﻿Feasibility of reconstructed ancestral H5N1 influenza
viruses for cross-clade protective
vaccine development
Mariette F. Ducateza
, Justin Bahlb,c
, Yolanda Griffina
, Evelyn Stigger-Rossera
, John Franksa
, Subrata Barmana
,
Dhanasekaran Vijaykrishnab,c
, Ashley Webba
, Yi Guanc,d
, Robert G. Webstera
, Gavin J. D. Smithb,c
, and Richard J. Webbya,1
a
Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, TN 38105-3678; b
Duke­National University of Singapore Graduate
Medical School, Republic of Singapore 169857; c
State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, University of Hong Kong,
Hong Kong, SAR, China; and d
International Institute of Infection and Immunity, Shantou University, Shantou 515031, China
Edited by Peter Palese, Mount Sinai School of Medicine, New York, NY, and approved November 16, 2010 (received for review August 23, 2010)
Since the reemergence of highly pathogenic H5N1 influenza
viruses in humans in 2003, these viruses have spread throughout
avian species in Asia, Europe, and Africa. Their sustained circula-
tion has resulted in the evolution of phylogenetically diverse
lineages. Viruses from these lineages show considerable antigenic
variation, which has confounded vaccine planning efforts. We
reconstructed ancestral protein sequences at several nodes of the
hemagglutinin (HA) and neuraminidase (NA) gene phylogenies
that represent ancestors to diverse H5N1 virus clades. By using the
same methods that have been used to generate currently licensed
inactivated H5N1 vaccines, we were able to produce a panel of
replication competent influenza viruses containing synthesized HA
and NA genes representing the reconstructed ancestral proteins.
We identified two of these viruses that showed promising in vitro
cross-reactivity with clade 1, 2.1, 2.2, 2.3.4, and 4 viruses. To
confirm that vaccine antigens derived from these viruses were
able to elicit functional antibodies following immunization, we
created whole-virus vaccines and compared their protective
efficacy versus that of antigens from positive control, naturally
occurring, and broadly reactive H5N1 viruses. The ancestral
viruses' vaccines provided robust protection against morbidity
and mortality in ferrets challenged with H5N1 strains from clades
1, 2.1, and 2.2 in a manner similar to those based on the control
strains. These findings provide proof of principle that viable, com-
putationally derived vaccine seed viruses can be constructed
within the context of currently licensed vaccine platforms. Such
technologies should be explored to enhance the cross reactivity
and availability of H5N1 influenza vaccines.
universal | cross-reactive | pandemic
Highly pathogenic avian influenza (HPAI) A H5N1 remains
a significant pandemic threat (1). The precursor of these
viruses, A/goose/Guangdong/1/1996, was first isolated from
a goose in Guangdong, China (2), and its derivatives have since
spread throughout Southeast Asia, Eurasia, and Africa. As of
June 17, 2010, the World Organization for Animal Health updates
of HPAI H5N1 outbreaks highlighted 6,731 outbreaks in poultry
in 51 countries (3). Reported human cases caused by this virus as
of August 3, 2010, numbered 505, of which 300 were fatal (4).
In conjunction with their spread, H5N1 viruses have diversified
into multiple lineages, many of which continue to concurrently
circulate and cause human infections. These lineages have been
classified into clades (0 to 9) and subclades on the basis of their
hemagglutinin (HA) gene genealogy (5). Viruses from these dif-
ferent clades also show considerable antigenic variation, which
confounds pandemic preparedness efforts. Additional complex-
ities have emerged as a result of the difficulty in handling and
shipping HPAI viruses and sovereignty issues surrounding iso-
lates. Antiviral drugs are effective against HPAI H5N1 infection if
they are administered early enough (6). However, acquired re-
sistance to these drugs can develop. Therefore, significant re-
search efforts have been directed toward developing broadly
reactive and alternative reagents, including vaccines, for pan-
demic preparedness.
Several approaches for alternative, broadly reactive vaccines
have been proposed. M2e peptide vaccines, based on the con-
served ectodomain of matrix protein 2, have shown promise to
provide broad protection against different influenza viruses when
used in murine models (7, 8). Another potential solution pre-
sented has been to mix antigens from multiple viruses in a single
vaccine. This approach was previously tested in animal models
with a mixture of inactivated whole viruses from different HPAI
H5N1 clades, which successfully protected ferrets against chal-
lenge with clades 1 and 2 (6). However, it requires the production
of multiple antigens, which will limit the number of doses pro-
duced. Consensus HA and neuraminidase (NA) synthetic DNAs
have also been explored as an approach to enhance H5N1 vaccine
efficacies. With such DNA vaccines, only partial protection was
observed in mice challenged with HPAI H5N1 (9­11). Although
promising, the consensus DNA methods underestimate the true
diversity of the circulating viruses and are based on influenza
vaccine platforms not yet approved for human use.
Here we propose an ancestral sequence reconstruction method
based on a resolved phylogenetic topology to produce vaccine
candidate viruses. Ancestral sequences are computationally de-
rived sequences that represent the most recent common ancestor
of a pool of viruses (i.e., the internal nodes of a phylogenetic tree)
(12). Despite their potential benefits, it is unknown if these an-
cestral HA and NA proteins could be used to construct viable,
antigenically representative viruses able to fit within the limi-
tations of currently licensed inactivated influenza vaccine plat-
forms (13, 14). To address these issues we generated the sequence
of putative ancestral HA and NA genes and created a panel of
replication competent attenuated influenza strains. The ability of
these viruses to elicit functionally useful antibodies in the context
of inactivated vaccines was then assessed in vitro and in vivo in the
ferret model alongside two of the broadly reactive vaccine seed
strains recommended by the World Health Organization (WHO).
