Swine flu

Swine influenza vaccines: current status and future perspectives

Wenjun Ma and Jürgen A. Richt*

Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State

University, Manhattan, KS, USA

Received 15 February 2010; Accepted 8 April 2010; First published online 13 May 2010

Abstract Swine influenza is an important contagious disease in pigs caused by influenza A viruses.

Although only three subtypes of influenza A viruses, H1N1, H1N2 and H3N2, predominantly

infect pigs worldwide, it is still a big challenge for vaccine manufacturers to produce efficacious

vaccines for the prevention and control of swine influenza. Swine influenza viruses not only

cause significant economic losses for the swine industry, but are also important zoonotic

pathogens. Vaccination is still one of the most important and effective strategies to prevent and

control influenza for both the animal and human population. In this review, we will discuss the

current status of swine influenza worldwide as well as current and future options to control this

economically important swine disease.

Keywords: swine influenza, vaccines

Introduction

Swine influenza is one of the most important respiratory

diseases in pigs. The recent pandemic H1N1 virus is

genetically very similar to influenza viruses that occur in

swine and has been transmitted from humans to other

species including pigs. Control of virus spread among

herds and prevention of possible transmission to humans

can be achieved through the vaccination of swine. Even

though only three subtypes of influenza A viruses, H1N1,

H1N2 and H3N2 predominantly infect pigs worldwide,

current commercially available inactivated vaccines for

swine are not highly efficacious (at least in North

America) due to the multitude of genetically diverse

viruses co-circulating in swine herds today. Since swine

are susceptible to both human and avian influenza

viruses, viral reassortment can occur in pigs allowing

the generation of novel viruses which might be the cause

of a human pandemic (Brown, 2008). These are major

concerns to both the swine industry and public health.

Control of influenza infection in swine is critical not

only for the reduction of disease symptoms and econ-

omic losses but also to limit potential viral reassortment,

cross-species adaptation and the spread of influenza

viruses. This review will discuss the current epidemi-

ological situation of swine influenza virus (SIV) world-

wide and the challenges faced by current commercially

available inactivated vaccines. We will focus on various

strategies to develop future swine influenza vaccines such

as live-attenuated, subunit, vectored and DNA vaccines.

Influenza A virus and swine influenza

Swine influenza is caused by influenza A virus, a genus

within the family Orthomyxoviridae. Influenza A virus

is a negative, single-stranded RNA virus whose genome

consists of eight gene segments that encode 10 or 11 viral

proteins. The glycoprotein hemagglutinin (HA) and

neuraminidase (NA) are encoded by segments 4 and 6,

respectively; both are located on the surface of the virus

and are responsible for viral entry (HA) and efficient viral

release from the infected cells (NA). In addition, these

surface proteins are highly variable and are the major

targets of the host humoral immune response. Segment 7

of influenza A viruses encodes the M1 and M2 proteins.

The M2 protein is integrated into the viral envelope

and its ion channel activity is required for efficient viral

uncoating during virus invasion of cells. The M1 protein*Corresponding author. E-mail: [email protected]

*c Cambridge University Press 2010 Animal Health Research Reviews 11(1); 81–96 ISSN 1466-2523 doi:10.1017/S146625231000006X

lies beneath the virus envelope and is thought to be

critical for virus assembly and budding. The eight viral

RNA segments together with the nucleoprotein (NP,

segment 5) and the viral polymerase proteins, PB2

(segment 1), PB1 (segment 2) and PA (segment 3), form

the ribonucleoprotein complex that participates in RNA

replication and transcription. Segment 8 encodes for the

non-structural protein 1 (NS1) and NS2 or nuclear export

protein (NEP). The major function of the NS1 is to

modulate the type I interferon (IFN) response of the host

(Garcia-Sastre et al., 1998). The viral NEP has been found

in the virus particle and is required for the export of the

viral RNA from the nucleus to the cytoplasm of infected

cells (O’Neill et al., 1998). In addition, PB1 using an

alternative open reading frame in many influenza A

viruses encodes a pro-apoptotic factor called PB1-F2

(Chanturiya et al., 2004), which modulates virulence and

severity of secondary bacterial infections (Zamarin et al.,

2006; Conenello et al., 2007; McAuley et al., 2007).

The most significant characteristic of influenza A virus

is its enormous genetic variability, which presents an

immense challenge in the control and prevention of dis-

ease. Two major mechanisms contribute to this: antigenic

drift (random mutations within individual genes) and

antigenic shift. Antigenic shift or reassortment occurs

when two or more different influenza A viruses infect the

same cell and a mixing of RNA segments results in novel

reassortant viruses.

Influenza A viruses are divided into subtypes based

on the antigenic nature of their HA and NA glycoproteins.

Currently, 16 HA subtypes (H1–H16) and 9 NA subtypes

(N1–N9) have been isolated from wild waterfowl and

seabirds (Webster et al., 2006; Wright et al., 2007).

Although aquatic birds are the major reservoir for influ-

enza A viruses, pigs play an important role in the

transmission of novel viruses to humans by acting as a

‘mixing vessel’ (Scholtissek, 1994; Brown, 2008; Ma et al.,

2009a), since human, avian and SIVs can replicate in pigs

(Ito et al., 1998; Ma et al., 2009a).

SIV worldwide

Swine influenza in pigs leads to fever, lethargy, sneezing,

coughing, labored breathing and decreased appetite;

it presents with high morbidity (approaching 100%) and

generally low mortality (<1%) rates. Despite the low

mortality in herds, it is still an economically important

infectious disease for the swine industry. The following

subtypes of influenza A virus predominantly infect pigs

worldwide.

Classical H1N1 virus

Swine influenza was first recognized in 1918 in the

USA, Hungary and China, coinciding with the 1918

Spanish pandemic in humans (Webster, 2002). The first

SIV isolate belonging to the H1N1 subtype was obtained

in 1930 from U.S. pigs (Shope, 1931); subsequently, a

similar virus was isolated from humans (Smith et al.,

1933). This H1N1 swine virus and closely related viruses

are designated classical H1N1 (cH1N1) viruses. For the

next 50 years, SIVs were almost exclusively cH1N1 virus

in swine populations worldwide. The cH1N1 viruses

began disappearing from the European pig populations

after 1979 with the emergence of the avian-like H1N1

virus (Pensaert et al., 1981). In North America, the

cH1N1 virus was relatively conserved as the predominant

virus until 1998 (Hinshaw et al., 1978; Chambers et al.,

1991; Olsen et al., 2000). To date, the cH1N1 viruses are

still the predominant viruses in Asian pigs (Liu et al.,

2009).

