Mucosal vaccines to prevent porcinereproductive and respiratory syndrome:a new perspective

Gourapura J. Renukaradhya*, Varun Dwivedi, Cordelia Manickam,Basavaraj Binjawadagi and David Benfield

Food Animal Health Research Program, Ohio Agricultural Research and Development Center,

Department of Veterinary Preventive Medicine, The Ohio State University, 1680 Madison Avenue,

Wooster, OH 44691, USA

Received 21 November 2011; Accepted 19 December 2011; First published online 2 May 2012

AbstractPorcine reproductive and respiratory syndrome (PRRS) is an economically important infectious

disease of swine. Constant emergence of variant strains of PRRS virus (PPRSV) and virus-

mediated immune evasion followed by viral persistence result in increased incidence and

recurrence of PRRS in swine herds. Current live and killed PRRSV vaccines administered by a

parenteral route are ineffective in inducing complete protection. Thus, new approaches in

design and delivery of PRRSV vaccines are needed to reduce the disease burden of the swine

industry. Induction of an effective mucosal immunity to several respiratory pathogens by direct

delivery of a vaccine to mucosal sites has proven to be effective in a mouse model. However,

there are challenges in eliciting mucosal immunity to PRRS due to our limited understanding of

safe and potent mucosal adjuvants, which could potentiate the mucosal immune response to

PRRSV. The purpose of this review is to discuss methods for induction of protective mucosal

immune responses in the respiratory tract of pigs. The manuscript also discusses how PRRSV

modulates innate, adaptive and immunoregulatory responses at both mucosal and systemic

sites of infected and/or vaccinated pigs. This information may help in the design of innovative

mucosal vaccines to elicit superior cross-protective immunity against divergent field strains

of PRRSV.

Keywords: immunosuppression, mucosal adjuvants, BALT, protective immunity, mucosal

vaccines

Introduction

Porcine reproductive and respiratory syndrome (PRRS)

causes an annual economic loss of an estimated

$664 million to the U.S. pork industry (Holtkamp and

Kliebenstein, 2011). According to the Animal and Plant

Health Inspection Service report of January 2009, 49.8% of

unvaccinated pigs in the U.S. are seropositive to PRRSV.

This is based on collective data from 94% of pork pro-

ducers in 17 states, suggesting the widespread prevalence

of PRRS in the U.S. Pigs in more than 50% of sites from all

four regions in the U.S. (North, South, West Central and

East Central) had PRRSV antibodies and approximately

60% of pig samples in northern and southern regions

were positive for anti-PRRSV antibodies. PRRSV causes

respiratory and reproductive disease (Wensvoort et al.,

1991; Collins et al., 1992; Christopher-Hennings et al.,

1995), with major losses from reproductive failure in

sows, including stillbirths, mummifications, weak born

piglets and high preweaning mortality (Mengeling et al.,

1998; Meulenberg, 2000; Rowland, 2007). PRRSV is

excreted from all the body secretions at low levels or

perhaps intermittently in saliva, nasal secretions, urine,

milk, colostrum and feces from infected pigs, and also in

semen of infected boars (Rossow et al., 1994; Voicu et al.,*Corresponding author. E-mail: gourapura.1@osu.edu

*c Cambridge University Press 2012 Animal Health Research Reviews 13(1); 21–37ISSN 1466-2523 doi:10.1017/S1466252312000023

1994; Albina, 1997; Christopher-Hennings et al., 1998;

Wagstrom et al., 2001; Zimmerman, 2003). Under natural

conditions in the absence of any intervention, transport

and transmission of PRRSV by fomites and personnel

occur between swine populations (Pitkin et al., 2009). In

addition, airborne transport of PRRSV may occur over

long distances with no change in the infectivity of virus

(Otake et al., 2010). Therefore, control of PRRSV trans-

mission within and between swine herds is a major

challenge to the swine industry.

PRRSV is divided broadly into two distinct genotypes,

type I (European) and type II (North American). Each

genotype contains several subtypes and strains, which are

genetically highly diverse and display significant differ-

ences in their virulence and pathogenicity (Kim et al.,

2007). PRRSV field isolates within the North American

genotype have genetic diversity ranging from 84 to 100%,

based on the amino acid homologies of ORFs 2–6 relative

to VR2332, a prototype North American strain of PRRSV

(Kim et al., 2007). Immunity to the initial infecting geno-

type of the PRRSV may therefore provide partial to no

protection against reinfection, reflecting not only the

complexity of the genetics but also immunological vari-

ation among strains (Botner et al., 1997; Kimman et al.,

2009; Martelli et al., 2009; Li et al., 2010).

Currently, commercial modified live virus-PPRS (MLV-

PRRS) and inactivated PRRSV vaccines are licensed

for use; although the MLV-PRRS offers good protection

versus homologous virus isolates, there continue to be

questions regarding the vaccine efficacy against anti-

genically variant or heterologous field strains (Botner

et al., 1997; Mengeling et al., 2003a, b; Cano et al., 2007;

Kimman et al., 2009; Martelli et al., 2009). The efficacy of

available killed PRRSV vaccines is inadequate to protect

pigs against even genetically closely related PRRSV, and

none of the current vaccines prevent respiratory infection

and pig-to-pig transmission of PRRSV (Kimman et al.,

2009). Administration of either field isolates of PRRSV

or vaccine including MLV-PRRS by the parenteral or

intranasal route suppresses the innate natural killer (NK)

cell cytotoxic function and IFN-a production (Albina

et al., 1998; Renukaradhya et al., 2010; Dwivedi et al.,

2011b, 2012).

The most efficient and rapid host response against

viruses consists of production of type I IFNs (IFN-a/b), an

essential part of the antiviral innate immune system (Thiel

and Weber, 2008). The IFN production is triggered by the

first contact of cells with the virus; the secreted IFN slows

down or even blocks virus multiplication, aiding in

establishment of an adaptive immune response (Thiel

and Weber, 2008). The NK cell is a lymphocyte subset that

can be activated to mediate significant levels of innate

anti-viral cytotoxic activity and also produce high levels of

IFN-g and chemokines (Biron et al., 1999; Gerner et al.,

2009). Thus, innate immune response mediated through

type I IFNs and NK cells is critical for induction of

protective immunity. PRRSV experts collectively agree

that only replicating PRRSV vaccines have the promise

to reduce the PRRS incidence in the field (http://vetmed.

illinois.edu/news/PRRSwhitepaper.pdf). Since the 1990s,

development of a safe and effective vaccine that induces

broad protective immunity to PRRS remains as a principal

goal of researchers and the swine industry.

PRRSV-induced modulation in cytokine production

Swine are the only known hosts susceptible to PRRSV,

and the virus enters the host through respiratory and

reproductive mucosal surfaces. Primary permissive cells

are the alveolar macrophages (Mfs) and the virus also

infects Mfs present in pulmonary intravascular spaces,

lymph nodes, thymus, heart, spleen, placenta and

umbilical cord (Halbur et al., 1995; Rossow et al., 1996;

Duan et al., 1997; Lawson et al., 1997; Beyer et al.,

2000). PRRSV elicits poor innate and adaptive immune

responses (Albina et al., 1998; Van Reeth et al., 1999;

Meier et al., 2003; Renukaradhya et al., 2010) associ-

ated with increased immune modulation (Read et al.,

2000; Suradhat et al., 2003; Sakaguchi et al., 2009;

Dwivedi et al., 2011a, b), resulting in incomplete viral

clearance and associated secondary microbial infections

(Thanawongnuwech et al., 2001; Murtaugh et al.,

2002a; Van Reeth et al., 2002; Zimmerman et al., 2006;

Renukaradhya et al., 2010). In infected pigs, PRRSV sup-

presses innate IFN-a secretion (Albina et al., 1998;

Buddaert et al., 1998; Murtaugh et al., 2002a, b), and

dampens the NK cell-mediated cytotoxicity (Jung et al.,

2009; Renukaradhya et al., 2010; Dwivedi et al., 2012).

Cytokine interleukin-10 (IL-10) possesses potent immu-

nosuppressive properties and its production is necessary

to dampen the virus-induced inflammatory response,

which otherwise causes severe tissue damage and lethal

disease (Sun et al., 2011; Trandem et al., 2011a, b).

However, viruses evolved methods to evade the host im-

munity by promoting early secretion of IL-10 to antag-

onize the protective antiviral Th1 immune response

(Suvas et al., 2004; Humphreys et al., 2007; Gomez-

Laguna et al., 2010; Avdic et al., 2011; Lee et al., 2011).

Pig monocyte/Mfs infected with four PRRSV isolates

of varying clinical virulence were found to induce

increased IL-10 gene expression associated with signifi-

cantly reduced IFN-g and TNF-a expression in vitro

(Charerntantanakul et al., 2006). In pigs, a significant

correlation was observed between PRRSV infection and

expression of cytokine IL-10, and up-regulated IL-10 was

associated with reduced expression of cytokines IFN-a,

IFN-g , IL-12 and TNF-a (Gomez-Laguna et al., 2010),

indicating that immune responses generated in pigs

to PRRSV are inadequate to completely clear the virus

during the first 8–12 weeks post-infection. This dys-

regulated immune response has a strong impact on

PRRSV pathogenesis and on severity of the disease. One

possible approach to overcome the PRRSV-induced

22 G. J. Renukaradhya et al.

immunosuppression is to consider alternative routes of

administration of vaccines such as intranasal delivery that

could help to stimulate protective mucosal immunity,

because a majority (>80%) of total body immune cells are

present at mucosal sites (Czerkinsky and Holmgren,

2010).

Administration of the MLV-PRRS vaccine intranasally

mimics the patho-immunology of infection with the field

isolates, with the induction of IL-10 and transforming

growth factor b (TGF-b) in pig lungs and blood. However,

if the commercial MLV-PRRSV is intranasally co-

administered with a potent adjuvant, production of

IL-10 and TGF-b is significantly reduced (Dwivedi et al.,

2011a). PRRSV-induced increased frequency of FoxP3-

expressing T-regulatory cells (Tregs) has been implicated

in mediating immunosuppression in pigs (Didierlaurent

et al., 2007). Infiltration of Tregs into infected pig lungs

also contributes to secretion of high levels of IL-10 and

TGF-b (Didierlaurent et al., 2007). The role of Tregs in the

establishment of chronic persistent diseases such as those

caused by HIV, hepatitis C and B viruses, cytomegalovirus

and Epstein–Barr virus have been reported (Kinter et al.,

2004; Vahlenkamp et al., 2005; Peng et al., 2008). Perhaps

there are similar mechanisms that explain persistent

infection of PRRSV in pigs, but this remains to be proved.