This study provides the proof of concept that viable vaccine seed
viruses can be generated by the synthesis of putative ancestral
sequences and that further evaluation and optimization of such
approaches is warranted.
Author contributions: M.F.D., G.J.D.S., and R.J.W. designed research; M.F.D., J.B., Y. Griffin,
E.S.-R., J.F., S.B., D.V., and A.W. performed research; M.F.D. and R.J.W. analyzed data; and
M.F.D., J.B., Y. Guan, R.G.W., G.J.D.S., and R.J.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The sequences for A/turkey/Egypt/7/2007 reported in this paper have
been deposited in the GenBank database (accession nos. CY055188­CY055195).
1
To whom correspondence should be addressed. E-mail: richard.webby@stjude.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1012457108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1012457108 PNAS | January 4, 2011 | vol. 108 | no. 1 | 349­354
MICROBIOLOGY
Results
Phylogenetic Reconstruction and Calculation of Ancestral Sequences.
To generate the putative ancestral viral antigens, phylogenetic
trees were produced from the H5 and N1 sequences available in
public databases. The phylogeny generated for the HA gene was
consistent with current H5N1 clade nomenclature (Fig. 1A). For
the NA gene, the phylogeny indicated that there were two major
groups, with and without the 20-aa deletion in the NA stalk (Fig.
1B). The putative HA ancestral aa sequences were calculated for
four nodes (Fig. 1A) and differed by only 3 to 9 aa (3 aa dif-
ference between B and C and between B and D; 5 aa between C
and D; 6 aa between A and B; and 9 aa between A and C and
between A and D). Putative NA ancestral amino acid sequences
were predicted for the root of the phylogeny (r; Fig. 1B) and at
the common ancestor for all NA genes with a 20-aa deletion in
the stalk region (d; Fig. 1B). Both NA ancestral genes differed
not only by presence or absence of 20 aa in the stalk region but
also by an additional 8 aa mutations. Sequences of the four HA
and two NA ancestral constructs are presented in Appendix S1.
Each of the four HA and two NA genes were synthesized and
cloned into pHW2000 (15).
Virus Rescue. Currently licensed H5N1 influenza vaccines are
based on the growth of vaccine seed strains in embryonated
chickens eggs or cultured cells. The seed viruses contain six gene
segments (PB2, PB1, PA, NP, M, NS) from A/Puerto Rico/8/34
and the HA and NA of the target virus (so-called 6+2 viruses).
As we wished to remain within the framework of currently li-
censed influenza vaccine platforms, we next attempted to create
replicating influenza viruses containing combinations of the
computed ancestral HA and NA genes by using plasmid-based
reverse genetics (rg). rg 6+2 viruses were successfully generated
for the different H5N1 clade representatives and for the eight
ancestral H5N1 vaccine candidates--A+d, A+r, B+d, B+r, C+d,
C+r, D+d, and D+r--showing that the computationally derived
sequences coded for functional proteins and could be used to
create a replicating virus. Moreover, the eight rescued ancestral
viruses grew to similar titers in Madin­Darby canine kidney cells
as similar strains derived from contemporary H5N1 viruses (as
shown by the end point of 50% tissue culture infectious dose;
Table S1). The four viruses containing the r N1 ancestral gene
grew to a higher titer in eggs than the viruses containing the
d N1, with a difference of as much as 3 log10 (Table S1). A+r,
B+r, C+r, and D+r were therefore selected for the work pre-
sented in this study, and are referred to as "A", "B", "C", and
"D" from now on.
Antigenic Properties of Ancestral Viruses. To assess the potential of
the four ancestral viruses A, B, C, and D, and the reference H5N1
isolates A/Vietnam/1203/04 (VN1203, clade 1), A/Hong Kong/
213/03 (HK213, clade 1), A/duck/Hunan/795/02 (DKHUN795,
clade 2.1), A/whooper swan/Mongolia/244/05 (WSM244, clade
2.2), A/turkey/Egypt/7/07 (TYEGY7, clade 2.2), A/Japanese
white-eye/Hong Kong/1038/06 (JWEHK1038, clade 2.3.4), A/
duck/Laos/3295/06 (DKLAO3295, clade 2.3.4), and A/goose/
Guiyang/337/06 (GG337, clade 4) as vaccine antigens, the cross-
reactivity of their homologous antisera toward various H5N1
viruses was studied by ELISA, hemagglutination inhibition (HI)
assay, and microneutralization (MN) assay. Table 1 summarizes
the results and shows the considerable diversity in the degree of
reactivity to the various reference viruses. Of the reference anti-
sera, only those from DKHUN795 and GG337 had ELISA titers
to the clade 1 VN1203 antigen that were within twofold of the
homologous serum values (i.e., VN1203 antiserum); three of
the four ancestral virus antisera (A, C, and D) fell within this
range. In comparison, reactivities to the clade 2.2 (A/Egypt/2321-
NAMRU3/06), 2.3.4 (JWEHK1038), and 4 (GG337) antigens
were somewhat reduced, with only the VN1203, DKHUN795, A,
and D-specific serum (and JWEHK1038 serum for GG337 anti-
gen) less than twofold lower than the homologous antiserum
values. With JWEHK1038, titers from VN1203, DKHUN795,
and D-specific serum were actually higher than that of the ho-
mologous. Of note, the clade 2.1 antigen, A/Indonesia/5/05, was
well recognized by all the sera tested (ELISA normalized titers
>0.5; Table 1). HI and MN assays both gave overall low values,
suggesting a modest cross-neutralization for ancestral as well as
reference H5N1 strains. A and D showed a slightly higher cross-
reactivity versus their ancestral counterparts in HI and MN
assays, respectively (Table 1). Overall, the postinfection ferret
antiserum raised to VN1203, DKHUN795, and ancestors A
and D were consistently the most cross-reactive, a trend also seen
in HI and MN assays and one confirming the ability of the syn-
thetic proteins to induce antibodies able to recognize a range of
H5 viruses.