Avian-like H1N1 virus

In 1979, an avian-like H1N1 virus emerged in European

swine populations. The virus is antigenically and geneti-

cally distinguishable from the cH1N1 SIVs (Scholtissek

et al., 1983; Brown et al., 1997), and has quickly re-

placed the cH1N1 viruses in European pigs (Brown,

2000). All eight gene segments of this avian-like H1N1

prototype virus are directly derived from Eurasian avian

influenza viruses without reassortment with other (human

or swine) viruses (Dunham et al., 2009). Currently, this

avian-like virus is co-circulating in the European pig

populations with swine influenza H3N2 and H1N2 sub-

types. Recently, European avian-like H1N1 viruses have

been isolated from pigs in China (Liu et al., 2009; Yu et al.,

2009).

Reassortant H3N2, H1N2 and H1N1 viruses

Asia Following the 1968 H3N2 human pandemic, human-like

H3N2 viruses and cH1N1 viruses were co-circulating

widely in Asian and European pig populations (Kundin,

1970; Haesebrouck et al., 1985), producing double

reassortant H1N2 viruses through reassortment; the latter

have become widespread in pigs and continue to

circulate in pigs in Asia (Ouchi et al., 1996; Jung and

Chae, 2004; Qi and Lu, 2006). Recently, double reassor-

tant H3N2 viruses containing human (HA and NA) and

avian genes (PB2, PB1, PA, NP, M and NS) and triple

reassortant H3N2 viruses carrying human (HA and NA),

swine (NP) and avian (PB2, PB1, PA, M and NS) genes

have emerged in pigs in China (Yu et al., 2008). Novel

triple reassortant H1N2 influenza viruses containing genes

from the classical swine (HA, NP, M and NS), human (NA

and PB1) and avian (PB2 and PA) lineages have been

reported in pigs in China (Yu et al., 2009).

82 W. Ma and J. A. Richt

Europe With the replacement of cH1N1 viruses, the avian-like

H1N1 has been the predominant virus in European pig

populations and has undergone a reassortment with the

human H3N2 virus, producing a human-like H3N2 virus

containing HA and NA genes from the human virus and

six internal genes from the avian-like virus (Castrucci

et al., 1993). Subsequently, an H1N2 virus, first isolated

from Great Britain swine herds, spread to the rest of

Europe (Van Reeth et al., 2000). The H1N2 virus con-

tained human-like H1 and N2 genes and avian-like

internal genes (Brown et al., 1998). In 2005, a novel

H1N2, which was a reassortant between swine H1N2 and

swine H3N2 virus, was identified in Germany (Zell et al.,

2008). Currently, the avian-like H1N1, human-like H3N2

and reassortant H1N2 SIVs have become widespread

among pigs in Europe (Van Reeth et al., 2008).

North America Since 1998, triple reassortant H3N2 viruses were iso-

lated from pigs and have been endemic in swine herds of

North America; they contain HA, NA and PB1 polymerase

genes from human influenza viruses, M, NS and NP genes

from classical swine viruses, and PA and PB2 polymerase

genes from avian viruses (Zhou et al., 1999; Webby et al.,

2000). Reassortment between triple reassortant H3N2

viruses and cH1N1 viruses has resulted in the subsequent

development of H1N2 (Karasin et al., 2000), reassortant

H1N1 (rH1N1) (Webby et al., 2004) and H3N1 viruses

(Lekcharoensuk et al., 2006; Ma et al., 2006). The rH1N1

viruses contain the HA and NA from the cH1N1 virus and

the internal genes from triple reassortant H3N2 viruses.

The H1N2 viruses contain the HA from the classical swine

virus and the NA and internal genes from the triple

reassortant H3N2 viruses (Karasin et al., 2002; Webby

et al., 2004). The H3N1 viruses contain the NA from

the classical swine virus and the HA and internal genes

from the triple reassortant H3N2 viruses. Also, novel

human-like H1N1 and H1N2 SIVs have been isolated

from swine herds across the U.S., representing a re-

assortment of triple reassortant SIVs with seasonal human

H1N1 viruses; therefore, the HA and/or NA genes are

human-like whereas the internal genes are derived from

triple reassortant SIVs (Vincent et al., 2009). The re-

assortant H3N2, H1N2 and H1N1 (including rH1N1 and

human-like H1N1) viruses are circulating in swine

populations in North America (Vincent et al., 2008b; Ma

et al., 2009b).

Immunity to influenza A viruses

Infection of influenza A virus triggers immune responses

of the host including innate immunity, mucosal immunity

and systemic immunity (both humoral and cell-mediated

immunity). Innate immunity is the first line of host

defense inhibiting influenza virus replication in a non-

specific manner and is therefore critical in the early con-

tainment of influenza virus infection (White et al., 2008).

The innate immune response is complex involving a

variety of soluble innate inhibitors in respiratory secre-

tions and strongly contributes to the promotion and

direction of the adaptive, pathogen-specific immune

response (White et al., 2008; McGill et al., 2009). There

are several excellent reviews on influenza innate immu-

nity (Ichinohe et al., 2008; White et al., 2008; McGill et al.,

2009). To start an infection, influenza viruses first attach

to the mucosal tissues of the respiratory tract. Cellular

recognition of viral products such as viral RNA by Toll-

like receptors or cytoplasmic sensors (e.g. retinoic acid-

inducible gene I and melanoma differentiation-associated

gene 5) results in induction of the type I IFN system to

establish an antiviral state in the cell.

If animals were previously exposed or vaccinated

against influenza viruses, the mucosal immune response

provides an important line of defense against influenza

infection apart from innate immunity. Specific IgA and

IgM secreted locally in the respiratory tract are the major

neutralizing antibodies that prevent influenza virus entry

and can inhibit influenza replication intracellularly (Cox

et al., 2004). The neutralizing antibodies detected in nasal

secretions specifically target the HA and NA surface

proteins of influenza virus. In the pig model, influenza-

specific mucosal antibodies have been detected and

demonstrated to contribute significantly to the clearance

of SIV from the respiratory tract (Larsen et al., 2000; Richt

et al., 2006). Mucosal immunity induced by natural

influenza infection at the respiratory tract is more effective

and protective against subsequent heterovariant virus

infection than systemic immunity induced by parenteral

immunization with inactivated vaccines (Ichinohe et al.,

2008).

During infection, the humoral immune system pro-

duces antibodies against all major influenza viral proteins.