However, our laboratory has reported that intranasal

co-administration of MLV-PRRS with an adjuvant Myco-

bacterium tuberculosis whole cell lysate (M. tb WCL)

significantly reduces the frequency of Tregs in the lungs,

blood and the tracheobronchial lymph nodes (TBLNs)

associated with a concomitant reduction in the produc-

tion of IL-10 and TGF-b (Dwivedi et al., 2011a). Overall,

these results all support the immunosuppressive nature

of PRRSV by induction of IL-10, TGF-b and Tregs (Read

et al., 2000; Johnsen et al., 2002; Suradhat et al., 2003;

Royaee et al., 2004; Diaz et al., 2005; Kaser et al., 2008;

Sakaguchi et al., 2009; Silva-Campa et al., 2009a, b;

Renukaradhya et al., 2010; Wongyanin et al., 2010;

Dwivedi et al., 2011a, b; Leroith et al., 2011).

Pigs infected with PRRSV (both low pathogenic and

virulent strains) are known to be susceptible to secondary

microbial (bacterial and viral) infections, probably due to

a PRRSV-induced immunosuppressive response resulting

in exacerbation of the respiratory disease (Done and

Paton, 1995; Van Reeth, et al., 2002; Renukaradhya et al.,

2010). In a co-infection study involving PRRSV and por-

cine respiratory coronavirus, a severe respiratory disease

was associated with elevated levels of cytokines IL-6,

IL-12, IL-10 and TGF-b, and reduced IFN-a and IFN-gproduction with synergistically dampened NK cell-

mediated cytotoxicity compared to infection by either

single virus (Jung et al., 2009; Renukaradhya et al., 2010).

Both live and inactivated PRRSV have the ability to up-

regulate IL-10 gene expression in porcine peripheral

blood mononuclear cells (PBMCs) (Suradhat et al., 2003).

Even after the clearance of PRRS viremia, the virus con-

tinues to induce IL-10 production for a prolonged

(>2 months) period of time (Drew, 2000; Johnsen et al.,

2002; Yoo et al., 2010; Dwivedi et al., 2011a). Induction of

IL-10 production in PBMCs and bronchoalveolar lavage

fluid (BAL) cells by both North American and European

strains of high and low virulence PRRSV was observed

(Aasted et al., 2002; Suradhat and Thanawongnuwech,

2003; Silva-Campa et al., 2009a).

Induction of IL-10 by PRRSV is mediated by interaction

between PRRSV proteins and Mfs/DCs, as these cells are

susceptible targets of PRRSV. The PRRSV N protein

induces IL-10 production by pig PBMCs and alveolar Mfs(Wongyanin et al., 2009; Yoo et al., 2010). In contrast,

results of a study involving pigs of all ages infected with

PRRSV suggested that virulent PRRSV infection and dis-

ease are markedly more severe and prolonged in younger

piglets than in finishers or sows. This is because porcine

innate and adaptive immune systems are not fully devel-

oped at birth and the prolonged infection may not be due

to IL-10-mediated immunosuppression (Klinge et al.,

2009). However, based on several other reports, the role

of IL-10 in delaying the onset and dampening the PRRSV-

targeted protective immunity is clearly evident. Thus,

based on our recent study and other published reports

eliciting a protective mucosal immune response with the

aid of potent adjuvant/s appears to be one of the viable

mechanisms for overcoming PRRSV vaccine-induced

immunosuppressive response to achieve a better control

over the viral clearance during outbreaks, especially in

growing pigs.

PRRSV-induced modulation in the immunecell population

PRRSV-modulated cytokines have a role in altering

the abundance of the immune cells that are essential for

viral clearance. Following natural or experimental infec-

tion of pigs with PRRSV, the virus-specific T-cell response

is delayed for 1–3 months post-infection (Bautista and

Molitor, 1999; Lopez Fuertes et al., 1999). Recently, we

evaluated the abundance of the immune cells present in

PRRSV (both virulent MN184 and vaccine strain VR2332)

infected pigs at post-infection days (PID) 15, 30 and 60 in

the lung parenchyma and TBLN (Dwivedi et al., 2011b;

Manickam et al., unpublished data). We evaluated im-

mune responses in the lungs and TBLN side-by-side,

because PRRSV causes lung pathology in infected pigs

and persists in the lymphoid tissues for long periods, up

to 200 days. Generally, for effective viral clearance from

the body, adequate adaptive immune responses have to

be generated and the sites of initiation of such response

are in the TBLN. Following infection of pigs with PRRSV

strain MN184, the total T-lymphocyte population was

gradually reduced over a period of time in the lungs, and

by PID 60 it was 20% of the mock-control pigs. In

contrast, the T-lymphocyte population was reduced 50%

at PID 30 and returned to near normal levels by PID 60

Mucosal vaccines to prevent porcine reproductive and respiratory syndrome 23

post-infection in the TBLN. Similarly, in PRRSV VR2332-

infected pigs the total T-lymphocyte population was

reduced in the lungs but at a slower rate, and by PID 60,

their population was 65% lower than in mock pigs. There

were no appreciable changes in the lymphocyte popu-

lation in TBLN of VR2332-infected pigs. Strikingly, the

number of Tregs was increased by greater than 2.5-fold

in the lungs, TBLN and PBMCs in pigs infected with

either PRRSV strain, and the number of Tregs in these

tissues remained high until PID 60 (Dwivedi et al., 2011b;

Manickam et al., unpublished data).

Others have also reported that PRRSV infection sig-

nificantly reduces the leukocyte population in blood,

wherein the levels of total leukocytes, lymphocytes,

monocytes and eosinophils, but not neutrophils, are

lower for several days shortly after infection (Lohse et al.,

2004; Che et al., 2011). Such a decline in total peripheral

blood leukocyte population during the early stage of

PRRSV infection may undermine the immune functions in

infected pigs, and promote susceptibility of pigs to se-

condary microbial infections. In addition, depending on

the virulence of the PRRSV strain, infected pigs have

varying degrees of modulation in the abundance of

lymphocytes in the lungs. However, we have demon-

strated that PRRSV-induced reduction in the lymphocyte

populations at both mucosal and systemic sites could be

restored to a physiological normal level when pigs were

vaccinated intranasally using MLV-PRRS combined with

M. tb WCL adjuvant prior to PRRSV challenge (Dwivedi

et al., 2011a).

PRRSV-induced modulation of antibody response

PRRSV-specific neutralizing antibodies (NAs) play a cri-

tical role in viral clearance and are considered as a major

component of the protective immune response (Murtaugh

et al., 2002a, b; Osorio et al., 2002; Lopez and Osorio,

2004; Lopez et al., 2007). PRRSV infection instigates abun-

dant production of antibodies in pigs of all ages (Loemba

et al., 1996; Lemke et al., 2004), and this is due to virus-

mediated polyclonal activation of B-cells (Drew, 2000;

Lamontagne et al., 2001). However, a majority of these

secreted antibodies are antibodies to host proteins and

PRRSV non-NAs (Lemke et al., 2004). Studies of the kin-

etics of humoral immune response induced in pigs exper-

imentally infected intranasally with a North American

strain (VR2332) of PRRSV identified that the earliest

antibodies are directed against viral nucleocapsid (N)

protein, followed by matrix (M) and surface glycoprotein

(GP) 5 (Nelson et al., 1994). Also, in MLV-PRRS vaccinated

pigs, enhanced production of non-NAs, directed mainly

against PRRSV-N protein and also against anti-neutralizing

epitopes (decoys) of the virus, was detected (Yoon et al.,

1995; Lopez and Osorio, 2004). PRRSV surface GP

contains immunodominant epitopes A and B, wherein

epitope A is a decoy epitope inducing an early and strong

non-NA response (Osorio et al., 2002; Ostrowski et al.,

2002). The neutralizing epitope B is surrounded by up to

four N-linked glycosylation sites, and variant strains

are lacking few of those important glycosylation sites

that potentially promote or assist escape from antibody-

mediated neutralization (Faaberg et al., 2006; Mateu et al.,

2006; Vu et al., 2011).

Overall, the PRRSV-NA response is vague, weak and

delayed (Mateu et al., 2006; Mateu and Diaz 2008).

Moreover, due to the low fidelity of the PRRSV RNA-

dependent–RNA polymerase, mutations are consistently

introduced into the viral genome resulting in the

emergence of antigenic heterogeneity in immunodomi-

nant epitopes recognized by both antibodies and T-cells

(Meng, 2000; Huang and Meng, 2010). This plasticity of

the PRRSV genome results in numerous “vaccine failures”

in the field, posing constant challenges to swine

veterinarians and producers.

Such genetic and antigenic variability supports the

evolution of PRRSV variants in the field and may explain

the need for a vaccine that can elicit immune response

to other conserved epitopes present on the viral surface

GP that induces broadly reactive immune response to

heterologous PRRSV. We have demonstrated that, in pigs

inoculated intranasally with MLV-PRRS along with M. tb

WCL virus, specific total antibody response was reduced

substantially by PID 30, and interestingly, the virus-

specific NA response was significantly increased com-

pared to pigs administered only MLV-PRRS intranasally.

These results suggest that mucosal immunization with

MLV-PRRS coupled with M. tb WCL adjuvant suppresses

the virus-induced total antibody response and at the same

time importantly enhances the required virus neutraliza-

tion titers for better viral clearance.

Mucosal adjuvants and MLV-PRRS

Until 2010 there were no published studies on the

development of a live PRRSV mucosal vaccine. This may

be due to limited research undertaken to identify potent

mucosal adjuvants for use with MLV-PRRS. However, the

ability of PRRSV to rapidly modulate the host innate

immune response suggests the need for a potent adjuvant

that has the inherent capacity to overcome the PRRSV

vaccine-induced immunosuppressive response, and at the

same time promote virus-specific cell-mediated immunity.