Ancestral Viruses as Vaccine Antigens. To determine the ability of
the computationally derived ancestral HA and NA proteins to
act as vaccine antigens, groups of ferrets were immunized twice
with inactivated whole virus vaccines made from A and D and
then challenged with H5N1 viruses representative of currently
circulating clades: clade 1 (VN1203), clade 2.1 (DKHUN795),
and clade 2.2 (TYEGY7). As a positive control, whole virus
vaccines to the most broadly in vitro reactive reference viruses,
VN1203 and DKHUN795, were also included. Because of the
relatively mild virulence of DKHUN795 in ferrets, a reassortant
virus was used as the challenge virus in these experiments (HA
and NA of DKHUN795 and internal genes from VN1203). Table
S2 shows the differences between the HA proteins of the vaccine
antigens DKHUN795, VN1203, A, and D. DKHUN795 and D
were the most similar strains with only 3 aa differences. A and
D, A and DKHUN795, D and VN1203, A and VN1203, and
VN1203 and DKHUN795 had 9, 10, 12, 13, and 13 aa differ-
ences, respectively. Whereas mutations at position 8 (signal
peptide) and positions 494 to 525 (transmembrane domain)
likely have little effect on the immunogenicity of the vaccine
antigens, the 12 mutations in the globular head of the HA (be-
tween positions 52 and 336; Table S2) likely play a more im-
portant role. Most notably, positions 145 and 172 (129 and 156
H5 numbering) were located within antigenic sites 3 and 2, re-
spectively. Mutations at these sites distinguish VN1203 and A,
and DKHUN795 and D.
Before challenge, ferrets were bled, and the development of
antibodies against the immunizing antigen was investigated by
HI assays (Table S3). MN titers were also determined, and they
correlated with HI titers. "Protective" serum HI titers of 40
against their respective homologous strains were reached in 33%
to 83% of the vaccinated ferrets. The most immunogenic antigens
(as measured in homologous HI) were those derived from D and
DKHUN795, for which 67% and 83% of ferrets, respectively, had
HI titers of at least 40 (whereas it was true for only one third of
the A- and VN1203-vaccinated ferrets). The arithmetic mean
titers of the D- and DKHUN795-vaccinated animals were 95 and
87, respectively: significantly higher than those of the A- and
VN1203-vaccinated ferrets (37 and 26, respectively; P = 0.011,
ANOVA). The titers against the challenge strains ranged from
undetectable to equivalent to those of the homologous strains.
Mock-vaccinated ferrets did not have any detectable HI titers
against the different H5N1 strains (Table S3).
After challenge with H5N1 strains VN1203, DKHUN795, and
TYEGY7, all vaccinated ferrets survived irrespective of their
vaccine regimen (Fig. 2), and they had no significant weight loss
(Fig. S1). Mock-vaccinated animals all died or were euthanized
by day 5 or by day 8 when infected with VN1203 or DKHUN795,
respectively. Although mock-vaccinated animals challenged with
TYEGY7 survived, they lost weight (Fig. S1), were lethargic, and
showed neurological signs.
There were significant differences between vaccinated and
control groups in the amount of virus detected in the nasal
washes of DKHUN795- and TYEGY7-infected animals 3, 5, and
350 | www.pnas.org/cgi/doi/10.1073/pnas.1012457108 Ducatez et al.
