Vaccine 28 (2010) 594–596
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Alphavirus replicon particle vaccines developed for use in humans induce high
levels of antibodies to influenza virus hemagglutinin in swine: Proof of concept
M.M. Erdmana, K.I. Kamrudb, D.L. Harrisc,∗, J. Smithb
aVeterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, 11 Kildee, Ames, IA 50011, United States
bAlphavax, Inc., 2 Triangle Drive, Research Triangle Park, NC 27709-0307, United States
cAnimal Science, College of Agriculture and Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine,
Iowa State University, Ames, IA 50011, United States
a r t i c l ei n f o
Received 6 July 2009
Received in revised form 2 October 2009
Accepted 5 October 2009
Available online 22 October 2009
a b s t r a c t
A propagation-defective, single-cycle, alphavirus replicon particle (RP) system was used to produce two
vaccines against human influenza virus A/Wyoming/03/2003 (H3N2). One vaccine was prepared from
Venezeulan equine encephalitis virus (VEEV) strain 3014 and the other from VEEV strain TC-83. Both
vaccines induced high antibody titers to the influenza hemagglutinin (HA) protein and illustrated the
potential of using alphavirus RP influenza vaccines in swine.
© 2009 Elsevier Ltd. All rights reserved.
outbreaks could include the preparation of alphavirus (Togaviri-
dae) replicon particle (RP) vaccines for prevention of disease in
defective, single-cycle vectors which cannot spread from cell to
cell. Alphavirus RP is devoid of the structural genes of the parent
of interest in cells of the vaccine recipient. Since alphaviruses have
a wide host range, they could make ideal vaccine vectors for rapid
response immunization of a variety of animal species as well as
humans in impending pandemic disease outbreaks .
Alphavirus RP vaccines have been studied extensively in recent
agents in a number of different hosts . Only one alphavirus is
known to infect swine, Sagiyama virus, but appears to be restricted
geographically to Asia . Venezeulan equine encephalitis virus
(VEEV) has been experimentally inoculated into swine, but only
∗Corresponding author. Tel.: +1 515 294 1664; fax: +1 515 294 5294.
E-mail address: email@example.com (D.L. Harris).
induced a transient viremia . Antibodies to VEEV have been
reported in feral swine after an epizootic of VEEV in horses in cen-
tral America and Texas in 1971 . Influenza virus hemagglutinin
(HA) expressed by a VEEV replicon system has been shown to pro-
tect chickens against H5N1 influenza virus . There have been no
reports of VEEV virus RP vaccines being evaluated in swine.
Whole virus inactivated vaccines for influenza virus are com-
monly used in swine . For many years, classic H1N1 influenza
virus was predominant in the United States swine population.
However, in 1998, double and triple reassortant strains began to
appear in swine which have increased the need for vaccines effec-
rently co-circulate in swine [8,9].
Both humans and swine are susceptible to the same influenza
A subtypes H1N1, H3N2, and H1N2 [11,12]. Antibodies to swine
tact with swine than those not exposed . A triple reassortant
swine influenza virus (H3N2) infected and caused disease in a child
living in a communal farm containing swine and seropositivity
occurred in household and non-household members . Thus the
potential for both direct and community transmission outbreaks of
human influenza originating from swine exists. Recently, a novel
0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
M.M. Erdman et al. / Vaccine 28 (2010) 594–596
H1N1 strain of influenza occurred in humans in Mexico and rapidly
spread to the U.S. . This strain of H1N1 was reported to be dif-
. On May1, 2009 the novel strain occurred in a swine herd in
Alberta, Canada causing disease in both swine and humans .
Experimentally, the novel H1N1 strain (A/CA/04/2009) obtained
from a human patient in California was found to infect and cause
disease in pigs . Takano et al. have shown that H5N1 strains of
influenza virus which have been transmitted to swine in Indonesia
became attenuated for mice during their replication in the pig .
These workers suggest that the attenuation of influenza virus by
pig passage may prepare the virus for swine to human transmis-
sion. In a pandemic situation, a vaccine prepared for prevention of
influenza in humans may be of benefit for use in swine.
developed for use in humans induce protective antibody levels to
H3N2 influenza virus in swine. One vaccine was derived from the
3014 strain of VEEV and the other from the TC-83 VEEV strain, a
live-attenuated vaccine strain with extensive use in both human
and veterinary applications .