Antibody to the HA is the most important for neutraliz-

ation of virus and therefore prevention of disease. In

contrast, antibody to the NA is less effective in preventing

infection, but it prevents the release of mature viruses

from infected cells. Antibodies to the conserved internal

proteins (M and NP) cannot provide protection from

infection (Cox et al., 2004; Wesley et al., 2004), although

there could be a role for the M2 protein in antibody-

mediated protection (Treanor et al., 1990; Wang et al.,

2008). The HA- and NA-specific antibodies in serum are

most important for protection against influenza; therefore,

the serum antibody level to HA and NA are considered to

correlate with the prevention and resistance to illness

(Cox and Subbarao, 1999). However, the humoral im-

mune response might fail to prevent influenza infections

if faced with antigenic shift and/or drift of the infecting

virus.

Cell-mediated immunity is believed to play an im-

portant role in clearance of influenza viruses from the

Swine influenza vaccines 83

respiratory tract and subsequent recovery from disease.

Influenza-specific cytotoxic T lymphocytes (CTLs) have

been found in the blood and the lower respiratory tract

of infected hosts and are able to lyse cells infected with

different subtypes of influenza A virus. In mice and

humans, specific CTL response is directed against in-

fluenza viral internal proteins, specifically against NP, M1,

NS1 and the polymerase proteins (PB1, PB2 and PA)

(Bennink et al., 1982, 1987; Gotch et al., 1987; Reay et al.,

1989; Jameson et al., 1998; Epstein et al., 2000). The NP

of influenza A viruses is an important target antigen for

both subtype-specific and cross-reactive CTLs in mice and

humans (Townsend et al., 1984; Yewdell et al., 1985;

McMichael et al., 1986). There is limited knowledge on

cellular immune responses in pigs after influenza infec-

tions (Heinen et al., 2001). Previous studies indicate that

the CTL response is cross-reactive between influenza A

strains providing heterovariant and heterosubtypic immu-

nity and is critical in reducing viral spread and clearing

virus in combination with neutralizing antibodies

(Nguyen et al., 2001). Therefore, an ideal vaccine is able

to induce a balanced immune response including mu-

cosal, humoral and cell-mediated immunity.

Swine influenza vaccines

Although pigs are susceptible to infection with many

subtypes of influenza A viruses (Kida et al., 1994), only

three subtypes (H1N1, H1N2 and H3N2) are consistently

isolated from swine herds worldwide (Webster et al.,

1992; Olsen, 2002; Landolt and Olsen, 2007). Despite this

limited repertoire of circulating subtypes, novel geno-

types within individual subtypes and novel reassortant

viruses (e.g. human-like H1N1) have been an enormous

challenge for the production of efficacious vaccines to

prevent and control swine influenza. Antigenic shift and

drift of SIVs are occurring constantly, and the present

system for the production and licensing of inactivated

SIV vaccines does not allow the industry to react in a

timely manner. To date, only inactivated whole-virus

vaccines are commercially available and widely used for

swine influenza worldwide.

Inactivated SIV vaccine

Current commercially available SIV vaccines are tra-

ditional, adjuvanted, inactivated bivalent whole-virus

vaccines containing H3N2 and H1N1 subtype SIVs

propagated in embryonated hen eggs. These vaccines

stimulate high titers of IgG in serum and lungs, which

are critical for ameliorating or preventing influenza virus

infection and protection against clinical disease. How-

ever, protection is to be expected only when the priming

HA antigen is antigenically matched or closely related to

the HA of the challenge virus. Since there is great genetic

and antigenic variety within currently circulating SIVs,

commercially available vaccines are not able to provide

optimal protection for pigs against SIVs. A number of

studies have shown only partial protection from inacti-

vated virus vaccines following a heterovariant or hetero-

subtypic influenza challenge (Brown and McMillen, 1994;

Bikour et al., 1996; Vincent et al., 2010a); they are only

efficacious when genetically similar viruses are used for

challenge. Other studies have revealed that previous

exposure of pigs to European H1N1 and H3N2 viruses

conferred complete protection against a novel H1N2

with an unrelated HA protein (Van Reeth et al., 2003). In

contrast, vaccination with commercially available in-

activated vaccines containing H1N1 and H3N2 viruses

does not protect against the H1N2 challenge (Van Reeth

et al., 2004), indicating that serum hemagglutination

inhibition (HI) or virus neutralizing antibodies are not

essential and that cell-mediated and/or mucosal immunity

are critical for heterosubtypic protection. One study has

shown that a killed cH1N1 SIV vaccine not only fails to

protect against a heterologous H1N2 infection but sur-

prisingly also potentiates pneumonia in challenged pigs

(Vincent et al., 2008a). These results indicate that in-

activated vaccines when faced with a heterovariant

challenge may enhance disease.

Interference by maternally derived antibodies (MDAs)

is another big challenge for vaccine (especially in-

activated vaccines) efficacy because passively acquired

antibodies from the sow’s colostrum can inhibit the

immunogenicity of a vaccine and interfere with the pig’s

immune response to the vaccine if they are still present

at the time of immunization. Kitikoon et al. (2006)

have shown that the MDA suppressed serum antibody

responses and the induction of SIV-specific memory

T-cells following the administration of a bivalent in-

activated vaccine in pigs. Enhancement of lung pneu-

monia was observed in pigs immunized with a bivalent

inactivated SIV vaccine in the presence of MDA when

challenged with a heterologous H1N1 virus (Kitikoon

et al., 2006).

In summary, there are three major difficulties with the

use of current commercially available inactivated SIV

vaccines: (1) SIV is antigenically changing faster than

traditional inactivated vaccines can be developed; (2) the

commercially available inactivated SIV vaccines do not

provide good cross-protection among different SIV

isolates, especially against heterovariant and heterosub-

typic viruses; and (3) passively acquired immunity (MDA)

can interfere with vaccine immunity in piglets. These

difficulties have led to a significant decrease in the sale

of commercially available SIV vaccines and a significant

increase in the production and use of autogenous vac-

cines (presently about 50% of the U.S. market). A priority

for novel SIV vaccine development is the improvement

of heterovariant and heterosubtypic immunity and the

selection of currently circulating SIV isolates as vaccine

seeds.

84 W. Ma and J. A. Richt

Live-attenuated swine influenza as vaccines

With the development of molecular biology technology,

influenza viruses can be rescued from plasmid DNA by a

technique called reverse genetics. This method makes it

possible to modify the viral genome for generation of

rationally designed novel live-attenuated influenza virus

vaccines as described in the following section. The sur-

face glycoprotein HA of influenza A virus mediates virus

entry into susceptible cells. HA is synthesized as a precur-

sor HA0 comprising HA1 and HA2. Cleavage of the HA0

into HA1 and HA2 by host proteases is a prerequisite to

gain access to cells by activating the fusion peptide; this

process is a major determinant of virus pathogenicity. The

cleavage site contains a conserved arginine or a multiple

basic amino acid motif. Mutation of the HA cleavage site,

which now requires cleavage by elastase instead of

trypsin, has led to the attenuation of influenza viruses

in mice (Stech et al., 2005). The polymerase proteins

PB2, PB1 and NP have been shown to contribute to

the virus ability to grow at a lower temperature in some

temperature-sensitive virus strains (Jin et al., 2003). The

NS1 protein of the influenza A virus is exclusively

expressed in virus-infected cells and not present in virus

particles. One of the major functions of the NS1 protein

of influenza viruses is the inhibition of the innate host

type I IFN-mediated antiviral response. Modifications of

either the HA, the polymerase proteins PB1 and PB2, or

the NS1 can be utilized to produce live-attenuated SIVs

which have a great potential as live-attenuated vaccines.