1
2.1
2.2
2.3
Denotes nodes for
sequence reconstruction
2.5
2.4
8
9
5,6
4
7
0
20-aa
Deletion
No
Deletion
0.0010
Ck/FJ/1042/05
Ck/ST/810/05
Ck/YN/447/05
Dk/GY/2231/05
Anhui/1/05
Ck/Kup1NTT/BPPV6/04
Dk/GY/293/06
BHGs/QH/5/05
BHGs/QH/12/05
Ck/Indonesia/BL/03
Dk/HN/15/04
Ck/ST/4231/03
pig/Anhui/ca/04
Dk/Vietnam/15/03
Ck/ZJ/24/05
Ck/Henan/12/04
Indonesia/CDC1047/07
Anhui/2/05
Indonesia/CDC326N/06
Ck/GY/846/06
Gs/GX/4513/05
Ck/HK/YU777/02
HK/156/97
GX/1/05
Dk/GY/1418/06
Ck/GHA/3158/N3/07
Ck/HK/YU562/01
Ck/CHN/1204/04
Dk/GX/3741/05
Ck/Laos/44/04
HK/483/97
Ph/HK/FY155/01
robin/HK/75/06
Dk/CHN/E3192/03
Dk/FJ/11094/05
Ck/GY/1218/06
Ck/Korea/ES/03
Dk/YN/5133/05
Dk/Vietnam/148/04
Ck/YN/115/04
Dk/Indonesia/MS/04
Indonesia/5/05
Indonesia/CDC370E/06
Dk/Vietnam/272/05
MDk/JX/2295/05
Dk/HN/303/04
Indonesia/CDC599/06
Dk/GX/1830/06
Vietnam/CL01/04
Dk/YN/4589/05
CPH/HK/18/05
KHM/JP52a/05
Mall/Vietnam/133/04
Gs/GX/3017/05
Ck/Vietnam/10/05
Ck/ST/3840/06
Gs/YN/1136/06
Gs/GX/914/04
Ph/ST/44/04
Ck/KulPro/BBVetXII2/04
teal/CHN/2978.1/02
Japanese white-eye/HK/737/07
black bird/HN/1/04
Bei/01/03
Dk/Vietnam/48/04
Ck/Vietnam/1/04
Hatay/04
civet/Vietnam/1/05
HK/213/03
Dk/HN/5106/05
egret/HK/7572/03
Ck/THA/1/04
Gs/ST/1621/05
Ck/GX/2461/04
Vietnam/1203/04
Dk/GX/50/01
Dk/HN/101/04
Dk/FJ/668/06
Dk/GY/3242/05
Dk/GX/668/04
Ck/Nigeria/641/06
Dk/Egypt/22533/06
Ck/Indonesia/5/04
Ck/Indonesia/PA/03
Gs/Vietnam/324/01
Ck/HK/3176.3/02
Ck/GY/3570/05
Indonesia/CDC1046/07
Dk/HN/152/05
Qa/THA/57/04
Dk/GX/2775/05
sw/Italy/808/06
Dk/GX/13/04
Vietnam/JP14/05
Ck/Nigeria/1047/8/06
MDk/JX/1653/05
Ty/Israel/345/06
Ck/AFG/1207/06
Dk/HN/856/06
Dk/YN/6445/03
Ck/HK/3123.1/02
whooper swan/Mongolia/244/05
Ck/Kyoto/3/04
Indonesia/CDC625/06
Ck/FJ/11933/05
Dk/Vietnam/286/05
Ck/HK/61.9/02
Gs/GX/1458/06
Ck/AFG/1573/7/06
MDk/JX/2136/05
Indonesia/CDC887/06
Gs/Vietnam/113/01
Gs/GX/345/05
cr0w/Osaka/102/04
Ck/THA/NP/172/06
Gs/YN/1396/06
Ck/FJ/584/06
Dk/Vietnam/317/05
Gs/ST/2086/06
Gs/YN/3315/05
Gs/GX/3316/05
Ck/Yam/7/04
GZ/1/06
Ck/Sala/BBVetI/05
Ck/Laos/7192/04
Dk/Laos/3295/06
Ck/Yog/BBVetIX/04
Ck/Nongkhai/400802/07
THA/16/04
Environment/HK/437.10/99
Dk/HN/127/05
Gs/YN/1143/06
Ck/HK/FY157/03
Ck/MYS/5858/04
Ck/HN/999/05
Ck/Henan/01/04
wDk/GD/314/04
crow/Kyoto/53/04
Dk/ST/13323/05
GF/ST/1341/06
Ck/Nakhonphanom/NIAH113718/06
Dk/HK/2986.1/00
Vietnam/CL100/04
Hanoi/30408/05
Ck/GX/604/05
Ck/HK/3169.1/02
Dk/HN/1265/05
Indonesia/CDC742/06
Dk/HN/5806/03
Ck/GY/29/06
Indonesia/CDC940/06
Gs/GX/2383/04
Gs/THA/79/04
Egypt/0636/N3/07
Dk/GX/1311/04
Ck/HK/282/06
Gs/GX/582/06
Ck/GX/12/04
Gs/GY/1175/06
Ck/YN/493/05
Gs/GX/532/06
Dk/HN/795/02
Qa/MYS/6309/04
Ck/GY/2147/05
Dk/Vietnam/12/05
Gs/ST/3265/06
Ck/Sima/BPPVI/05
Ck/Egypt/1300/N3/07
Vietnam/1194/04
Ck/ST/1233/06
Ck/HK/31.4/02
Gs/GY/337/06
Ck/YN/374/04
Ck/Vietnam/398/05
Ck/GY/441/06
Gs/GD/1/96
Dk/ST/4003/03
Ty/Kedaton/BPPV3/04
Dk/YN/6255/03
Gs/ST/239/06
Ck/Wajo/BBVM/05
Dk/Vietnam/258/04
THA/676/05
Dk/GX/1681/04
Dk/Pare/BBVM/05
Ty/Egypt/7/07
Ck/HK/YU324/03
Ck/Vietnam/19/03
SCk/HK/SF189/01
Dk/GX/1378/04
Dk/Vietnam/568/05
Dk/Novosibirsk/02/05
Ck/HK/WF157/03
Ty/Turkey/1/05
Ck/GunKid/BBVW/05
Dk/YN/5236/05
CHN/GD01/06
Qa/Indonesia/BPPV4/04
Ck/HB/326/05
Ck/GY/1018/06
Dk/Vietnam/283/05
Dk/YN/4400/05
Dk/Yokohama/aq10/03
Ck/GY/3055/05
Ck/Henan/210/04
Sw/Astrakhan/1/05
Gs/Suzdalka/10/05
Ck/KHM/013LC1b/05
Gs/GY/3422/05
Gs/GX/52/06
Ty/15/06
Egypt/14724/N3/06
D
C
B
A
0.0010
Gs/ST/2086/06
magpie robin/HK/75/06
Ck/Vietnam/133/04
Thailand/676/05
Dk/YN/5133/05
Vietnam/CL01/04
Vietnam/1203/04
Ck/GY/3570/05
Vietnam/1194/04
China/GD01/06
MDk/JX/2136/05
Dk/Egypt/2253-3/06
Ck/HK/YU324/03
Ck/Korea/ES/03
Ck/HK/282/06
Ck/Henan/210/04
Gs/GX/582/06
Gs/ST/239/06
Ck/Henan/01/04
Dk/Vietnam/48/04
Dk/HN/152/05
Ck/Vietnam/10/05
Ck/HK/3176.3/02
Dk/Vietnam/317/05
Ck/Afghanistan/1207/06
crow/Kyoto/53/04
Dk/Vietnam/272/05
Ck/Indonesia/BL/03
Ck/FJ/10567/05
Hatay/04/
Qa/Boyolali/BPPV4/04