Therefore, in our attempt to search for a potent mucosal

adjuvant to used with MLV-PRRS intranasally, we evalu-

ated the elicited immune responses in the respiratory tract

of pigs inoculated intranasally with a panel of candidate

adjuvants. We chose a panel of nine bacterial prepara-

tions belonging to Mycobacterium, Vibrio and Strepto-

coccus species, and all known to possess adjuvant

properties in rodent models (Bonavida et al., 1986; Foss

and Murtaugh, 1999; Kuroki et al., 2003; Barral and

Brenner, 2007; Beetz et al., 2007). Pigs were inoculated

24 G. J. Renukaradhya et al.

with adjuvants alone, an adjuvant with MLV-PRRS and

MLV-PRRS alone intranasally; the pigs were euthanized

on day 7 post-inoculation and immune responses were

assessed. Based on PRRSV-specific mucosal and systemic

immune responses mediated by a battery of candidate

adjuvants, we chose three preparations for use along with

MLV-PRRS in viral challenge studies, they were M. tb WCL,

Cholera toxin B subunit (Sigma) and OK-432 (a product of

Streptococcus pyogenes) (Dwivedi and Renukaradhya,

unpublished data). Results of this study demonstrated that

among these three adjuvants, only M. tb WCL significantly

dampened the PRRSV-induced immunosuppressive re-

sponse, in addition to inducing enhanced virus-specific

innate and adaptive immune responses (Dwivedi et al.,

2011a, b).

Heat-killed M. tb is an excellent candidate adjuvant

used in the preparation of Freund’s complete adjuvant

(FCA) and has been used extensively in experimental

animals (Roberts et al., 1992). However, its use in humans

and in food animals is constrained due to a severe granulo-

matous inflammatory reaction induced at the injection

site. The adverse effect due to FCA results from toxic

cell wall components of M. tb, such as mycolic acids,

arabinogalactan and wax D (Bekierkunst 1968). However,

adjuvanticity of various purified components of M. tb

have been evaluated individually with satisfactory results

(Bekierkunst et al., 1971; Harmala et al., 2002). Particu-

larly, water soluble purified fraction (M. tb WCL) free from

bacterial toxic cell wall components has been shown to

possess superior adjuvanticity in rodents, guinea pigs and

rabbits (White et al., 1955; Werner et al., 1975; Srivastava,

2002; Choudhary et al., 2003; Bansal et al., 2010). In our

recently published study, pigs that received M. tb WCL did

not have any toxic symptoms or any adverse inflamma-

tory response at the local inoculation site, and there was

no fever (Dwivedi et al., 2011a, b). At present, it is not

known what component of the M. tb WCL made this

candidate adjuvant more potent than the other tested

candidates in the respiratory tract of pigs that also

received MLV-PRRS. A vector-based recombinant vaccine

expressed in M. tb strain BCG containing PRRSV GP5 and

M protein helped to reduce clinical PRRS disease.

Immunized pigs challenged with virulent homologous

PRRSV had decreased viremia and viral load in tissues,

suggesting that a favorable immune response to PRRSV

could be induced using M. tb preparation as an adjuvant

(Bastos et al., 2004).

Pigs vaccinated intranasally with MLV-PRRS along with

M. tb WCL and challenged with the virulent heterologous

PRRSV strain MN184 had significantly increased PRRSV-

specific NA response compared to pigs vaccinated with

MLV-PRRS without any adjuvant (Dwivedi et al., 2011a).

In addition, significantly higher levels of IFN-g and IL-12

(P<0.05) and significantly lower amounts of IL-10 were

detected in pigs given the combination of MLV-PRRS and

M. tb WCL (Dwivedi et al., 2011a). However, both groups

of vaccinated pigs were viremic and the virus was not

eliminated until day 60 post-challenge (albeit relatively less

virus was detected in pigs that received the adjuvant).

Interestingly, significantly reduced lung lesions with no

clinical signs were associated with increased anti-PRRSV-

specific Th1 immune response in pigs that received the

adjuvanted MLV-PRRS vaccine; this indicates the benefits

of intranasal vaccination of commercially available MLV-

PRRS with M. tb WCL (Dwivedi et al., 2011a, b).

The study described above did not include a group of

pigs inoculated with MLV-PRRS alone by the parenteral

route for a side-by-side comparison (Dwivedi et al.,

2011b). However, there have been several previous

studies of pigs inoculated with MLV-PRRS by the paren-

teral route and challenged with heterologous PRRSV

(Botner et al., 1997; Mengeling et al., 2003a, b; Cano et al.,

2007; Kimman et al., 2009; Martelli et al., 2009), and one

study performed during evaluation of mucosal adjuvan-

ticity of a panel of adjuvants along with MLV-PRRS

administered by the intranasal route (Dwivedi et al.,

2011b, Dwivedi and Renukaradhya, unpublished data).

Based on these studies, we strongly hypothesize that

mucosal vaccination with a potent adjuvant such as M. tb

WCL is advantageous in reducing the heterologous PRRSV-

induced disease burden. However, similar studies using a

large number of pigs under field conditions and in pigs of

diverse genetic background are essential to advance this

promising mucosal immunization approach to the field.

Mucosal surfaces and associated lymphoid tissuesin the respiratory tract

Mucous membranes cover the largest surface area in

the body, with 80% of total body immune cells present

at mucosal surfaces and mucosa-associated lymphoid

tissues (MALT) (Czerkinsky and Holmgren, 2010). The

MALT is a highly compartmentalized immune system

that functions independently of the systemic immune

system, including the skin-associated lymphoid tissues

(Holmgren and Czerkinsky, 2005). It is highly populated

with phenotypically and functionally distinct B-cell, T-cell

and accessory cell subpopulations comparable to sys-

temic lymphoid tissues. Also, it possesses well-developed

strong restrictions upon lymphoid cell recirculation be-

tween mucosal sites. MALT comprises anatomically de-

fined lymphoid micro-compartments such as the Peyer’s

patches, the mesenteric lymph nodes, the appendix and

solitary follicles in the intestine called the gut-associated

lymphoid tissues (GALT); nasopharynx-associated lym-

phoid tissues (NALT), tonsils and adenoids at the entrance

of the aerodigestive tract; and bronchus-associated

lymphoid tissues (BALT) in the lower respiratory tract.

The NALT is spread over the nasal cavity, nasopharynx

and the middle ear (Kiyono and Fukuyama, 2004; Canessa

et al., 2010; Randall, 2010), which also includes tonsils

that form the Waldeyer’s ring. A Waldeyer’s ring consists

of five different tonsils made of lymphoepithelial tissue,

Mucosal vaccines to prevent porcine reproductive and respiratory syndrome 25

and are present in a similar pattern in both humans and

pigs at the oropharyngeal and nasopharyngeal openings

(Davis, 2001). Recently, we studied the abundance of

various immune cell populations in the tonsils of pigs and

detected T-lymphocytes (42%), NK cells (4%), myeloid

cells (CD172+) (10%), dendritic cells (DCs) (3%) and gd T

cells (6%). Further, the population of subsets of T-

lymphocytes include CD8+ T-cells (CD3+CD4�CD8+;

22%); memory/T-helper cells (CD3+CD4+CD8+, 16%); T-

helper cells (CD3+CD4+CD8�; 47%); and FoxP3+ Tregs

(CD4+CD25+ FoxP3+, 25%) (Renukaradhya et al., unpub-

lished data). The MALT also contains diffuse accumula-

tions of large numbers of lymphoid cells in the

parenchyma of mucosal organs and exocrine glands

which form the mucosal effector sites where immune

responses are manifested. Considering the relative immu-

nological similarities of humans and pigs, we expect

comparable structure, distribution and function of

immune cells present in the pig MALT.

Immune cells present at mucosal sites are involved in

providing pathogen-specific mucosal immunity. Thus,

effective mucosal delivery of vaccines has been gaining

increased attention for the control of pathogens which

primarily cause pathology and disease at mucosal sites,

for example, influenza, parainfluenza, HIV, Rotavirus and

PRRSV. Stimulating the immune system systemically (by

parenteral injection) results mainly in systemic protection

with low mucosal immune responses. In contrast,

optimum stimulation of the mucosal immune system

provides both mucosal and systemic immunity, resulting

in inhibition of replication and/or colonization of

infectious agents before they replicate and cause disease

(Holmgren et al., 1992; Etchart Wild et al., 1996; Singh

et al., 2001; Holmgren and Czerkinsky, 2005). NALT

and BALT contain T- and B-cell zones with an entire

repertoire of accessory immune cells strategically located

to orchestrate regional immune functions against airborne

infections (Mann et al., 2009; Randall, 2010). Immune

cells at mucosal sites are primarily involved in the regu-

lation of inflammatory responses, but at the same time

guard the body against the entry of harmful pathogens.

Therefore, it is difficult to elicit protective mucosal

immunity using conventional vaccine preparations deliv-

ered directly to mucosal sites. However, administration of

conventional vaccines in the presence of suitable adju-

vants by the intranasal route has been shown to modulate

the immune regulatory response with simultaneous

upregulation of protective antibody and cell-mediated

immune response (Shephard et al., 2003; Kamijuku et al.,

2008; Guillonneau et al., 2009; Dwivedi et al., 2011b).

Therefore, mucosal vaccination offers an advantage in

inducing immune protection against viruses that typically

invade and cause disease at mucosal sites. The respiratory

tract is a portal of entry for many viruses and it is a

challenge for the mucosal immune system to completely

clear the virus-infected cells. NALT, BALT, upper and

lower respiratory tract draining lymph nodes such as the

cervical and TBLNs, help in the clearance of pathogens by

initiating specific adaptive immune responses. Consider-

ing the inherent advantages of potent mucosal immunity,

researchers have been finding innovative strategies to

develop effective mucosal vaccines.

Inducible BALT structures in pigs

BALT is a secondary lymphoid organ embedded in the

walls of large airways and at airway bifurcations; it is

similar to Peyer’s patches in function (Sminia et al., 1989).

In addition to normally present BALT tissues, additional

BALT structures in bronchi are inducible and are called

“inducible BALT” (iBALT). iBALT is considered to be a

tertiary lymphoid structure that develops either in re-

sponse to microbial infection or under chronic inflamma-

tory conditions (Tschernig and Pabst, 2000; Foo and

Phipps, 2010), and persists for several months, even after

clearance of pathogens (Moyron-Quiroz et al., 2007).