The advantage of modified live-attenuated vaccines is

enhanced stimulation of cell-mediated immunity, directed

most likely against the conserved NP (Yewdell et al.,

1985), thus providing more heterovariant and hetero-

subtypic protection (Xie et al., 2009). A major concern

with live-attenuated vaccines would be a possible

reassortment between field viruses and vaccine strains,

producing novel reassortant viruses.

Live-attenuated swine influenza vaccine with modified NS1 protein Attenuated SIVs expressing NS1-truncated proteins with

73, 99 or 126 amino acids (Tx/98 NS1n73, Tx/98 NS1n99

and Tx/98 NS1n126) with promising vaccine potential

have been generated via modification of the viral NS1

gene of an H3N2 (A/Swine/Texas/4199-2/98, Tx/98) virus

using reverse genetics (Solorzano et al., 2005). The Tx/98

NS1n126 virus is the most attenuated virus displaying the

lowest level of NS1 expression and decreased replication

in vitro and in vivo compared to the wild-type and Tx/98

NS1n73, Tx/98 NS1n99 viruses (Solorzano et al., 2005).

Intratracheal infection of pigs with Tx/98 NS1n126 virus

induces minimal macroscopic and histopathologic lung

lesions. Pigs vaccinated with Tx/98 NS1n126 virus were

completely protected against a challenge with the homo-

logous Tx/98 virus and partially protected against a

challenge with a heterosubtypic H1N1 virus (Richt et al.,

2006). All vaccinated pigs developed a detectable level of

HI titers, serum IgG, and mucosal IgG and IgA antibodies

against parental H3N2 antigens (Richt et al., 2006).

Subsequent studies showed that the intranasal route

was more efficient than the intramuscular route at eliciting

mucosal anti-influenza virus antibodies (Vincent et al.,

2007). A single dose of Tx/98 NS1n126 virus admin-

istered intranasally conferred complete protection against

a homologous virus challenge and nearly complete

protection against a heterovariant challenge with an

antigenically distant H3N2 SIV (A/Sw/CO/23619/99).

Moreover, intranasal vaccination reduced clinical symp-

toms (fever) and virus titers in lungs of pigs which were

challenged with a heterosubtypic H1N1 SIV (A/Swine/

Iowa/00239/2004). These studies indicate that a complex

host response including both cellular and humoral mech-

anisms contributes to the broad efficacy of the Tx/98

NS1n126 modified live virus (MLV) after intranasal

delivery, and this efficacy appears to be superior to that

induced by inactivated influenza vaccines (Vincent et al.,

2007). In addition, a study using the Tx/98 NS1D126 virus as an MLV vaccine in piglets with MDA revealed that

Tx/98 NS1D126 virus can provide good immunity against homologous and heterovariant viruses without disease

enhancement (Vincent et al., 2010b). The series of

experiments described above demonstrate that the NS1-

truncated MLV vaccine appears to be more efficacious

when compared to the inactivated vaccine, indicating that

they are promising vaccine candidates against SIVs.

Elastase-dependent live attenuated swine influenza vaccine Avian- or mouse-adapted influenza viruses can be at-

tenuated by modification of the HA cleavage site from

a trypsin-sensitive motif to an elastase-sensitive motif.

Recently, Masic et al. (2009a) used the same strategy to

generate two elastase-dependent mutant SIVs derived

from A/Sw/Saskatchewan/18789/02 (H1N1) called A/Sw/

Sk-R345V (R345V) and A/Sw/Sk-R345A (R345A). These

two viruses displayed similar growth properties in vitro to

the wild-type virus, but were highly attenuated in pigs.

This was demonstrated by significantly decreased macro-

scopic lung lesions and virus titers in lungs and no nasal

virus shedding (Masic et al., 2009a) when compared to

the wild-type virus. Administration of either the R345V or

R345A via an intratracheal route induced antigen-specific

humoral and cell-mediated immunity. Pigs immunized

with the R345V virus had significantly higher HI titers

than the R345A-vaccinated animals (Masic et al., 2009b).

Therefore, the R345V virus was selected to further test

its efficacy against a challenge from homologous and

heterologous viruses. After pigs were vaccinated and

boosted with this virus intratracheally, they were subse-

quently challenged with either the wild-type homolog-

ous A/Sw/Saskatchewan/18789/02 (H1N1), heterovariant

A/Sw/Indiana/1726/88 (H1N1) or heterosubtypic Tx/98

Swine influenza vaccines 85

H3N2 virus. Pigs vaccinated with R345V virus were com-

pletely protected against a challenge with the homo-

logous and heterovariant H1N1 SIVs and partially

protected against a challenge with the heterosubtypic

H3N2 SIV. This protection was measured by significantly

reduced macroscopic and microscopic lung lesions, lower

virus titers from the respiratory tract, and lower levels

of pro-inflammatory cytokines (Masic et al., 2009b). It can

be concluded that elastase-dependent SIV mutants are

promising candidates as live-attenuated virus vaccines

against SIVs in pigs; however, the safety (reversion and

re-assortment) and efficacy employing practical vaccina-

tion routes need to be further investigated.

Cold-adapted live attenuated vaccine Cold-adapted (temperature sensitive, ts) influenza viruses

replicate efficiently at a cooler temperature (25�C or

26�C), but their growth is restricted at normal body

temperature. The nature of ts live-attenuated viruses leads

to their efficient multiplication in the cooler environment

of the upper respiratory tract where they induce local and

systemic immune responses, and inefficient replication

in the warmer environment of the lower respiratory tract

where wild-type viruses may cause severe lung damage.

A ts influenza vaccine (FluMist1) has been approved

in the U.S. for intranasal use in humans (Belshe, 2004) and

a ts modified-live equine influenza virus vaccine (Flu

Avert1 I.N. Vaccine) derived from the wild-type A/Eq/

Kentucky/1/91 (H3N8) influenza virus has been licensed

and is commercially available in North America (Paillot

et al., 2006).