crow/Osaka/102/04
Dk/HN/795/02
Ck/Ghana/3158-NAMRU3/07
Ck/Afghanistan/1573-47/06
Dk/FJ/668/06
Dk/YN/4589/05
Dk/GY/3242/05
Ck/Kyoto/3/04
Dk/ST/13323/05
Chinese pond heron/HK/18/05
Gs/GY/337/06
Dk/GX/13/04
Indonesia/546bH/06
Ck/GX/2461/04
Ck/HK/YU777/02
Gs/GX/4513/05
Vietnam/PEV16T/05
Dk/GX/3741/05
cygnus olor/Italy/808/06
Ck/Malaysia/5858/04
Dk/Vietnam/148/04
Turkey/15/06
Dk/Vietnam/15/03
Dk/Novosibirsk/02/05
Ck/GY/2147/05
Dk/Vietnam/12/05
Ck/YN/447/05
Dk/Vietnam/568/05
Ty/Egypt/7/07-PCR
Dk/GX/2775/05
Dk/GX/1681/04
Ck/Pangkalpinang/BPPV3/04
Guangzhou/1/06
Ck/ST/3840/06
blackbird/HN/1/04
Dk/GY/2231/05
Ck/GY/29/06
Ck/HK/3169.1/02
Gs/GY/3422/05
Ck/HK/3123.1/02
Egypt/14724-NAMRU3/06
Ck/Indonesia/5/04
Thailand/16/04
Ck/GY/3055/05
Dk/YN/5236/05
Dk/HK/2986.1/00
Ck/Vietnam/19/03
Ck/Nigeria/1047-8/06
Ck/GY/441/06
Ck/ST/4231/03
Ck/HK/YU562/01
Indonesia/5/05
Gs/ST/3265/06
Vietnam/CL100/04
Ck/Henan/12/04
Ck/YN/374/04
MDk/JX/1653/05
Ck/Cambodia/013LC1b/05
Ck/YN/493/05
Ck/Zhejiang/24/05
Ck/Thailand/1/04
Ck/HK/WF157/03
Ck/Simalanggang/BPPVI/05
Hanoi/30408/05
Guinea fowl/ST/1341/06
Dk/GX/1830/06
Cambodia/JP52a/05
Dk/YN/4400/05
Gs/GD/1/96
Ph/ST/44/04
Ck/GX/12/04
Ck/FJ/1042/05
Dk/Yokohama/aq10/03
Ck/HN/999/05
Dk/GX/1378/04
BHGs/QH/5/05
swan/Astrakhan/1/05
Ck/Nigeria/641/06
Ck/Indonesia/PA/03
HK/213/03
Dk/YN/6445/03
Ck/ST/810/05
Dk/Laos/3295/06
Ck/HK/61.9/02
Ck/GX/604/05
Ty/Israel/345/06
Ck/HK/31.4/02
Dk/GX/1311/04
Ck/FJ/11933/05
Ck/Thailand/NP-172/06
Dk/HN/101/04
Gs/ST/1621/05
Dk/HN/5106/05
Gs/Vietnam/113/01
BHGs/QH/12/05
whooper swan/Mongolia/244/05
Ck/ST/1233/06
Gs/YN/3315/05
WDk/GD/314/04
Dk/HN/127/05
Owston's civet/Vietnam/1/05
Dk/FJ/11094/05
Gs/GX/914/04
Ck/HK/FY157/03
Dk/HN/5806/03
Gs/GX/1458/06
Egypt/0636-NAMRU3/07
Gs/Suzdalka/10/05
Gs/GX/532/06
Dk/GY/293/06
Ck/China/1204/04
Beijing/01/03
Ck/Vietnam/398/05
Gs/GX/2383/04
Qa/Malaysia/6309/05
Gs/Thailand/79/04
Japanese white-eye/HK/1038/06
Dk/Vietnam/258/04
teal/China/2978.1/02
SCk/HK/SF189/01
Dk/ST/4003/03
MDk/JX/2295/05
Dk/Vietnam/283/05
Dk/HN/1265/05
Dk/GX/50/01
Anhui/1/05
Environment/HK/437-10/99
Dk/China/E319-2/03
Vietnam/JP14/05
Gs/GX/345/05
Qa/Thailand/57/04
Ck/Hebei/326/05
Ph/HK/FY155/01
Ck/Vietnam/1/04
Ck/Yamaguchi/7/04
Dk/HN/303/04
Dk/Vietnam/286/05
swine/Anhui/ca/04
Ck/YN/115/04
Gs/GX/668/04
Ck/Gunung Kidal/BBVW/05
Ty/Turkey/1/05
Dk/YN/6255/03
Gs/GX/3017/05
Gs/GX/3316/05
r
d
97.1
72.1
100
74
79.2
65.8
80.3
100
98.5
94.9
87.3
99.6
91.8
100
99.8
94.7
81.5
90.8
98.3
100
95.2
99
100
100
96.3
72.7
72.8
99.3
100
97.5
82.3
100
93.6
100
99.7
89
94
89.3
100
88.4
100
54
97.9
100
96
100
69.7
100
98.3
99.4
82
98.2
97.4
99.4
90.7
92.5
95.4
95.9
99.3
100
97.5
99.8
100
97.6
54.2
70.4
96.3
99.9
82.3
98.3
92.8
98
86.3
98
99.2
100
60.8
81.6
86.7
94.8
58.3
96.5
100
98.5
99
Fig. 1. Phylogenetic relationship of influenza A viruses used to calculate putative ancestral sequences. Four nodes on the HA gene (a) were chosen to
represent (A) the midpoint root of the tree; (B) the ancestral node common to clades 1, 2.1, 2.2, 2.3, 2.4, 2.5, and 8; (C) the ancestral node to clade 1 viruses;
and (D) the ancestral node for all clade 2 viruses. For the NA gene (b), two nodes were predicted, one at the root of the phylogeny (r) and another at the
common ancestor for all NA genes with a 20-aa deletion in the stalk region (d). Numbers at branch nodes indicate neighbor-joining bootstrap values of at
least 70% for major clades. Analyses were based on nucleotides 1 to 1,707 and 1 to 1,407 of the HA and NA genes, respectively. The HA tree was rooted to
A/goose/Guangdong/1/96 and the NA tree was midpoint rooted. Numbers to the right of the figure refer to WHO H5N1 clade designations (5). (Scale bar,
0.001 substitutions per site.) Viruses used in the present study are in red font.
Ducatez et al. PNAS | January 4, 2011 | vol. 108 | no. 1 | 351
MICROBIOLOGY
7 d after infection (P < 0.01; Fig. 3). Despite a trend for lower