2. Materials and method
Pigs were obtained from a farm free of swine influenza virus
and porcine reproductive and respiratory syndrome virus (PRRSV)
at 2 weeks of age, weighed, identity tagged, and randomized into 3
groups of 4 pigs each. The animal experiments were approved by
the Iowa State University Institutional Animal Care and Use Com-
mittee. Prior to vaccination, serum was collected to assure that
the pigs were free of influenza hemagglutination inhibition (HI)
antibodies as determined against swine strain H3N2 and human
strain A/Wyoming/3/2003 H3N2 (CDC# 2003714420). Sera were
collected throughout the study for antibody detection using the
HI assay and an HA ELISA using a purified, homologous HA anti-
gen (Protein Sciences, Meriden, CT) as described . An alphavirus
replicon plasmid containing the HA gene from A/Wyoming/3/2003
(H3N2) strain of influenza virus was constructed as previously
described [1,20]. An alphavirus replicon plasmid containing the
ORF6 gene of porcine reproductive respiratory syndrome virus
particles were generated using VEEV vectors derived from either
Design of alphavirus replicon particle (RP) vaccine study in pigs. Pigs received either
RP expressing the influenza hemagglutinin (HA) protein or negative control RP
expressing M protein of porcine reproductive respiratory syndrome virus (PRRSV).
All vaccines were given intramuscular.
Group (n) TreatmentDose Vaccination schedule
PRRSV M (Neg control)
TC-83 HA RP (IM)
3014 HA RP (IM)
Day 0 prime, day 14 boost
Day 0 prime, day 14 boost
Day 0 prime, day 14 boost
V3014 or TC-83 VEEV strains as previously described [21,22]. A
prime/boost immunization schedule of 2ml of 108RP per intra-
muscular injection was performed (Table 1) with pigs receiving
the first dose at 5 weeks of age. Pigs received either RP express-
ing the influenza hemagglutinin (HA) protein or negative control
RP expressing M protein of PRRSV.
A robust HI antibody response was induced in pigs receiving
RPs prepared from either VEEV strains 3014 or TC-83 (Table 2). HI
antibodies were maintained through day 62 of the experiment at
similar levels (data not shown). This response was initiated follow-
virus, based on previous reports this level of HI antibody would
in Table 2 and were elevated prior to the booster injection.
These results indicate that in the event of a need to prevent
the spread of a human pandemic strain of influenza virus or the
spread of a novel animal subtype among the animal population, an
alphavirus RP vaccine designed for use in humans could be used in
swine. These RP vaccines induce both humoral and cell mediated
responses to the heterologous protein expressed, and have been
protective in many animal models of infectious, toxin-mediated,
and neoplastic diseases . In addition, these results indicate that
Hemagglutination inhibition (HI) and HA ELISA antibody results (inverse dilutions) of pigs immunized with RP expressing the influenza HA protein (groups 2 and 3 in Table 1)
as compared to negative control vaccinated pigs. Pigs were immunized on day 0 at 5 weeks of age, boosted on day 14 and necropsied on day 62.
Pre-vaccination 14 days post-prime7 days post-boost*
HIELISA HIELISA HIELISA
HA RP (3014)
HA RP (TC-83)
Negative control RP
GMT: geometric mean titer.
*Both HA RP groups had significantly different HI and ELISA titers compared to the negative control group at 7 days post-boost (ANOVA, p<0.05).
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M.M. Erdman et al. / Vaccine 28 (2010) 594–596
RP derived from VEEV strains TC-83 and V3014 induce equivalent
immune responses in swine suggesting that either system would
be effective for vaccine development against a variety of infectious
Gray and Kayali recently suggested that swine and poultry
workers be included in preparedness plans for future influenza
pandemics . Robinson et al. suggest that surveillance for swine
influenza virus cases among swine and poultry workers may be the
best approach for early warning of an impending pandemic .
The reproductive rate R0for an H1N1 swine influenza outbreak in
humans in 1976 was calculated to be only 1.1–1.2 . However,
future reassortant strains of swine influenza virus when transmit-
ted to humans could occur with a higher R0and be detected in time
for the rapid development of alphavirus RP vaccines to be used in
humans, swine, and poultry. To date, the novel H1N1 strain has not
been reported to occur in swine in any other country but Canada;
however, it is possible that pre-emptive immunization of swine
prior to novel H1N1 introduction into the swine herd in a particu-
The authors thank Brenda Crabtree, Stephen Gaul, Ryan Vander
Veen, and Mark Mogler of Iowa State University, Ames, Iowa, USA
and Kim Alterson and Max Custer of AlphaVax Inc, Research Trian-
gle Park, North Carolina, USA for technical assistance. The authors
also thank Drs. Amy Vincent and Isabel Harris for their respective
the National Pork Board, the Iowa Pork Producers Association, and
the Grow Iowa Values Fund.
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