FluMist1 vaccine strains contain six internal gene

segments (PB1, PB2, PA, NP, M and NS) from the master

donor virus (MDV), a cold-adapted human H2N2 (A/Ann

Arbor/6/60) influenza virus, along with two external

surface gene segments (HA and NA) derived from

currently circulating human influenza viruses. The cold-

adapted MDV for influenza A strains of FluMist1 was

generated using serial passages in primary chicken kidney

cell culture at a temperature gradually reduced to 25�C

(Maassab, 1967). Molecular analysis showed that the PB1,

PB2 and NP protein of the MDV each contributes to viral

temperature sensitivity and the combination of all three

gene segments results in the expression of the ts pheno-

type of the MDV (Jin et al., 2003). Site mutagenesis

analysis revealed that five loci [PB1 (K391E, E581G,

A661T), PB2 (N265S) and NP (D34G)] are responsible for

the ts phenotype of the MDV (Jin et al., 2003).

Flu Avert1 vaccine derived from A/Eq/Kentucky/1/91

(H3N8) influenza virus was created by serial passage in

embryonated chicken eggs at consecutively lower tem-

peratures (Youngner et al., 2001). This ts vaccine rep-

licates efficiently at 26�C while its growth is restricted at

38�C and 39�C. Intranasal immunization of ponies with a

single dose of Flu Avert1 provided full protection from

clinical signs of the disease following a challenge with the

parental A/Eq/Kentucky/1/91 influenza virus at 5 weeks

and 6 months post vaccination. At late time points post-

immunization, the duration of nasal virus shedding was

significantly shorter when compared to unvaccinated

control ponies (Townsend et al., 2001). This vaccine also

protects horses against infection with a heterovariant

equine influenza virus (Chambers et al., 2001). Ts live-

attenuated equine influenza vaccines elicit long-term

immunity that ameliorates duration and severity of clinical

signs and nasal shedding of virus after challenge.

Solórzano et al. (2010) have generated an attenuated

ts H3N2 SIV (A/Swine/WI/14094/99; Sw99) by changing

the viral PB1 and PB2 genes (four loci) based on

sequences observed in the cold-adapted human H2N2

(A/Ann Arbor/6/60) influenza virus. The mutated virus,

called Sw99ts, was partially attenuated in vitro and

in vivo. In order to make a fully attenuated virus, the

PB1 and PB2 mutations were combined with the insertion

of an HA epitope (eight amino acids derived from the

influenza virus H3 HA protein sequence) into the

C-terminus of the PB1 protein. The virus, named Sw99att,

showed no replication at the restrictive temperature

(39�C) but replicated efficiently at 33�C in cell culture.

To show the potential of Sw99att as a live-attenuated

vaccine virus, the surface genes were substituted with

the HA and NA genes of a cH1N1 (A/Swine/IA/15/30,

IA30) virus. Vaccination of mice with this virus provided

complete protection in a homologous challenge (IA30)

and partial protection (no clinical signs) following a

heterovariant challenge with the 2009 pandemic H1N1

virus. The potential of this ts live-attenuated vaccine for

pigs needs to be evaluated (Solórzano et al., 2010).

Baculovirus-derived influenza subunit vaccines

The baculovirus-insect cell expression system was devel-

oped over 20 years ago and has become one of the

most widely used systems for production of recombinant

proteins for both veterinary and human vaccines. The

baculovirus system is a good method for producing

recombinant glycoprotein due to the eukaryotic nature

of the insect cells. Currently, more than 10 baculovirus

expression system-derived vaccines are either commer-

cially available (e.g. classical swine fever, porcine

circovirus associated disease) or in clinical trials (hepatitis

B and C viruses) (Meghrous et al., 2009). In addition to

the ease and safety of production, this system is an ideal

platform for producing recombinant proteins owing to

effective post-translational modification and high yields

(He et al., 2009; Meghrous et al., 2009). Therefore,

recombinant influenza proteins produced by the baculo-

virus expression system used as a subunit vaccine might

be an alternative strategy to overcome the limitations

and drawbacks of traditional killed influenza vaccines

produced by the egg-based manufacturing system. The

main advantage of a subunit vaccine derived from the

86 W. Ma and J. A. Richt

baculovirus expression system is that the manufacturing

of the HA proteins does not require the handling of live

influenza viruses as required for embryonated eggs or

mammalian cell production systems (Cox and Hollister,

2009).

Numerous studies have been conducted on the

immunogenicity and safety of baculovirus expression

system-derived recombinant HA vaccines in the last two

decades. A recombinant HA influenza vaccine provided

equivalent or better immunogenicity than an egg-derived

inactivated vaccine and was safe and efficacious in human

clinical trials (Treanor et al., 1996, 2006, 2007; King et al.,

2009). Subunit HA vaccines for avian and human

influenza viruses have been studied in animal models

(Powers et al., 1997; Crawford et al., 1999; Gambotto

et al., 2008) and some are in clinical trials (Powers et al.,

1997; He et al., 2009). However, to our knowledge, no

baculovirus-derived subunit vaccines based on SIV

antigens have been produced and tested in the pig

model. One disadvantage of this technology is that it

produces a highly hydrophobic recombinant HA protein,

which makes purification difficult, resulting in a decrease

of its effectiveness as a vaccine (He et al., 2009). In

addition, a large amount of recombinant HA is required

for vaccination in order to achieve an equivalent immune

response to that of traditional inactivated influenza

vaccines (Gambotto et al., 2008). The biggest challenge

for subunit vaccines derived from the baculovirus system

is the frequent antigenic drift and/or shift of the HA,

leading to a mismatch between the immunogen and

circulating viruses (Carrat and Flahault, 2007) and there-

fore vaccine failure.

Vectored vaccines

A vector is a biological carrier of genes of other patho-

gens. The viral antigens expressed by vectored vaccines

are produced in host cells in vivo and can induce both

humoral and cellular immunity. The following vectored

vaccines for swine influenza are currently under investi-

gation or might be studied in the near future.

Adenovirus-based SIV vaccine A human adenovirus serotype 5 (hAd5) vector has been

utilized to express various genes of interest for molecu-

lar therapy and vaccine development. Vaccination with

human adenovirus vectors induced both humoral and

cell-mediated immunity, making them potentially more

effective than inactivated or subunit vaccines and more

similar to the response elicited from MLV vaccines

(Gamvrellis et al., 2004). In addition, administration of

hAd5 vectored vaccines via the mucosal route induced

superior, long-lasting mucosal immunity (Baca-Estrada

et al., 1995). hAd5 viruses have broad host ranges and

accommodate large segments of foreign DNA. Normally,

livestock does not have pre-existing immunity against

hAd5 virus that can interfere with vaccine efficacy

(Wesley et al., 2004). A series of studies have shown

that certain disadvantages of inactivated vaccines can be

overcome by using recombinant hAd5-vector vaccines

that can stimulate cell-mediated and mucosal immunity

(Baca-Estrada et al., 1995; Monteil et al., 2000; Wesley

et al., 2004).