viral titers in vaccinated versus control animals 3 d after infection
in the VN1203 challenge, the differences were not significant. All
VN1203 control animals died by 5 d after infection. No statisti-
cally significant differences were observed among the different
vaccine groups for any of the three challenge viruses, indicating
that the ancestral vaccine antigens were able to induce anti-
bodies that were indeed protective and generally similar in na-
ture to those induced by reference viruses.
Discussion
The challenge in influenza vaccination is that the most potent
form of immunity, neutralizing antibodies, targets the most
variable viral protein, HA. This dilemma has proven to be es-
pecially challenging in the context of developing H5N1 stockpile
vaccines, because the virus has evolved into several antigenically
and genetically distinct clades in the 14 y it is known to have
circulated (16). The current solution of the WHO to this di-
versification is the creation of additional vaccine seed strains to
cover this diversity; the current tally of such seed stocks is 13,
with six more in various stages of development (17). Newer
approaches must be evaluated, as not only is it economically
restrictive to produce vaccines from each seed virus, but it is also
becoming less clear which are the most useful seed strains for
a given region. Our hypothesis that viable viruses derived from
computationally generated ancestral HA and NA proteins could
be generated was validated.
We found that each of the four computationally derived HA
proteins and the two NA proteins were functional and able to be
incorporated into replicating influenza viruses through the use of
the eight-plasmid reverse genetics system (15). We did find,
however, that viruses containing the NA protein with a stalk
deletion were less able to replicate in chicken eggs. Castrucci and
Kawaoka demonstrated in 1993 that mutated A/WSN/33 (H1N1)
strains with a shortened stalk replicated poorly in eggs and did
not cause systemic disease (18). In 2009 Matsuoka et al. (19) and
Zhou et al. (20) both showed higher virulence of H5N1 viruses
with longer NA stalks in chickens, but their studies reported
varying effects in mice (19, 20). Although isolates with the NA
stalk deletion did grow, we did not proceed with them because of
the importance of egg growth for current manufacturing of in-
fluenza vaccines. In addition, the immunogenicity of the varying
Table 1. ELISA, HI and MN titers in infected ferret serum
Antigen
Antiserum/
titer
Clade 1
(VN1203)
Clade 2.1 (A/Indonesia/5/05 or
DKHUN795)
Clade 2.2 (A/Egypt/2321-NAMRU3/06 or
WSM244)
Clade 2.3.4
(JWEHK1038)
Clade 4
(GG337)
VN1203
ELISA 1 (135) 0.75 0.95 2.1 3.6
HI 1 (26) 0.33 0.29 0.09 0.79
MN 1 (113) 0.13 0.50 0.02 0.63
DKHUN795
ELISA 0.71 1 (80) 1.76 1.9 1.1
HI 0.16 1 (160) 0.88 0.08 0.06
MN 0.16 1 (842) 0.90 0.03 0.10
WSM244
ELISA 0.27 0.63 1 (113) 0.27 0.33
HI 0 0.24 1 (74) 0.02 0.05
MN 0.53 0.94 1 (125) 0.17 0.43
JWEHK1038
ELISA 0.25 0.56 0.40 1 (57) 1.1
HI 0 0.13 0.06 1 (37) 0
MN 0.10 0.12 0.29 1 (258) 0.26
GG337
ELISA 0.89 1.23 0.18 0.47 1 (40)
HI 0.12 0 0 0 1 (226)
MN 0.13 0.02 0.11 0.01 1 (1,417)
A
ELISA 0.79 0.75 0.60 1 0.55
HI 0.28 0.03 0.53 1.28 0.31
MN 0.16 0.12 0.21 0.12 0.19
B
ELISA 0.32 0.56 0.12 0.4 0.33
HI 0.02 0.09 0.11 0.70 0.05
MN 0.10 0.11 0.19 0.08 0.11
C
ELISA 0.57 0.69 0.02 0.23 0.22
HI 0.25 0.22 0.23 0.34 0.04
MN 0.11 0.08 0.11 0.05 0.08
D
ELISA 0.79 0.88 0.88 3.9 4.9
HI 0.02 0.21 0.35 0.63 0
MN 0.13 0.21 1.18 0.17 0.17
Normalized titer: ratio of heterologous titer to homologous titer, ratio calculated animal per animal. Clade-homologous titers have both normalized and
geometric mean titers (e.g., "1 (135)" for VN1203 ELISA titer) and are indicated in italics. Four ferret sera were tested against each antigen. Geometric mean
titers are indicated in parentheses.
