facilitate their
stockpiling and thus provide an immediate availabil-
ity and simple distribution in a pandemic situation.

10

The effect of PCPP on thermal stability of H5N1

in aqueous formulations was studied in degradation
experiments at 40

◦

C (Fig. 5). Activity was monitored

by SRID and formulations stored at 4

◦

C were used

as standards. PCPP showed a strong stabilizing ef-
fect on the antigen formulation under these condi-
tions, which is illustrated by an almost fourfold in-
crease in half-life and a substantial raise in a period
of 20% activity loss (Fig. 5). To explain this effect, it
is important to note that the ability of polymers to

Figure 5.

Effect of PCPP and PCPP-surfactant system

on thermal stability of H5N1 antigen in aqueous solutions
as demonstrated by changes in half-life (white bars) and
time of 20% activity loss (black bars) (40

◦

C, 0.025 mg/mL

of antigen, 1 mg/mL of PCPP, 0.025 mg/mL of Tween, PBS,
pH 7.4).

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ROLE OF PCPP-FORMULATED H5N1 INFLUENZA VACCINE IN LETHAL CHALLENGE STUDIES

1441

stabilize proteins has been previously studied

26

and,

in case of polyelectrolytes, it has been generally at-
tributed to their self-organization in solutions.

27

Ionic

macromolecules have been reported to display a slight
stabilizing effect on proteins or suppress their ther-
moaggregation through the formation of polyelec-
trolyte complexes.

28–31

In particular, stabilization of

proteins in such systems is generally attributed to
preferential exclusion, surface activity, steric hin-
drance of protein–protein interactions, or restriction
of protein structural movement.

26

In context of this

knowledge, it is reasonable to suggest that the abil-
ity of PCPP to form macromolecular complexes with
H5N1 antigen, as discussed above, provides a key to
the observed phenomenon. Interestingly, addition of
surfactant appears to amplify this stabilizing effect
(Fig. 5). This needs further investigation; however,
it can be reasonable to assume more sophisticated
molecular self-organization in a tertiary system, such
as formation of polymer bound micelles, typically de-
scribed as a “necklace” model.

27,32

PCPP-Formulated H5N1 Vaccine Tolerates Ambient
Temperature Drying Techniques

The ability of PCPP-formulated H5N1 vaccine to
tolerate an ambient temperature drying process
was also investigated because such technique plays
an important role in the development of some
advanced delivery system, particularly in micro-
fabrication of coated microneedles for intradermal
immunization.

22,33

The PCPP–HA formulation was

deposited on titanium foil using microdip-coating
technique, which involved multiple cycles of solution
deposition and drying with nitrogen gas at ambient
temperature.

20,22

Solid coatings were then dissolved

in PBS and in vitro potency of encapsulated anti-
gen was measured by SRID as a percent of its ini-
tial antigenicity in solution. The results are summa-
rized in Table 1. As seen from Table 1, the PCPP–HA
formulation was compatible with the technique with
no loss of potency detected even after 50 coating cy-
cles. This is especially important in view of dramatic
loss of potency reported recently for other H5N1 for-
mulations during similar procedures,

34

which could

only be eradicated by the addition of a stabilizer,
trehalose.

34

Present results demonstrate that intro-

Table 2.

Lethal Challenge Studies of PCPP-Adjuvanted and

Nonadjuvanted H5N1 Vaccine Formulations in Ferrets

Dose (:g)

Vaccination Regimen

HA

PCPP

% Survival (Dead/Total)

HA

1

–

0 (4/4)

HA

10

–

0 (4/4)

HA adjuvanted with
PCPP

1

66

100 (0/4)

Single-dose intramuscular immunization, challenge at 9 weeks, 10

6

of

EID50 of VN/1203/04 virus.

HA, hemagglutinin; PCPP, poly[di(carboxylatophenoxy)phosphazene].

duction of PCPP in the formulation during the ambi-
ent temperature drying process stabilizes the antigen
formulation without the need for an additional stabi-
lizing agent.

PCPP-Adjuvanted H5N1 Vaccine Provides Protection
and Dose-Sparing Effect in Lethal Challenge Studies in
Ferrets

PCPP was studied for its ability to adjuvant H5N1
influenza vaccine. As it has been previously reported
that ferret model provides the basis for developing
pandemic influenza vaccines,

35,36

the performance of

PCPP was evaluated using this animal model in lethal
challenge studies. The immune correlates of protec-
tion against influenza are not well understood and
assays for different aspects of the immune response
to avian influenza viruses, in particular cell-mediated
immune responses, are not available.

2,3

Animals

were immunized using single-dose i.m. adminis-
tration of adjuvanted and nonadjuvanted formula-
tions and then were challenged with 10

6

of EID

50

of VN/1203/04 virus 9 weeks after immunization
(Table 2). As seen from Table 2, PCPP formulation
provided full protection from mortality at a dose of
antigen of 1 :g of HA. Because nonadjuvanted for-
mulation was not protective at a dose of up to 10 :g
of HA, it can be concluded that PCPP allowed for at
least 10-fold dose sparing.

CONCLUSIONS

We found that PCPP, a well-defined molecular ad-
juvant, displays both strong stabilizing and potent
immunoadjuvant effects on H5N1 influenza vaccine

Table 1.

Ambient Temperature Drying of Antigen–PCPP Formulations

Coating Content (:g/array)

Number of Coating/
Drying Cycles

PCPP

Antigen

In Vitro Potency of H5N1

Antigen after Drying

a

(%)

38

16.0

0.4

100

50

34.6

0.8

100

a

Relative to antigenicity in solution before drying.

PCPP, poly[di(carboxylatophenoxy)phosphazene].

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ANDRIANOV ET AL.

formulations. Physicochemical and immunological
characterization of formulations in vitro suggests that
these important properties are linked to macromolec-
ular interactions and water-soluble complex forma-
tion observed in the system. The use of the same
compound as a stabilizing and dose-sparing agent
can have important practical implications potentially
facilitating rapid production and stockpiling of pan-
demic vaccines.

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