A hAd5 recombinant virus expressing the HA of the

H3N2 Tx/98 virus has shown partial protection in mice

after a challenge with a heterovariant virus, A/HK/1/68

(H3N2) (Tang et al., 2002). Subsequently, a hAd5 re-

combinant virus expressing the NP of the Tx/98 H3N2 SIV

was also generated and challenging experiments in

pigs were conducted to test the efficacy of both hAd5

recombinant viruses as SIV vaccines (Wesley et al., 2004).

Pigs vaccinated with the recombinant hAd5 expressing

HA alone or HA plus NP developed high levels of virus-

specific HI antibody by 4 weeks post vaccination,

whereas the administration of the recombinant hAd5

expressing NP alone induced no detectable HI antibody.

Pigs immunized with both recombinant viruses (HA and

NP) in a mixture were completely protected as demon-

strated by a lack of nasal virus shedding and lung lesions

following a homologous challenge. Vaccination with

the recombinant virus expressing HA induced nearly

complete protection with a low viral titer in nasal swabs

and minimal lung lesions. In contrast, vaccination with

the recombinant virus expressing NP only reduced lung

lesions when compared to non-vaccinated controls.

Subsequent studies demonstrated that the recombinant

hAd5-vectored SIV vaccines are able to prime the immune

system in the presence of MDA that often interfere with

conventional inactivated vaccines (Wesley and Lager,

2006). Piglets with H3N2-specific MDA were either sham-

immunized with an empty hAd5 vector or immunized

with recombinant hAd5 SIV vaccines expressing the

HA and NP. The HI titer of sham-immunized animals

displayed continued antibody decay whereas piglets

vaccinated with the recombinant hAd5 SIV vaccine

developed an active immune response by the second

week post vaccination. When the HI titer of sham-

immunized piglets had decayed, the sham-immunized

group and half of hAd5 SIV vaccinates were boosted with

a commercial inactivated SIV vaccine and subsequently

challenged with a heterovariant virulent H3N2 SIV. The

pigs primed with the hAd5 SIV vaccine in the presence of

MDA had a strong anamnestic response to the booster

immunization while sham-immunized pigs did not re-

spond to the commercial inactivated vaccine. The pigs

primed with the hAd5-SIV vaccine and boosted with

inactivated vaccine had a reduction of clinical signs,

reduced virus loads in the respiratory tract and no lung

lesions. In contrast, MDA positive pigs immunized with

the inactivated vaccine alone exhibited a vaccine failure

(Wesley and Lager, 2006).

In addition, the route of administration (needle-free

device versus traditional intramuscular injection) for the

Swine influenza vaccines 87

recombinant hAd5 SIV vaccines was evaluated (Wesley

and Lager, 2005). The results showed that a traditional

intramuscular injection induced consistently higher HI

responses than vaccination via a needle-free device, but

the differences were not significant. Administration of

high doses of the recombinant hAd5 SIV vaccine (HA and

NP) using either method prevented nasal shedding after

challenge. In these experiments, the hAd5 SIV vaccine

virus was not transmitted to sentinel pigs (Wesley and

Lager, 2005).

Alphavirus-based SIV vaccines Alphaviruses are positive-stranded RNA viruses belonging

to the family of Togaviridae. Although alphaviruses

have a broad host range including humans, their sero-

prevalence in many mammalian host species is rather

low, making them potentially useful for vaccine devel-

opment (Rayner et al., 2002). Three alphavirus [Sindbis

virus (SINV), Semliki Forest virus (SFV) and Venezuelan

equine encephalitis virus (VEEV)] replicon expression

vectors have been developed (Xiong et al., 1989;

Liljestrom and Garoff, 1991; Pushko et al., 1997). VEEV

has a unique lymph node tropism (Walker et al., 1976;

Jackson et al., 1991), resulting in effective antigen

presentation and induction of a strong and balanced

immune response. Alphavirus replicon expression vectors

are propagation-defective (without alphavirus structural

genes), single-cycle vectors incapable of spreading from

infected to non-infected cells. However, these replicons

are self-replicating and can efficiently express foreign

antigens (Rayner et al., 2002). To date, numerous vaccine

candidates based on alphavirus replicons have been

developed and shown to induce protection against a

variety of infectious pathogens in a number of hosts

(Rayner et al., 2002).

Alphavirus replicon vectors have also been utilized to

express influenza antigens and their efficacy in animal

models was evaluated. Immunization of mice with SFV

based-replicons expressing the NP and HA of influenza

A virus provided protection against a challenge with the

homologous virus (Berglund et al., 1999). A VEEV

replicon vector has been used to express HA from the

human Hong Kong H5N1 influenza A isolate (A/HK/

156/97) and shown to protect chickens against a chal-

lenge with the homologous H5N1 virus (Schultz-Cherry

et al., 2000). So far, Sagiyama virus is the only alphavirus

found in swine and is geographically restricted to Asia

(Chang et al., 2006). Importantly, pigs can be infected

by VEEV and display a transient viremia (Dickerman

et al., 1973), suggesting that the VEEV replicon vector can

be used to develop vaccine candidates for the swine

industry. Recently, a VEEV replicon vector expressing the

HA from a human influenza virus A/Wyoming/03/2003

(H3N2) was developed to immunize pigs (Erdman et al.,

2010). The results revealed that this VEEV replicon vector

induced a robust HI antibody response in vaccinated pigs.

The VEEV replicon vector has also been used to express

the HA of the pandemic H1N1 A/California/04/2009 virus.

Pigs were vaccinated and challenged with the homo-

logous pandemic virus. Vaccinated pigs showed a sig-

nificantly higher specific antibody response, reduced lung

lesions and viral shedding, and higher average daily

weight gain compared to non-vaccinated control infected

animals, indicating that the VEEV replicon vaccine is

efficacious for swine against the pandemic H1N1 virus

(Vander Veen et al., 2009). This VEEV replicon vaccine

has obtained a conditional license in the USA. Although

the VEEV replicon particles can induce humoral, cell-

mediated and mucosal immune responses and provide

protection for a number of infectious agents, it is not

clear whether this vector expressing an HA molecule can

protect against heterovariant and heterosubtypic influ-

enza viruses.