352 | www.pnas.org/cgi/doi/10.1073/pnas.1012457108 Ducatez et al.
ancestral NA proteins was not tested here because of the greater
importance of HA-specific antibodies in the protective response.
Although we found a growth advantage for viruses containing the
ancestral NAs without deletions, it is interesting that this was not
necessarily seen with reassortant viruses containing WT NAs; good
egg growth properties were seen with reassortant viruses contain-
ing WT NAs with stalk deletions. The reasons underlying egg
growth phenotypes remain unclear, but it is clear that factors other
than the length of the NA stalk can also play important roles.
The premise of the ancestral virus approach is that such
viruses may be broadly representative of the population of in-
fluenza viruses circulating. Even though this study was primarily
designed as a proof of principle of the approach, we did evaluate
the ability of the ancestral proteins to induce antibodies able to
bind to H5N1 influenza strains. Despite some heterogeneity,
antiserum specific to the A and D antigens was able to recognize
each of the reference antigens with less than a 50% ELISA titer
decrease compared with the values for the homologous antigens.
Interestingly, antigen A induced cross-reactive antiserum as this
virus contains the putative ancestor HA antigen of all circu-
lating clades. Given the basal position of virus A, the broad cross-
reactivity of its antiserum appears to confirm the rationale for
these experiments.
Although the approach used in this study incorporates the
evolutionary history of the virus population, the method is de-
pendent on sampled virus sequences, a large portion of which
have been collected from disease outbreaks and not systematic
surveillance. As a result, we often see long branches in the
phylogenetic trees that indicate extended periods of unsampled
diversity. This sampling bias may potentially affect ancestral se-
quence reconstruction to, in some instances, produce a sub-
optimal antigen. However, advanced methods that incorporate
the phylogenetic uncertainty along with antigenic cartography
into the vaccine design could overcome these limitations. The
biological factors underlying the antigenic differences of the
antigens tested in this study are unknown but likely related to
differences in immunodominant antigenic site residues or gly-
cosylation patterns, which should be more deeply evaluated. A,
B, C, and D all contain the same number of potential glycosyl-
ation sites (n = 6), although differences were observed in anti-
genic site 1 residues 140 and 145 (H5 numbering in ref. 21).
In addition to the ability of computationally derived HAs to
induce antibodies that react with H5N1 viruses, we were also
able to show that these antibodies were indeed protective. Our
ferret challenge experiments were not powered to detect dif-
ferences between any of the tested antigens but rather to provide
support for their immunogenic nature. Whole-virus vaccines
based on A and D viruses conferred protection to ferrets against
morbidity and mortality when challenged with clade 1 (VN1203),
clade 2.1 (DKHUN795), and clade 2.2 (TYEGY7) viruses that
was of at least equal efficacy to that induced by the most cross-
reactive reference viruses. Although it is possible that all four
antigens are equally effective, further analysis with split vaccines
(whole-virus preparations are inherently more immunogenic but
also rarely used in humans in view of associated febrile respon-
ses), titrations of antigen dose, and more divergent challenge
viruses are warranted and required to test the hypotheses that
the ancestral antigens are more cross-reactive than some or all
naturally occurring counterparts. Nevertheless, the ability of
A B C
Fig. 2. Ferrets' survival rates after infection with VN1203 (A), DKHUN795 (B), and TYEGY7 (C). Control (mock-vaccinated) ferrets are represented by red open
squares. Ferrets vaccinated with ancestral strains are represented by closed blue triangles: A in light blue and D in dark blue. Ferrets vaccinated with positive
control isolate-based strains are represented by green closed diamonds: VN1203 in light green and DKHUN795 in dark green.
Fig. 3. Ferrets' nasal wash titers after infection with VN1203 (A),
DKHUN795 (B), and TYEGY7 (C). The mean virus titers of nasal washes for
the four animals per group (same vaccine and same challenge virus) are
expressed in log10 EID50/mL ± SEM (st er). Titers are grouped by vaccination
regimen on the x axis. Black, white, and gray filled bars represent titers 3, 5,
and 7 d after infection, respectively. (*P  0.05 and ***P  0.001 by ANOVA
comparing the five vaccine regimen groups virus titers.) Slashes separate
ANOVA P values at days 3, 5, and 7. [#
All mock-vaccinated (control) VN1203-
infected ferrets were dead before nasal washes could be collected at days 5
and 7 after infection.]