Both BALTs serve as principal mucosal inductive sites

to initiate immune responses against pathogens (Mowat,

2003; Kiyono and Fukuyama, 2004; Holmgren and

Czerkinsky, 2005). Influenza-specific memory CD8 T-cells

and plasma cells which contribute to the generation of

effector T-cells and NAs, respectively, aid in clearance of

both homologous and heterologous strains of influenza

virus and were identified in iBALT (Moyron-Quiroz et al.,

2007). Pigs immunized intranasally with MLV-PRRS in

combination with M. tb WCL and challenged with highly

virulent heterologous strain (MN184) of PRRSV had

increased formation of iBALT structures (Fig. 1). The

formation and persistence of iBALT for a long period of

time is dependent on the critical recruitment of DCs into

the lungs (GeurtsvanKessel et al., 2009). In contrast, Tregs

present in the lymph nodes draining the lung attenuate

iBALT formation by inhibiting the activation and prolif-

eration of lymphocytes and other unidentified popu-

lations of cells (Foo and Phipps, 2010). Consistent with

that, there was an increased frequency of DC-rich fraction

(CD172+ CD11c+ SLAII+ cells) in both blood and the

lungs, and significantly reduced frequency of Tregs in

TBLN and the lungs of pigs intranasally immunized (MLV-

PRRS+M. tb WCL) and challenged with the MN184 strain

of PRRSV (Dwivedi et al., 2011a). Interestingly, iBALT

contains DCs in higher frequency in the T-cell zone

compared to other lymphoid tissues and also DCs are

scattered throughout the B-cell zone (Woodland and

Randall, 2004). Thus, formation of increased iBALT

structures in intranasally immunized pigs has the potential

to induce effective mucosal immunity against PRRSV.

For the mucosal immune system to recognize microbes

or vaccines, specialized epithelial cells called “microfold

cells” (M-cells) located in follicle-associated epithelium

overlying the MALT should capture and transport the

antigens across the mucosal barrier (transcytosis). This

will lead to subsequent uptake and processing by

26 G. J. Renukaradhya et al.

professional antigen-presenting cells (APCs), DCs,

Mfs and B-cells for initiation of specific adaptive immune

response (Jepson et al., 1996; Neutra et al., 1999;

Kraehenbuhl and Neutra, 2000; Neutra and Kozlowski,

2006; Corr et al., 2008; Suzuki et al., 2008). APCs prime

the naı̈ve T-cells to clonally expand and differentiate into

T-cell subsets (Th1, Th2, Th17, or Tregs). Subsequently,

activated T-cells migrate to submucosal regions where

they perform effector functions. In particular, particulate

antigens have an inherent affinity for mucosal M-cells and

also for underlying APCs (Brayden et al., 2005). In pigs,

M-cells are present (Gebert et al., 1994), and their fre-

quency in follicle-associated epithelium is highly variable;

they constitute greater than 20% of the cells, which is

higher than in rodents (�10%) and humans (<5%) (Buda

et al., 2005). Recently, an abundant population of M-cells

in the nasopharyngeal mucosa of rodents was identified

(Kim et al., 2011). M-cells express MHC class II molecules

(Allan et al., 1993), thus fulfilling many of the criteria for

APCs (Brayden et al., 2005). Overall, M-cell-targeted

delivery of vaccines to successfully stimulate the mucosal

immune system has become an innovative strategy to

develop effective mucosal vaccines (Plotkin, 2005).

Mucosal vaccination is an effective alternativeto control infections

Research on mucosal vaccine development has demon-

strated that it is possible to activate immune cells present

in the MALT, using vaccine antigens co-administered with

appropriate adjuvants to mucosal sites. This approach

potentiates the generation of long-lasting memory im-

mune response against specific pathogens. There are

many potent adjuvants available but a majority of them

lack the ability to augment the mucosal immune re-

sponse. From among a few identified mucosal adjuvants

the majority are of bacterial origin (reviewed in Lawson

et al., 2011). Mucosal vaccines co-delivered with suitable

adjuvants have proved to be effective in controlling many

diseases, particularly against pathogens which cause dis-

ease primarily at mucosal sites. In this regard, there are

several studies, for example: (a) gastrointestinal infections

caused by Helicobacter pylori, Vibrio cholerae, enterotoxi-

genic Escherichia coli (ETEC), Shigella spp., Clostridium

difficile, polio virus, rotaviruses and calici viruses; (b) re-

spiratory infections caused by Mycoplasma pneumoniae,

influenza virus, parainfluenza-3 virus, respiratory syncitial

virus and PRRSV; (c) sexually transmitted genital infec-

tions caused by HIV, simian immunodeficiency virus,

Chlamydia trachomatis, Neisseria gonorrhoeae and

herpes simplex virus-1 (Ogra, 1984; Karron et al., 1995;

Van der Poel et al., 1995; Imaoka et al., 1998; Schmidt

et al., 2002; Rodriguez et al., 2003; Petrovsky and Aguilar,

2004; Holmgren and Czerkinsky, 2005; Ichinohe et al.,

2007a, b; Kamijuku et al., 2008; Guillonneau et al., 2009;

Dwivedi et al., 2011b). The protective mucosal immunity

against the above pathogens or their toxins has been

more effective in reducing the disease burden than have

conventional parenteral routes of immunization.

Activated innate immune response at mucosal sites

plays a major role in mucosal immunity against enteric

and respiratory infections (Mann et al., 2009). Intranasal

administration of influenza and parainfluenza-3 vaccines

with a potent adjuvant generated enhanced cytotoxic

T-lymphocyte and central memory immune response

to conserved internal viral proteins, resulting in cross-

protective immunity against a wide range of heterologous

influenza and parainfluenza viruses (Shephard et al.,

2003; Kamijuku et al., 2008; Guillonneau et al., 2009).

Currently, a licensed nasal-spray flu vaccine (FluMist,

MedImmune, Gaithersburg, Maryland, USA) containing

cold-adapted, temperature-sensitive, live-attenuated influ-

enza virus (LAIV) reassortant strains was found to be

effective (Belshe et al., 2004). FluMist induces an immune

response that closely resembles natural immunity com-

pared to conventional injectable vaccines (Cox et al.,

2004). In one large study among children aged 15–85

months, FluMist reduced the chance of influenza illness

by 92% compared with placebo. In yet another study

among adults vaccinated with FluMist vaccine, there were

19% fewer severe febrile respiratory tract illnesses, 24%

fewer respiratory tract illnesses with fever, 23–27% fewer

days of illness, 13–28% fewer lost work days, 15–41%

Mock PRRSV-challenged

Fig. 1. iBALT in pigs. Pigs were unvaccinated (mock) or vaccinated with MLV-PRRS plus Mtb WCL and challenged withvirulent heterologous PRRSV strain MN184, and euthanized on day 15 post-challenge. Representative photographs of the lungsof pigs from the mock and the virus challenged groups are shown. Arrow indicates iBALT structures.

Mucosal vaccines to prevent porcine reproductive and respiratory syndrome 27

fewer health care provider visits and 43–47% less use

of antibiotics compared with placebo (http://pediatrics.

about.com/cs/immunizations/a/flumist_qa.htm). Nasal or

tonsillar immunization in humans results in protective

mucosal antibody response in the upper respiratory tract

and the lungs but not in the gastrointestinal tract

(Johansson et al., 2001, 2004). In addition, nasal immuniz-

ation has been found to generate enhanced secretory-IgA

and IgG antibody responses in the human reproductive

tract mucosa (Kozlowski et al., 1997; Johansson et al.,

2001; Nardelli-Haefliger et al., 2003). This suggests that

intranasal delivery of PRRSV vaccine in the presence of a

suitable adjuvant has the potential to augment protective

immunity against PRRS.

Mucosal PRRSV vaccination in pigs and itsimplications

The pig is an important food animal species, and it

possesses a number of immunologically unique features,

such as an inverted lymph node structure (Binns, 1982),

and an unusual route for lymphocyte circulation (Pabst

and Binns, 1989). Porcine lymphocyte populations are

also unusual and feature abundant CD4CD8 double

positive T-cells (Zuckermann, 1999), NK (Yang and

Parkhouse, 1996; Yang and Parkhouse, 1997) and gdT-cells (Pabst and Binns, 1989; Yang and Parkhouse,

1996, 1997). In pigs, classical cytotoxic T-cells are fewer in

number, but large numbers of lymphocytes are CD8a+

(NK, gd T-cells, NKT cells) with a strong potential for

innate cytotoxic activity.

PRRSV gains entry through respiratory and reproduc-

tive mucosal surfaces and also causes disease primarily at

mucosal sites. Therefore, a protective mucosal vaccine

to PRRSV may be an appropriate strategy to control PRRS

outbreaks effectively. Since the development of

MLV-PRRS in the 1990s, the vaccine has been delivered

to pigs by the intramuscular route. MLV-PRRS induces

both humoral and cell-mediated immune responses (Foss

et al., 2002; Meier et al., 2003) associated with reduced

lung lesions (Mengeling et al., 2003a, b) and reduced

viremia in younger pigs challenged with a homologous

virus (Foss et al., 2002). However, incomplete protection

induced by MLV-PRRS against reinfections and against

genetically variant viruses indicates a need for alternate

vaccination approaches, such as mucosal immunization,

to effectively control PRRS.

Mucosal live PRRSV vaccines

Our research group and many others have demonstrated

that, in both PRRSV infected and MLV-PRRS vaccinated

pigs, the virus-specific cell-mediated immune responses

are either delayed or dampened. The poor immune re-

sponse is attributed to virus-induced immunosuppression

(Johnsen et al., 2002; Suradhat et al., 2003; Royaee et al.,

2004; Diaz et al., 2005; Renukaradhya et al., 2010). In a

study to enhance the MLV-PRRS-induced cell-mediated

immune response, pigs were administered four injections

of recombinant porcine IL-1 and IL-6 together, IL-12

alone, or cholera toxin alone within 1 week of intra-

muscular administration of MLV-PRRS. Results indicated

that IL-12 and cholera toxin have the potential to enhance

immune response to MLV-PRRS (Foss et al., 2002).

However, there was no difference in the level of viremia

between pigs that received vaccine with or without

adjuvant followed by homologous PRRSV challenge. In

addition, due to lack of data on NA titers and lack of

information on heterologous PRRSV challenge in vacci-

nated pigs, it is difficult to assume that this vaccination

strategy induces protective immunity. Moreover, we need

an MLV-PRRS that permits differentiation of animals that

have been infected by field viruses from animals that

have been vaccinated (DIVA). The importance of marker

(DIVA) vaccines has been recognized and such vaccines

have been constructed, and companion diagnostic tests

have been developed that allow infected animals to be

distinguished from vaccinated animals (van Oirschot

et al., 1986). Pseudorabies and bovine herpesvirus 1

DIVA vaccines have been demonstrated to reduce

transmission of wild-type virus in populations of pigs

and cattle in the field (van Oirschot et al., 1986; Kit, 1990).

The implementation of regional elimination projects has

overtaken immunology and epidemiology research,

necessitating the development of tests to differentiate

infected and vaccinated animals (DIVA).