Pseudorabies virus (PRV)-based SIV vaccines PRV is an alpha-herpesvirus with a linear double-stranded

DNA. PRV has a broad host range and its large DNA

genome is capable of accommodating a large segment of

foreign DNA. The PRV genome consists of many non-

essential regions, such as genes encoding thymidine

kinase (TK), gE, gG and gC, which can be deleted or

replaced by other genes without affecting virus replica-

tion. For example, a commercially available attenuated

DIVA (differentiating infected from vaccinated animals)

vaccine for PRV containing a gE deletion has been widely

used in the PRV eradication program (van Oirschot

et al., 1986; White et al., 1996; Muller et al., 2003). Due

to its good safety record and broad host spectrum, PRV is

a promising vaccine vector for expressing antigens of

choice from other pathogens (Thomsen et al., 1987;

Whealy et al., 1988; van Zijl et al., 1991; Tian et al., 2006;

Yuan et al., 2008) including the HA from SIVs. To express

the HA of an H3N2 SIV (A/Swine/Inner Mogolian/547/

2001), the PRV Bartha-K61 vaccine strain was utilized

(Tian et al., 2006). Mice were immunized with this

recombinant PRV expressing HA and challenged with a

heterovariant H3N2 SIV at 4 weeks post vaccination.

Vaccinated mice showed HI antibodies, reduced lung

lesions, and an absence of virus from the lungs when

compared to non-vaccinated control infected animals.

These results demonstrate that the recombinant PRV

expressing SIV HA gene can protect mice from a

heterovariant challenge and might be used as a candidate

vaccine against SIV (Tian et al., 2006). However, the

efficacy of a recombinant PRV as a swine influenza

vaccine needs to be further evaluated in pigs. The major

disadvantage of recombinant PRV as an SIV vaccine is that

it might interfere with the surveillance for the control and

eradication of PRV, because PRV has been eradicated

from many countries.

Vaccinia virus-based vaccines Vaccinia virus is a large, complex, enveloped double-

stranded DNA virus belonging to the poxvirus family.

88 W. Ma and J. A. Richt

Vaccinia virus is well known as the vaccine used to

eradicate human smallpox. The modified vaccinia Ankara

(MVA) is a highly attenuated vaccinia strain created by

serial passages in chicken embryo fibroblast cells and

has been safely and successfully used to vaccinate over

120,000 humans against smallpox. The MVA has been

engineered as a viral vector expressing foreign genes

under control of a vaccinia virus promoter. To date,

various recombinant MVA viruses have been shown to

be immunogenic and induce protective immunity against

viruses, bacteria and parasites in animal models and

clinical trials (Sutter et al., 1994; Moss et al., 1996;

Goonetilleke et al., 2003; Moorthy et al., 2003; Cosma

et al., 2003, 2007; Bisht et al., 2004; Drexler et al., 2004;

McShane et al., 2004; Wang et al., 2004). The MVA has

an excellent safety profile, the ability to elicit highly

effective virus neutralizing antibody responses, is highly

stable and replication deficient. It can be produced on a

large scale in chicken embryo fibroblasts under BSL-1

conditions. Pre-existing immunity is unlikely to affect the

immunogenicity of foreign antigens delivered by this

vector (Ramirez et al., 2000; Drexler et al., 2004).

Recombinant MVA has been used to develop influ-

enza vaccines by expressing various antigens from

influenza A viruses. The MVA expressing the HA from

H3N8 (A/Equine/Kentucky/1/81) equine influenza virus

induced protective immunity in horses whereas vaccina-

tion with MVA expressing the NP provided very limited

protection from clinical disease (Breathnach et al., 2006).

Vaccination of mice (two times 108 pfu/dose) with MVA

expressing the HA from the pandemic H5N1 (A/Vietnam/

1194/04) virus induced protective immunity against infec-

tion with homologous and antigenically distinct hetero-

variant H5N1 (A/Indonesia/5/05; A/Hongkong/156/97)

viruses (Kreijtz et al., 2007). Subsequent studies showed

that this recombinant MVA provided cross-clade protec-

tion in mice after a single immunization when challenged

with different H5N1 viruses at low doses (105 pfu/dose)

(Kreijtz et al., 2009a). In addition, the H5 MVA vaccine

induced cross-reactive antibodies and prevented virus

replication in the upper and lower respiratory tract and

the development of severe necrotizing bronchointerstitial

pneumonia in H5N1 infected macaques (Kreijtz et al.,

2009b, c). Since MVA can be used to express the HA from

influenza A viruses, it might be a promising future influ-

enza vaccine (Rimmelzwaan and Sutter, 2009). However,

to our knowledge no studies have been reported on a

MVA-based SIV vaccine.

Virus-like particle (VLP) vaccines

VLPs as vaccines have been discussed as promising alter-

natives for a variety of animal viral pathogens (Antonis

et al., 2006; Elmowalid et al., 2007; Yang et al., 2008) and

are approved as vaccines in humans (Keating and Noble,

2003; Reisinger et al., 2007). Influenza VLPs can be easily

produced by simultaneously expressing the HA and NA

along with a viral core protein, such as influenza M1 or

retroviral Gag protein using a baculovirus-insect cell

system (Haynes, 2009). The VLPs based on the M1 or Gag

are highly immunogenic (Pushko et al., 2005; Szecsi

et al., 2006) and can provide protection against virulent

influenza viruses of the H1, H3, H5, H7 and H9 subtypes

(Pushko et al., 2005; Szecsi et al., 2006; Matassov et al.,

2007; Quan et al., 2007; Bright et al., 2007, 2008;

Mahmood et al., 2008; Haynes et al., 2009; Kang et al.,

2009; Perrone et al., 2009; Ross et al., 2009) and cross-

protection from a heterovariant challenge in mouse and

ferret models (Bright et al., 2008; Mahmood et al., 2008).

The VLP vaccine based on baculovirus-insect cell system

might offer advantages over traditional killed vaccines:

improved immunogenicity and production systems with-

out handling live virus. The above studies suggest that

this technology could also be applied for SIVs.

Plasmid DNA-based vaccines

DNA vaccines are naked DNA plasmids that have

been genetically engineered to produce defined antigens

within transfected cells. Intracellular antigens can be pres-

ented by Major histocompatibility complex (MHC) class I

and II molecules, leading to stimulation of both humoral

and cellular immune responses. DNA vaccines are an

alternative to conventional killed vaccines and offer many

advantages of the attenuated live vaccines without their

potential risks (Olsen, 2000). The stable plasmid DNA can

be easily produced on a large-scale at low costs. DNA

vaccines have been tested for a wide variety of viral,

bacterial and protozoal infectious pathogens (Kim and

Jacob, 2009; Olsen, 2000). DNA vaccines for human and

avian influenza viruses have been developed and good

immune responses have been demonstrated in mice,

chickens, ferrets, horses and non-human primates follow-

ing the administration of HA, NP, NA and M constructs

(Fynan et al., 1993; Webster et al., 1994; Liu et al., 1997;

Chen et al., 2008, 1999a, b; Okuda et al., 2001; Zhang

et al., 2005; Oveissi et al., 2009; Yager et al., 2009); clinical

trials of DNA-based influenza virus vaccines are under-

way in humans (Drape et al., 2006).