Ducatez et al. PNAS | January 4, 2011 | vol. 108 | no. 1 | 353
MICROBIOLOGY
single antigens to protect across a range of challenge viruses is
encouraging from the standpoint of vaccine stockpiling, as has
been suggested by other ferret (22, 23, 6, 24) and human studies
(25). Although we observed no mortality or morbidity in H5N1
cross-clade ferret challenges, Govorkova et al. (22) challenged
HK213 (clade 1)-vaccinated animals with A/Hong Kong/156/97
(clade 0, 96% HA aa identity with the vaccine strain, excluding
the cleavage peptide) and reported morbidity but no mortality.
Baras et al. (23) used an adjuvanted VN1203 (clade 1)-based
split vaccine and were able to show that it protected 96% of
ferrets against mortality when challenged with homologous or
clade 2.1 (A/Indonesia/5/05, 97% HA aa identity with the vac-
cine) strains. Finally, Forrest et al. (6) showed ferrets immunized
with inactivated whole JWEHK1038 (clade 2.3) were partially
protected from VN1203 (clade 1, 96% HA aa identity with the
vaccine) challenge. One of the difficulties of evaluating and
comparing vaccine studies using different H5N1 challenge viru-
ses is the substantial differences in virus lethality. In our study,
this was overcome by generating a reassortant virus carrying
the HA and NA of DKHUN795 on the backbone of VN1203.
The parental DKHUN795 virus is not lethal in ferrets, whereas
this reassortant virus was. Similarly, it has been previously shown
that substitution of the polymerase genes (PB2, PB1, and PA) of
A/chicken/Vietnam/C58/04 for those of VN1203 was able to
convert this virus from sublethal to lethal in ferrets (26).
In summary, we were able to provide proof of concept that
a most recent common ancestor computational method can be
used to design H5N1 vaccine by using methodologies consistent
with currently licensed vaccine platforms. We obtained repli-
cating viruses able to induce antibodies consistent with that of
reference strains that should be further examined as a means to
increase the breadth of such responses.
Materials and Methods
Generation of Ancestral Sequences. Ancestral amino acid sequences were
calculated from nucleotide alignments used to generate the HA and NA
phylogenetic trees. A marginal reconstruction method as implemented in
ANCESCON (26) was used with default settings except that a maximum
likelihood rate factor was used. Marginal reconstruction compares the
probabilities of different amino acids at an internal node at a site, and the
amino acid that yields the maximum likelihood for the tree at that site is
then selected (26). This method also takes into account the variability of
substitution rates among sites. By using this method, ancestral protein
sequences were determined at four nodes of the HA phylogenetic tree and
two nodes of the NA phylogenetic tree. To generate HA and NA nucleotide
sequences that expressed the putative ancestral proteins, the proteins were
compared with sequences available in GenBank using BLAST and then the
minimum mutations to code for phenotypic changes were made. The con-
necting peptide was modified to match that of low-pathogenicity viruses so
that the reverse genetic strains could be used in biosafety level (BSL)-2+
laboratories.
Ferrets Immunization and Challenge. Vaccines in two doses given at a 3-wk
interval were injected intramuscularly into 3- to 4-mo-old ferrets. Each dose
of vaccine contained 7.5 g of rg VN1203, DKHUN795, A, or D HA (15 g of
HA total per ferret). Twelve ferrets were vaccinated with each vaccine: rg
6+2 A, D, VN1203, and DKHUN795. Twelve control ferrets were given PBS
solution only. Three weeks after the second (i.e., boost) dose of vaccine,
ferrets were anesthetized with isoflurane and inoculated intranasally with
106
50% egg infectious doses (EID50s) of a HPAI H5N1 challenge virus
(VN1203, DKHUN795, and TYEG7, representative of different clades) in
a BSL-3+ laboratory. Ferrets were then monitored daily for 14 d for weight
change, temperature, and clinical disease signs. Body temperature was
measured via transponders s.c. implanted between the shoulder blades
(BioMedic Data Systems).
A more complete description of the materials and methods used in the
present study is available in SI Materials and Methods.
ACKNOWLEDGMENTS. From St. Jude Children's Research Hospital, we thank
J. DeBeauchamp, D. Carey, and Dr. V. R. Pagala for excellent technical assis-
tance; S. Krauss and D. Walker for providing reagents; A. C. Boon for critically
reviewing the manuscript; and D. Galloway and K. Spelshouse for editorial
and graphic assistance. We thank Drs. M. A. Ali, M. A. Kutkat, and M. Bahgat
(Virology Laboratory, Center of Excellence for Advanced Sciences, National
Research Center, Giza, Egypt) for providing A/Turkey/Egypt/7/2007. This work
and G.J.D.S.'s career development award were supported by Contract
HHSN266200700005C from the National Institute of Allergy and Infectious
Disease, National Institute of Health, Department of Health and Human
Services, and by the American Lebanese Syrian Associated Charities. J.B., D.V.,
and G.J.D.S. are supported by the Duke­National University of Singapore Sig-
nature Research Program funded by the Agency for Science, Technology and
Research, Singapore, and the Ministry of Health, Singapore.
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