In our study, pigs vaccinated intranasally with MLV-

PRRS along with Mtb WCL were challenged with a

heterologous PRRSV strain MN184 (Kim et al., 2007); we

detected a significant rescue in body weight gain, reduced

lung pathology, enhanced PRRSV NA titers and reduced

viremia at early time points post-challenge (Dwivedi

et al., 2011a). Immunologically, in pigs intranasally vac-

cinated with M. tb WCL were compared to those

vaccinated without adjuvant or unvaccinated and chal-

lenged with strain MN184; an increased frequency of

Th cells, Th/memory cells, gd T-cells, DCs and activated

Th cells, and a reduced frequency of Tregs were detected

at both mucosal and systemic sites. Further, reduced

secretion of immunosuppressive cytokines (IL-10 and

TGF-b) and up-regulation of the Th1 cytokine IFN-g in the

blood and the lungs were detected (Dwivedi et al.,

2011a). Similar and more robust immune responses were

detected in pigs vaccinated using MLV-PRRS in combina-

tion with M. tb WCL and challenged with a homologous

PRRSV VR2332 (Dwivedi et al., unpublished data).

PRRSV-specific lymphocyte responses in pigs vacci-

nated with M. tb WCL and challenged with strain MN184

or VR2332 were also generated against internal viral

protein of PRRSV, indicated by significantly increased M

protein-specific IFN-g and IL-12 cytokine response in re-

stimulated lung MNC and PBMCs (Dwivedi et al., 2011a).

28 G. J. Renukaradhya et al.

This result has provided the evidence that PRRSV internal

viral protein-specific epitopes-targeted response is

augmented by intranasal vaccination of MLV-PRRS with

M. tb WCL, and that could be responsible for increased

cross-protective immune response. Our results are con-

sistent with those from an intranasal influenza vaccination

study in mice wherein adjuvanted live or inactivated

influenza virus vaccine enhanced the virus-specific

cytotoxic T-lymphocyte and central memory immune

cell response against influenza internal viral proteins, re-

sulting in cross-protective immunity (Guillonneau et al.,

2009). Intranasal immunization with adjuvant a-GalCer

and inactivated hemagglutinin vaccine of influenza

derived from H1N1, H3N2 and H5N1 strains induce

effective cross-protection against both heterologous and

heterosubtypic influenza viral strains through invariant

NKT cell-mediated immune activation in the lungs

(Kamijuku et al., 2008).

Previous studies related to passive protection provided

by PRRSV (NAs indicated that: an NA titer of 1/16 protects

pregnant sows against reproductive failure and also

blocks transplacental infection (Osorio et al., 2002); an

NA titer of 1/8 or higher protects piglets against develop-

ment of viremia; and NA titers of 1/32 provide sterilizing

immunity (Lopez et al., 2007)). Thus, vaccines capable of

inducing NA titers of more than 1/16 should protect pigs

from clinical disease and may help in PRRS clearance

from the body. Pigs immunized intranasally with MLV-

PRRS along with M. tb WCL had NA titers of more than

1/32 from day 7 until day 35 post-immunization (Dwivedi

et al., 2011b). In contrast, pigs inoculated with MLV-PRRS

alone (without any adjuvant), had NA titers of less than

1/16 at multiple time points post-immunization (Dwivedi

et al., 2011b). In pigs that were immunized with MLV-

PRRS plus M. tb WCL and challenged with the hetero-

logous PRRSV MN184 strain, NA titer of more than 1/16

persisted until PID 60. This was associated with signifi-

cantly reduced viral load in the blood of challenged pigs

(Dwivedi et al., 2011b). Moreover, the enhanced NA titer

in pigs which received MLV-PRRS plus M. tb WCL

was associated with augmented Th1 and suppressed

immunosuppressive responses.

Following intranasal vaccination of pigs with MLV-PRRS

and no adjuvant, the numbers of virus-specific IFN-gsecreting cells were in the range of 50–100 cells

per million PBMCs and they persisted until 10 weeks

post-challenge (Dwivedi et al., 2011a). In contrast, pigs

vaccinated against Aujeszky’s disease virus, had 200–300

IFN-g secreting cells per million PBMCs (Meier et al.,

2003). In these pigs IFN-g secreting cells were mainly

CD4+CD8+ T-cells. This observation indicates that PRRSV

induces relatively weak IFN-g response in infected pigs.

However, pigs immunized intranasally with MLV-PRRS

plus M. tb WCL and challenged with PRRSV MN184

had greater than 300 IFN-g secreting cells per million

PBMCs, associated with greater than a 2-fold increase

in the frequency of total CD4+CD8+ T-cells at day 15

post-challenge (Dwivedi et al., 2011a). These results

strongly indicate that immunization of pigs with MLV-

PRRS in combination with a potent adjuvant administered

by the intranasal route has the potential to induce en-

hanced IFN-g response which could reduce PRRSV

replication and dampen the severity of clinical PRRS.

Optimal production of reactive oxygen species (ROS)

by immune cells is essential for antimicrobial activity

(Martin and Edwards, 1993; Quinn and Gauss, 2004;

Acker, 2005; Chvanov et al., 2005), and excess production

of ROS causes apoptosis/necrosis of infected and

bystander cells (Clutton, 1997; Vaughan, 1997; Halliwell

and Gutteridge, 2006). ROS are one of the important

mediators of diffused PRRSV-mediated pathology in the

lungs and lymph nodes of infected pigs (Sirinarumitr

et al., 1998). BAL cells and PBMCs in pigs intranasally

vaccinated (MLV-PRRS plus M. tb WCL) and challenged

with either PRRSV VR2332 or MN184 strain had higher

levels of ROS production compared to mock control pigs;

but the ROS levels in pigs that received MLV-PRRS plus M.

tb WCL were significantly lower than in unvaccinated

challenged animals (Binjawadagi et al., 2011). This result

suggests that M. tb WCL adjuvant effects optimally

induced ROS production which helped to boost the

immunity to PRRSV without causing lung pathology in

virus-challenged pigs.

Enhanced antigen-specific proliferation of lung lympho-

cytes in vaccinated (MLV-PRRS plus Mtb WCL) MN184

challenged pigs, and increased antigen-specific prolifera-

tion of CD8+ T-cells in the blood of similarly vaccinated

PRRSV VR2332 challenged pigs have been detected

(Manickam et al., unpublished data). Nitric oxide (NO0)

has been shown to inhibit the viral replication in influ-

enza and herpes simplex virus type 1 infections (Croen,

1993; Rimmelzwaan et al., 1999; Akerstrom et al., 2005).

Consistent with this, the level of NO0 was significantly

higher in the lungs of intranasally vaccinated (MLV-PRRS

plus M. tb WCL) virus-challenged pigs (Manickam et al.,

unpublished data). These results support the conclusion

that elicited protective humoral and cell-mediated im-

mune responses were associated with suppression of

ROS-mediated lung damage and also enhanced NO0

production in pigs vaccinated with MLV-PRRS in com-

bination with a potent adjuvant. Thus, it is possible to

generate protective anti-PRRSV immunity against reinfec-

tions caused by genetically divergent strains of PRRSV.

Mucosal killed PRRSV vaccines

Although many reports demonstrated satisfactory immu-

nity induced by MLV-PRRS in growing pigs, others

reported reversion of the vaccine virus and the presence

of reverted virulent PRRSV in unvaccinated sows main-

tained in the same farm premises (Madsen et al., 1998;

Nielsen et al., 2001, 2002). Also recombination between

vaccine and field strains associated with revertant PRRSV

Mucosal vaccines to prevent porcine reproductive and respiratory syndrome 29

strains that are pathogenic in sows and piglets has been

reported (Nielsen et al., 2001; Mengeling et al., 2002;

Murtaugh et al., 2002a, b; Li et al., 2009). In addition,

identification of mutations in multiple vaccine-derived

isolates in identical positions of the viral genome has

suggested a strong parallel selective pressure on critical

positions in the vaccine virus in the field (Nielsen et al.,

2001). Vaccine-derived virus has been isolated from

fetuses, stillborn and dead piglets, indicating the spread

of live vaccine virus from the vaccinated growing pigs in

the neighboring herd to non-vaccinated sows, and the

involvement of vaccine virus in reproductive problems

(Nielsen et al., 2002). Also, vaccine-derived PRRSV isolates

are common in swine populations (Key et al., 2003), and

some vaccine-derived field strains of PRRSV are actually

causing diseases, at least under experimental conditions

(Opriessnig et al., 2002). Therefore, safety of widely used

MLV-PRRS remains questionable as the vaccine-derived

mutated virus can spread to unvaccinated herds and

increase the PRRS incidence by many-fold in the future.

Since pregnant sows are not protected by vaccination,

prevention of PRRSV infection in sows is a big challenge

to swine producers. Control of PRRS in sows is very

important because this prevents transmission of the virus

to susceptible pigs through vertical and horizontal routes.

Therefore, live PRRSV vaccine is not safe for use where

growing pigs and pregnant sows are maintained in the

same premises, and the use of live PRRSV vaccine in

sows, gilts and breeding boars is not practiced. Unfortu-

nately, none of the available killed PRRSV vaccines elicits

protective immunity. All these limitations made research

on the development of a cross-protective killed PRRSV

vaccine a high priority.

Several attempts have been made to develop protec-

tive killed PRRSV vaccines using recombinant PRRSV

protein-expressing plasmid DNA and inactivated PRRSV

administered with or without adjuvant by a parenteral

route, but the results are not satisfactory (reviewed in

Charerntantanakul, 2009). Studies performed to deter-

mine the efficacy of killed PRRSV vaccine administered

intranasally along with adjuvants CpG ODN (TLR-9

ligand) (Zhang et al., 2007) and oligodeoxynucleotides

containing synthetic immunostimulatory motifs (Zhang

et al., 2006) resulted in augmented PRRSV-specific anti-

body response, enhanced antigen-specific T-cell prolif-

eration, and increased secretion of cytokines (IFN-g and

IL-6). It is possible to induce PRRSV-specific NA response

when the vaccine virus undergoes proper inactivation

processes, such as by UV irradiation or treatment with

binary ethylenimine; these treatments help to preserve

virus-NA-specific epitopes. In addition, such an inacti-

vated vaccine candidate co-administered with incomplete

Freund’s adjuvant elicits a protective immune response

to a homologous viral challenge (Vanhee et al., 2009;

Darwich et al., 2010). It is noteworthy that the suppressive

effect on IFN-a production was lost when the PRRSV was

inactivated by UV light (Vanhee et al., 2009).