For swine influenza studies, two different DNA vaccine

constructs have been used (Eriksson et al., 1998; Macklin

et al., 1998). One study showed that the administration

of the NP from the human influenza virus A/PR8/34

(H1N1) in pigs induced a strong humoral response but no

detectable protection from virus challenge (Macklin et al.,

1998). In contrast, when pigs were administered a DNA

vaccine with the HA gene from a H1N1 SIV (A/Swine/

Indiana/1726/88), a decrease of virus shedding after

challenge was observed (Macklin et al., 1998). Larsen

and Olsen (2002) showed that HA DNA vaccination in-

duced strong priming of the humoral immune responses

in pigs which can be significantly enhanced by increasing

Swine influenza vaccines 89

the vaccine dose. Co-administration of interleukin-6 DNA

to pigs did not significantly improve immune responses to

HA DNA vaccination or protection from challenge ex-

posure (Larsen and Olsen, 2002). These results indicate

that the administration of DNA plasmids encoding the HA

gene from influenza viruses is an effective method for

priming and/or inducing virus-specific immune re-

sponses, and for providing partial protection from a chal-

lenge infection in pigs (Macklin et al., 1998; Olsen, 2000;

Larsen and Olsen, 2002).

Several safety concerns have been raised regarding the

use of DNA vaccines. It was argued that DNA vaccines

might integrate into host genomes, increasing the risk of

malignancy and production of auto-antibodies against

double stranded DNA leading to autoimmune disease

(Kim and Jacob, 2009). However, to date, there has been

no evidence of vaccine DNA integration into the host

genome or the induction of anti-DNA antibodies. DNA

vaccines are able to elicit broad-spectrum, long-lasting

immunity through both humoral and cell-mediated im-

mune reactions against influenza virus. DNA vaccines

could be good candidates for swine vaccines, since they

might provide heterosubtypic immunity and the inter-

nalization of DNA inside host cells would minimize

interference by MDA (Thacker and Janke, 2008). How-

ever, a large amount of DNA is needed for vaccination

and experimental trials of DNA vaccines in pigs have not

been proven very successful. DNA vaccines might be

useful as primer vaccines when followed by conventional

inactivated vaccines (Larsen and Olsen, 2002). The need

to develop more efficient delivery strategies that allow

administration of DNA to easily accessible sites on the

pig’s body is a critical challenge for this technology and its

clinical use in veterinary medicine.

What is an ideal vaccine for swine influenza?

Each vaccine formulation has its own advantages and

disadvantages. For example, killed vaccines are safe and

provide good protection from genetically similar viruses,

but lack heterovariant and heterosubtypic protection,

might enhance disease and experience interference by

MDA. Modified live-virus vaccines are able to provide

good homosubtypic and partial heterosubtypic protec-

tion, do not enhance disease, but have the potential to

reassort with circulating viruses. Antigenic shift and drift

can cause vaccine failure in animals immunized with

subunit vaccines. In most cases, vectored vaccines can

only be applied once, i.e. the main target animals are only

grow-finish pigs, not sows. Taken together, the choice

of vaccines and immunization program is dependent on

the epidemiological status in a swine herd and the age

and future use of individual animals.

An ideal vaccine for swine influenza must overcome

the difficulties encountered by traditional killed vaccines.

Novel strategies have to keep up with the ever-evolving

influenza viruses via updating virus seeds, overcoming

interference from MDA, and providing broad homosub-

typic and heterosubtypic protection. An ideal vaccine for

swine influenza should be safe, easy to apply, cheap and

able to prevent disease and virus shedding. In addition,

one should be able to store the vaccine indefinitely at

room temperature. An ideal vaccine should also have the

following features: capacity of inducing effective herd

immunity, one dose requirement, administration with-

out a hypodermic syringe, and DIVA compatibility. To

develop an ideal vaccine for swine influenza, future

research is needed to address each of these areas.

Currently, a priority for novel SIV vaccine development

should focus on improvement of heterovariant and

heterosubtypic immunity. There is only limited infor-

mation on the extent of cross-protection between influ-

enza virus variants or subtypes in humans and swine.

In comparison to humans and mice, there is a big knowl-

edge gap in swine immunology. Therefore, the cell-

mediated and humoral immune responses at the systemic

and mucosal levels need to be analyzed in future pig

studies in order to develop better vaccines for the swine

industry.

Vaccine licensure

Since SIV is rapidly changing, continuous production and

licensing of novel inactivated SIV vaccines is imperative.

Because the efficacy of currently available killed influenza

vaccines in swine is questionable, it is urgent to select

novel vaccine seeds from currently circulating SIVs based

on SIV surveillance data. It might be necessary to employ

a similar vaccine strain selection system as used by

WHO for human influenza vaccines to produce effective

national and regional swine vaccines. However, there is

currently no systemic surveillance for SIV in swine

populations and no support by governments. In order

to control swine influenza, procedures for new vaccine

licensure need to be updated to keep pace with the fast

changes in influenza virus genetics.

A swine influenza vaccine under U.S. Department of

Agriculture (USDA) licensure procedures often takes up

to 5 years to be licensed, which is much more laborious

and expensive than for human influenza vaccines

(Thacker and Janke, 2008). Therefore, national agencies

[e.g. Center for Veterinary Biologics (CVB), part of USDA]

need to streamline vaccine approval methods to enable

timeliness of market entry for novel vaccines and up-

dating of already existing vaccines. Recently, CVB

changed its guidance on licensed killed swine influenza

vaccines (Veterinary Services Memorandum No. 800.111),

allowing up to two strain substitutions for each subtype

at any one time without full-scale field safety tests. How-

ever, antigen concentration of each new strain must be

not less than the strains in the licensed vaccine and manu-

facturing methods must be similar. Also, immunogenicity

90 W. Ma and J. A. Richt

and efficacy must be demonstrated in an acceptable host

challenge model.

Lessons from the current H1N1 virus pandemic teach us

that influenza viruses are important zoonotic pathogens

and surveillance for SIVs in pigs is necessary to prevent

and control future pandemics. Therefore, national and

international government agencies need to adjust policies

on influenza surveillance and vaccine licensing in order to

protect the public health and the swine industry.

Acknowledgments

The authors would like to acknowledge grant sup-

port from the National Institute of Allergy and Infecti-

ous Diseases, National Institute of Health, Department

of Health and Human Services (Contract No.

HHSN266200700005C).

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