Oral immunization with PRRSV nucleocapsid as a gen-

etic fusion with cholera toxin resulted in virus-specific

local intestinal mucosal antibody response with undetect-

able immune response in vaginal secretions (Hyland

et al., 2004). Therefore, the development of a protective

killed PRRSV vaccine to replace widely used MLV-PRRS to

eliminate issues with revertant vaccine viruses remains as

a challenge to researchers and to the swine industry.

Lack of success in developing a protective killed PRRSV

vaccine reflects the inability to properly present killed

PRRSV antigens to the immune system, or like live PRRSV,

killed virus may also be immunosuppressive. In addition,

the antigenic mass present in the killed PRRSV vaccine

may be an issue in inducing protective immunity, suggest-

ing the need for potent adjuvants and/or novel delivery

systems to improve the immune response to killed PRRSV

vaccines. One promising area of technology developed

over the past few years is nanotechnology based vaccine

delivery, an important area of research in the 21st century

(Panyam and Labhasetwar, 2003; Duncan, 2005; Nel et al.,

2006). Nanoparticles offer the advantage of increasing the

efficiency of drug and vaccine delivery, and also possess

adjuvant properties (Gupta et al., 1998). In addition, due

to the particulate nature of nanoparticles and the inherent

ability of APCs to passively phagocytize particulate matter

(Inaba et al., 1993), nanoparticles protect killed vaccine

antigens from degradation by proteases present at mu-

cosal surfaces and aid in pulse release of the vaccine

(Gupta et al., 1998). The development of nanoparticle-

based mucosal vaccines is gaining increased attention

with respect to the induction of protective mucosal

immunity against infectious pathogens. Therefore, nano-

particles may serve as appropriate vehicles to deliver

killed vaccines to mucosal surfaces, and the presence of

abundant APCs (Mfs and DCs) at mucosal sites could

facilitate uptake of nanoparticles and induce protective

immunity against diseases like PRRS.

A killed influenza virus vaccine entrapped in nanopar-

ticles containing E. coli heat labile toxin as an adjuvant

administered intranasally to mice, rabbits and pigs elicited

protective immunity (Singh et al., 2001). Immune re-

sponses elicited in pigs by intranasal delivery of nano-

particles based vaccine was significantly better than those

elicited by immunization by intramuscular administration

of the vaccine (Singh et al., 2001). Nanoparticles prepared

from PLGA [poly (DL-lactide-co-glycolide)] containing

hepatitis B, rotavirus, influenza or parainfluenza virus

delivered to mucosal sites of mice generated protective

immunity (Singh et al., 2001; Shephard et al., 2003; Nayak

et al., 2009; Thomas et al., 2009). Biodegradable and bio-

compatible PLGA nanoparticles are free from any toxicity

in animals and humans, they are safe for use in humans,

and are approved by the U.S. Food and Drug Adminis-

tration (Duncan, 2005; McNeil, 2005; Semete et al., 2010).

The use of nanoparticles as a vaccine delivery system

allows flexibility in the size, charge and surface properties

of the formulations for targeted delivery of vaccine to

30 G. J. Renukaradhya et al.

mucosal immune cells (Lawson et al., 2011). At present

our laboratory is working on a nanoparticles-based killed

PRRSV vaccine to augment protective immunity to PRRSV.

Our results are encouraging in reducing gross and

microscopic lung pathology as well as in reducing PRRSV

load in the lungs and blood of immunized, homologous

and heterologous PRRSV challenged pigs (Dwivedi et al.,

manuscript in preparation).

Conclusion

The mucosal immune system in the pig respiratory tract

is only partially understood compared to information

known in rodents and humans. There is, therefore, a need

for additional research aimed at improving our knowl-

edge of the pig respiratory mucosal immune system.

There are positive outcomes of mucosal vaccinology re-

search in effective control of several respiratory viral

pathogens, while a need exists for improved vaccines

against PRRS, a porcine respiratory disease of major impor-

tance in the pig industry. It therefore seems important to

conduct research aimed at adequately activating the pig

respiratory immune system to generate protective immu-

nity against PRRS. Importantly, experimental studies have

demonstrated that it is possible to generate heterologous

protection by effective mucosal immunization strategies.

We note that protective mucosal immunity against a

vaccine could be achieved only when the vaccine was

co-administered with a suitable adjuvant and/or a deli-

very system; this is critical for overcoming the immune

regulatory system at mucosal sites. Pilot studies per-

formed by us on intranasal delivery of PRRSV vaccines in

pigs, and by others in rodent models, to control other

respiratory viral pathogens using both live and killed viral

vaccines, have provided ample evidence of feasibility and

effectiveness of mucosal vaccines. Thus, adaption of inno-

vative strategies to control PRRS by mucosal immuniza-

tion may help to control PRRS more effectively than by

using conventional parenteral immunization.

References

Aasted B, Bach P, Nielsen J and Lind P (2002). Cytokine profilesin peripheral blood mononuclear cells and lymph nodecells from piglets infected in utero with porcine reproduc-tive and respiratory syndrome virus. Clinical and Diag-nostic Laboratory Immunology 9: 1229–1234.

Acker H (2005). The oxygen sensing signal cascade under theinfluence of reactive oxygen species. Philosophical Trans-actions of the Royal Society B: Biological Sciences 360:2201–2210.

Akerstrom S, Mousavi-Jazi M, Klingstrom J, Leijon M, Lundkvist Aand Mirazimi A (2005). Nitric oxide inhibits the replicationcycle of severe acute respiratory syndrome coronavirus.Journal of Virology 79: 1966–1969.

Albina E (1997). Epidemiology of porcine reproductive andrespiratory syndrome (PRRS): an overview. VeterinaryMicrobiology 55: 309–316.

Albina E, Carrat C and Charley B (1998). Interferon-alpharesponse to swine arterivirus (PoAV), the porcine repro-ductive and respiratory syndrome virus. Journal of Inter-feron and Cytokine Research 18: 485–490.

Allan CH, Mendrick DL and Trier JS (1993). Rat intestinal M cellscontain acidic endosomal–lysosomal compartments andexpress class II major histocompatibility complex determi-nants. Gastroenterology 104: 698–708.

Avdic S, Cao JZ, Cheung AK, Abendroth A and Slobedman B(2011). Viral IL-10 expressed by human cytomegalovirusduring the latent phase of infection modulates latentlyinfected myeloid cell differentiation. Journal of Virology 85:7465–7471.

Bansal K, Elluru SR, Narayana Y, Chaturvedi R, Patil SA,Kaveri SV, Bayry J and Balaji KN (2010). PE_PGRS antigensof Mycobacterium tuberculosis induce maturation andactivation of human dendritic cells. Journal of Immunology184: 3495–3504.

Barral DC and Brenner MB (2007). CD1 antigen presentation:how it works. Nature Reviews Immunology 7: 929–941.

Bastos RG, Dellagostin OA, Barletta RG, Doster AR, Nelson E,Zuckermann F and Osorio FA (2004). Immune responseof pigs inoculated with Mycobacterium bovis BCG expres-sing a truncated form of GP5 and M protein of porcinereproductive and respiratory syndrome virus. Vaccine 22:467–474.

Bautista EM and Molitor TW (1999). IFN gamma inhibits porcinereproductive and respiratory syndrome virus replication inmacrophages. Archives of Virology 144: 1191–1200.

Beetz S, Marischen L, Kabelitz D and Wesch D (2007). Humangamma delta T cells: candidates for the development ofimmunotherapeutic strategies. Immunologic Research 37:97–111.

Bekierkunst A (1968). Acute granulomatous response producedin mice by trehalose-6,6-dimycolate. Journal of Bacteriology96: 958–961.

Bekierkunst A, Yarkoni E, Flechner I, Morecki S, Vilkas E andLederer E (1971). Immune response to sheep red bloodcells in mice pretreated with mycobacterial fractions.Infection and Immunity 4: 256–263.

Belshe R, Lee MS, Walker RE, Stoddard J and Mendelman PM(2004). Safety, immunogenicity and efficacy of intranasal,live attenuated influenza vaccine. Expert Review of Vaccines3: 643–654.

Beyer J, Fichtner D, Schirrmeier H, Polster U, Weiland Eand Wege H (2000). Porcine reproductive and respiratorysyndrome virus (PRRSV): kinetics of infection in lymphaticorgans and lung. Journal of Veterinary Medicine BInfectious Diseases Veterinary Public Health 47: 9–25.

Binjawadagi B, Dwivedi V, Manickam C, Torrelles JB andRenukaradhya GJ (2011). Intranasal delivery of an adju-vanted modified live porcine reproductive and respiratorysyndrome virus vaccine reduces the ROS production. ViralImmunology 24: 475–482.

Binns RM (1982). Organisation of the lymphoreticular systemand lymphocyte markers in the pig. Veterinary Immunol-ogy and Immunopathology 3: 95–146.

Biron CA, Nguyen KB, Pien GC, Cousens LP and Salazar-MatherTP (1999). Natural killer cells in antiviral defense: functionand regulation by innate cytokines. Annual Review ofImmunology 17: 189–220.

Bonavida B, Katz J and Hoshino T (1986). Mechanism of NKactivation by OK-432 (Streptococcus pyogenes). I. Sponta-neous release of NKCF and augmentation of NKCF pro-duction following stimulation with NK target cells. CellularImmunology 102: 126–135.

Botner A, Strandbygaard B, Sorensen KJ, Have P, Madsen KG,Madsen ES and Alexandersen S (1997). Appearance of acute

Mucosal vaccines to prevent porcine reproductive and respiratory syndrome 31

PRRS-like symptoms in sow herds after vaccination witha modified live PRRS vaccine. Veterinary Record 141:497–499.

Brayden DJ, Jepson MA and Baird AW (2005). Keynote review:intestinal Peyer’s patch M cells and oral vaccine targeting.Drug Discovery Today 10: 1145–1157.

Buda A, Sands C and Jepson MA (2005). Use of fluorescenceimaging to investigate the structure and function of intes-tinal M cells. Advanced Drug Delivery Reviews 57: 123–134.

Buddaert W, Van Reeth K and Pensaert M (1998). In vivo andin vitro interferon (IFN) studies with the porcine reproduc-tive and respiratory syndrome virus (PRRSV). Advances inExperimental Medicine and Biology 440: 461–467.

Canessa C, Vierucci S, Azzari C and Vierucci A (2010). Theimmunity of upper airways. International Journal ofImmunopathology and Pharmacology 23: 8–12.

Cano JP, Dee SA, Murtaugh MP and Pijoan C (2007). Impact of amodified-live porcine reproductive and respiratory syn-drome virus vaccine intervention on a population of pigsinfected with a heterologous isolate. Vaccine 25: 4382–4391.

Charerntantanakul W (2009). Adjuvants for porcine reproductiveand respiratory syndrome virus vaccines. Veterinary Immu-nology and Immunopathology 129: 1–13.

Charerntantanakul W, Platt R and Roth JA (2006). Effectsof porcine reproductive and respiratory syndrome virus-infected antigen-presenting cells on T cell activationand antiviral cytokine production. Viral Immunology 19:646–661.

Che TM, Johnson RW, Kelley KW, Van Alstine WG, Dawson KA,Moran CA and Pettigrew JE (2011). Mannan oligosaccharideimproves immune responses and growth efficiency ofnursery pigs experimentally infected with porcine repro-ductive and respiratory syndrome virus. Journal of AnimalScience 89: 2592–2602.

Choudhary RK, Mukhopadhyay S, Chakhaiyar P, Sharma N,Murthy KJ, Katoch VM and Hasnain SE (2003). PPE antigenRv2430c of Mycobacterium tuberculosis induces a strong B-cell response. Infection and Immunity 71: 6338–6343.

Christopher-Hennings J, Nelson EA, Hines RJ, Nelson JK,Swenson SL, Zimmerman JJ, Chase CL, Yaeger MJ andBenfield DA (1995). Persistence of porcine reproductiveand respiratory syndrome virus in serum and semen ofadult boars. Journal of Veterinary Diagnostic Investigation7: 456–464.

Christopher-Hennings J, Nelson EA, Nelson JK, Rossow KD,Shivers JL, Yaeger MJ, Chase CC, Garduno RA, Collins JEand Benfield DA (1998). Identification of porcine repro-ductive and respiratory syndrome virus in semen andtissues from vasectomized and nonvasectomized boars.Veterinary Pathology 35: 260–267.

Chvanov M, Petersen OH and Tepikin A (2005). Free radicalsand the pancreatic acinar cells: role in physiology andpathology. Philosophical Transactions of the Royal SocietyB: Biological Sciences 360: 2273–2284.

Clutton S (1997). The importance of oxidative stress in apoptosis.British Medical Bulletin 53: 662–668.

Collins JE, Benfield DA, Christianson WT, Harris L, Hennings JC,Shaw DP, Goyal SM, McCullough S, Morrison RB, Joo HS,Gorcyca D and Chladek D (1992). Isolation of swineinfertility and respiratory syndrome virus (isolate ATCC VR-2332) in North America and experimental reproduction ofthe disease in gnotobiotic pigs. Journal of VeterinaryDiagnostic Investigation 4: 117–126.

Corr SC, Gahan CC and Hill C (2008). M-cells: origin,morphology and role in mucosal immunity and microbialpathogenesis. FEMS Immunology and Medical Microbiology52: 2–12.

Cox RJ, Brokstad KA and Ogra P (2004). Influenza virus:immunity and vaccination strategies. Comparison ofthe immune response to inactivated and live, attenuatedinfluenza vaccines. Scandinavian Journal of Immunology59: 1–15.

Croen KD (1993). Evidence for antiviral effect of nitric oxide.Inhibition of herpes simplex virus type 1 replication.Journal of Clinical Investigation 91: 2446–2452.

Czerkinsky C and Holmgren J (2010). Topical immunizationstrategies. Mucosal Immunology 3: 545–555.

Darwich L, Diaz I and Mateu E (2010). Certainties, doubtsand hypotheses in porcine reproductive and respiratorysyndrome virus immunobiology. Virus Research 154:123–132.

Davis SS (2001). Nasal vaccines. Advanced Drug DeliveryReviews 51: 21–42.

Diaz I, Darwich L, Pappaterra G, Pujols J and Mateu E (2005).Immune responses of pigs after experimental infection witha European strain of porcine reproductive and respiratorysyndrome virus. Journal of General Virology 86: 1943–1951.

Didierlaurent A, Goulding J and Hussell T (2007). The impactof successive infections on the lung microenvironment.Immunology 122: 457–465.

Done SH and Paton DJ (1995). Porcine reproductive andrespiratory syndrome: clinical disease, pathology andimmunosuppression. Veterinary Record 136: 32–35.

Drew TW (2000). A review of evidence for immunosuppressiondue to porcine reproductive and respiratory syndromevirus. Veterinary Research 31: 27–39.

Duan X, Nauwynck HJ and Pensaert MB (1997). Effects of originand state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductiveand respiratory syndrome virus (PRRSV). Archives ofVirology 142: 2483–2497.

Duncan R (2005). Nanomedicine gets clinical. Materials Today 8:16–17.

Dwivedi V, Manickam C, Binjawadagi B, Linhares D, MurtaughMP and Renukaradhya GJ (2012). Evaluation of immuneresponses to porcine reproductive and respiratory syn-drome virus in pigs during early stage of infection underfarm conditions. Virology Journal 9: 45.

Dwivedi V, Manickam C, Patterson R, Dodson K, Murtaugh M,Torrelles JB, Schlesinger LS and Renukaradhya GJ (2011a).Cross-protective immunity to porcine reproductive andrespiratory syndrome virus by intranasal delivery of a livevirus vaccine with a potent adjuvant. Vaccine 29: 4058–4066.

Dwivedi V, Manickam C, Patterson R, Dodson K, Weeman M andRenukaradhya GJ (2011b). Intranasal delivery of whole celllysate of Mycobacterium tuberculosis induces protectiveimmune responses to a modified live porcine reproductiveand respiratory syndrome virus vaccine in pigs. Vaccine 29:4067–4076.

Etchart N, Wild F and Kaiserlian D (1996). Mucosal and systemicimmune responses to measles virus haemagglutinin in miceimmunized with a recombinant vaccinia virus. Journal ofGeneral Virology 77 (Pt 10): 2471–2478.

Faaberg KS, Hocker JD, Erdman MM, Harris DL, Nelson EA,Torremorell M and Plagemann PG (2006). Neutralizingantibody responses of pigs infected with natural GP5N-glycan mutants of porcine reproductive and respiratorysyndrome virus. Viral Immunology 19: 294–304.

Foo SY and Phipps S (2010). Regulation of inducible BALTformation and contribution to immunity and pathology.Mucosal Immunology 3: 537–544.

Foss DL and Murtaugh MP (1999). Mucosal immunogenicityand adjuvanticity of cholera toxin in swine. Vaccine 17:788–801.

32 G. J. Renukaradhya et al.

Foss DL, Zilliox MJ, Meier W, Zuckermann F and Murtaugh MP(2002). Adjuvant danger signals increase the immuneresponse to porcine reproductive and respiratory syndromevirus. Viral Immunology 15: 557–566.

Gebert A, Rothkotter HJ and Pabst R (1994). Cytokeratin 18 is anM-cell marker in porcine Peyer’s patches. Cell and TissueResearch 276: 213–221.

Gerner W, Kaser T and Saalmuller A (2009). Porcine Tlymphocytes and NK cells – an update. Developmental andComparative Immunology 33: 310–320.

Geurtsvankessel CH, Willart MA, Bergen IM, Van Rijt LS,Muskens F, Elewaut D, Osterhaus AD, Hendriks R,Rimmelzwaan GF and Lambrecht BN (2009). Dendriticcells are crucial for maintenance of tertiary lymphoidstructures in the lung of influenza virus-infected mice.Journal of Experimental Medicine 206: 2339–2349.

Gomez-Laguna J, Salguero FJ, Barranco I, Pallares FJ, Rodriguez-Gomez IM, Bernabe A and Carrasco L (2010). Cytokineexpression by macrophages in the lung of pigs infectedwith the porcine reproductive and respiratory syndromevirus. Journal of Comparative Pathology 142: 51–60.

Guillonneau C, Mintern JD, Hubert FX, Hurt AC, Besra GS,Porcelli S, Barr IG, Doherty PC, Godfrey DI and Turner SJ(2009). Combined NKT cell activation and influenza virusvaccination boosts memory CTL generation and protectiveimmunity. Proceedings of the National Academy of Sciencesof the USA 106: 3330–3335.

Gupta RK, Chang AC and Siber GR (1998). Biodegradablepolymer microspheres as vaccine adjuvants and deliverysystems. Developments in Biological Standardization 92:63–78.

Halbur PG, Miller LD, Paul PS, Meng XJ, Huffman ELand Andrews JJ (1995). Immunohistochemical identificationof porcine reproductive and respiratory syndrome virus(PRRSV) antigen in the heart and lymphoid system of three-week-old colostrum-deprived pigs. Veterinary Pathology32: 200–204.

Halliwell B and Gutteridge JMC (2006). Free Radicals in Biologyand Medicine. 4th edn. Clarendon Press, Oxford.

Harmala LA, Ingulli EG, Curtsinger JM, Lucido MM, Schmidt CS,Weigel BJ, Blazar BR, Mescher MF and Pennell CA (2002).The adjuvant effects of Mycobacterium tuberculosis heatshock protein 70 result from the rapid and prolongedactivation of antigen-specific CD8+ T cells in vivo. Journalof Immunology 169: 5622–5629.

Holmgren J and Czerkinsky C (2005). Mucosal immunity andvaccines. Nature Medicine 11: S45–53.

Holmgren J, Czerkinsky C, Lycke N and Svennerholm AM (1992).Mucosal immunity: implications for vaccine development.Immunobiology 184: 157–179.

Holtkamp D and Kliebenstein J (2011). PRRS costs industry$664 million annually. Pork Checkoff Study, http://www.pork.org/News/1265/PRRSCostsIndustry664Million.aspx

Huang YW and Meng XJ (2010). Novel strategies and approachesto develop the next generation of vaccines against porcinereproductive and respiratory syndrome virus (PRRSV).Virus Research 154: 141–149.

Humphreys IR, De Trez C, Kinkade A, Benedict CA, Croft M andWare CF (2007). Cytomegalovirus exploits IL-10-mediatedimmune regulation in the salivary glands. Journal ofExperimental Medicine 204: 1217–1225.

Hyland K, Foss DL, Johnson CR and Murtaugh MP (2004). Oralimmunization induces local and distant mucosal immunityin swine. Veterinary Immunology and Immunopathology102: 329–338.

Ichinohe T, Kawaguchi A, Tamura S, Takahashi H, Sawa H,Ninomiya A, Imai M, Itamura S, Odagiri T, Tashiro M, ChibaJ, Sata T, Kurata T and Hasegawa H (2007a). Intranasal

immunization with H5N1 vaccine plus Poly I:Poly C12U, aToll-like receptor agonist, protects mice against homo-logous and heterologous virus challenge. Microbes andInfection 9: 1333–1340.

Ichinohe T, Tamura S, Kawaguchi A, Ninomiya A, Imai M,Itamura S, Odagiri T, Tashiro M, Takahashi H, Sawa H,Mitchell WM, Strayer DR, Carter WA, Chiba J, Kurata T,Sata T and Hasegawa H (2007b). Cross-protection againstH5N1 influenza virus infection is afforded by intranasalinoculation with seasonal trivalent inactivated influenzavaccine. Journal of Infectious Diseases 196: 1313–1320.

Imaoka K, Miller CJ, Kubota M, Mcchesney MB, Lohman B,Yamamoto M, Fujihashi K, Someya K, Honda M, McGhee JRand Kiyono H (1998). Nasal immunization of nonhumanprimates with simian immunodeficiency virus p55gag andcholera toxin adjuvant induces Th1/Th2 help for virus-specific immune responses in reproductive tissues. Journalof Immunology 161: 5952–5958.

Inaba K, Inaba M, Naito M and Steinman RM (1993). Dendriticcell progenitors phagocytose particulates, including bacillusCalmette–Guerin organisms, and sensitize mice to myco-bacterial antigens in vivo. Journal of Experimental Medicine178: 479–488.

Jepson MA, Clark MA, Foster N, Mason CM, Bennett MK,Simmons NL and Hirst BH (1996). Targeting to intestinalM cells. Journal of Anatomy 189(Pt 3): 507–516.

Johansson EL, Bergquist C, Edebo A, Johansson C andSvennerholm AM (2004). Comparison of different routesof vaccination for eliciting antibody responses in the humanstomach. Vaccine 22: 984–990.

Johansson EL, Wassen L, Holmgren J, Jertborn M and Rudin A(2001). Nasal and vaginal vaccinations have differentialeffects on antibody responses in vaginal and cervicalsecretions in humans. Infection and Immunity 69: 7481–7486.

Johnsen CK, Botner A, Kamstrup S, Lind P and Nielsen J (2002).Cytokine mRNA profiles in bronchoalveolar cells of pigletsexperimentally infected in utero with porcine reproductiveand respiratory syndrome virus: association of sustainedexpression of IFN-gamma and IL-10 after viral clearance.Viral Immunology 15: 549–556.

Jung K, Renukaradhya GJ, Alekseev KP, Fang Y, Tang Y and SaifLJ (2009). Porcine reproductive and respiratory syndromevirus modifies innate immunity and alters disease outcomein pigs subsequently infected with porcine respiratorycoronavirus: implications for respiratory viral co-infections.Journal of General Virology 90: 2713–2723.

Kamijuku H, Nagata Y, Jiang X, Ichinohe T, Tashiro T, Mori K,Taniguchi M, Hase K, Ohno H, Shimaoka T, Yonehara S,Odagiri T, Tashiro M, Sata T, Hasegawa H and Seino KI(2008). Mechanism of NKT cell activation by intranasalcoadministration of alpha-galactosylceramide, which caninduce cross-protection against influenza viruses. MucosalImmunology 1: 208–218.

Karron RA, Wright PF, Hall SL, Makhene M, Thompson J, BurnsBA, Tollefson S, Steinhoff MC, Wilson MH, Harris DO,Clements ML and Murphy BR (1995). A live attenuatedbovine parainfluenza virus type 3 vaccine is safe, infectious,immunogenic, and phenotypically stable in infants andchildren. Journal of Infectious Diseases 171: 1107–1114.

Kaser T, Gerner W, Hammer SE, Patzl M and Saalmuller A (2008).Phenotypic and functional characterisation of porcineCD4(+)CD25(high) regulatory T cells. Veterinary Immunol-ogy and Immunopathology 122: 153–158.

Key KF, Guenette DK, Yoon KJ, Halbur PG, Toth TE andMeng XJ (2003). Development of a heteroduplex mobilityassay to identify field isolates of porcine reproductiveand respiratory syndrome virus with nucleotide sequences

Mucosal vaccines to prevent porcine reproductive and respiratory syndrome 33

closely related to those of modified live-attenuatedvaccines. Journal of Clinical Microbiology 41: 2433–2439.

Kim DY, Sato A, Fukuyama S, Sagara H, Nagatake T, Kong IG,Goda K, Nochi T, Kunisawa J, Sato S, Yokota Y, Lee CH andKiyono H (2011). The airway antigen sampling system:respiratory M cells as an alternative gateway for inhaledantigens. Journal of Immunology 186: 4253–4262.

Kim WI, Lee DS, Johnson W, Roof M, Cha SH and Yoon KJ(2007). Effect of genotypic and biotypic differences amongPRRS viruses on the serologic assessment of pigs for virusinfection. Veterinary Microbiology 123: 1–14.

Kimman TG, Cornelissen LA, Moormann RJ, Rebel JM andStockhofe-Zurwieden N (2009). Challenges for porcinereproductive and respiratory syndrome virus (PRRSV)vaccinology. Vaccine 27: 3704–3718.

Kinter AL, Hennessey M, Bell A, Kern S, Lin Y, Daucher M, PlantaM, McGlaughlin M, Jackson R, Ziegler SF and Fauci AS(2004). CD25(+)CD4(+) regulatory T cells from the periph-eral blood of asymptomatic HIV-infected individualsregulate CD4(+) and CD8(+) HIV-specific T cell immuneresponses in vitro and are associated with favorable clinicalmarkers of disease status. Journal of Experimental Medicine200: 331–343.

Kit S (1990). Genetically engineered vaccines for control ofAujeszky’s disease (pseudorabies). Vaccine 8: 420–424.

Kiyono H and Fukuyama S (2004). NALT- versus Peyer’s-patch-mediated mucosal immunity. Nature Reviews Immunology4: 699–710.

Klinge KL, Vaughn EM, Roof MB, Bautista EM and Murtaugh MP(2009). Age-dependent resistance to Porcine reproductiveand respiratory syndrome virus replication in swine.Virology Journal 6: 177.

Kozlowski PA, Cu-Uvin S, Neutra MR and Flanigan TP (1997).Comparison of the oral, rectal, and vaginal immunizationroutes for induction of antibodies in rectal and genital tractsecretions of women. Infection and Immunity 65: 1387–1394.

Kraehenbuhl JP and Neutra MR (2000). Epithelial M cells:differentiation and function. Annual Review of Cell andDevelopmental Biology 16: 301–332.

Kuroki H, Morisaki T, Matsumoto K, Onishi H, Baba E, Tanaka Mand Katano M (2003). Streptococcal preparation OK-432: anew maturation factor of monocyte-derived dendritic cellsfor clinical use. Cancer Immunology, Immunotherapy 52:561–568.

Lamontagne L, Page C, Larochelle R, Longtin D and Magar R(2001). Polyclonal activation of B cells occurs in lymphoidorgans from porcine reproductive and respiratorysyndrome virus (PRRSV)-infected pigs. Veterinary Immu-nology and Immunopathology 82: 165–182.

Lawson LB, Norton EB and Clements JD (2011). Defendingthe mucosa: adjuvant and carrier formulations for mucosalimmunity. Current Opinion in Immunology 23: 414–420.

Lawson SR, Rossow KD, Collins JE, Benfield DA and Rowland RR(1997). Porcine reproductive and respiratory syndromevirus infection of gnotobiotic pigs: sites of virus replicationand co-localization with MAC-387 staining at 21 days post-infection. Virus Research 51: 105–113.

Lee CH, Yeh TH, Lai HC, Wu SY, Su IJ, Takada K and Chang Y(2011). Epstein–Barr virus Zta-induced immunomodulatorsfrom nasopharyngeal carcinoma cells upregulate inter-leukin-10 production from monocytes. Journal of Virology85: 7333–7342.

Lemke CD, Haynes JS, Spaete R, Adolphson D, Vorwald A, LagerK and Butler JE (2004). Lymphoid hyperplasia resulting inimmune dysregulation is caused by porcine reproductiveand respiratory syndrome virus infection in neonatal pigs.Journal of Immunology 172: 1916–1925.

Leroith T, Hammond S, Todd SM, Ni Y, Cecere T and Pelzer KD(2011). A modified live PRRSV vaccine and the pathogenicparent strain induce regulatory T cells in pigs naturallyinfected with Mycoplasma hyopneumoniae. VeterinaryImmunology and Immunopathology 14: 312–316.

Li B, Fang L, Xu Z, Liu S, Gao J, Jiang Y, Chen H and Xiao S(2009). Recombination in vaccine and circulating strains ofporcine reproductive and respiratory syndrome viruses.Emerging Infectious Diseases 15: 2032–2035.

Li Y, Xue C, Wang L, Chen X, Chen F and Cao Y (2010). Genomicanalysis of two Chinese strains of porcine reproductive andrespiratory syndrome viruses with different virulence. VirusGenes 40: 374–381.

Loemba HD, Mounir S, Mardassi H, Archambault D and Dea S(1996). Kinetics of humoral immune response to themajor structural proteins of the porcine reproductiveand respiratory syndrome virus. Archives of Virology 141:751–761.

Lohse L, Nielsen J and Eriksen L (2004). Temporary CD8+ T-celldepletion in pigs does not exacerbate infection withporcine reproductive and respiratory syndrome virus(PRRSV). Viral Immunology 17: 594–603.

Lopez Fuertes L, Domenech N, Alvarez B, Ezquerra A,Dominguez J, Castro JM and Alonso F (1999). Analysis ofcellular immune response in pigs recovered from porcinerespiratory and reproductive syndrome infection. VirusResearch 64: 33–42.

Lopez OJ, Oliveira MF, Garcia EA, Kwon BJ, Doster A and OsorioFA (2007). Protection against porcine reproductive andrespiratory syndrome virus (PRRSV) infection throughpassive transfer of PRRSV-neutralizing antibodies isdose dependent. Clinical and Vaccine Immunology 14:269–275.

Lopez OJ and Osorio FA (2004). Role of neutralizing antibodiesin PRRSV protective immunity. Veterinary Immunology andImmunopathology 102: 155–163.

Madsen KG, Hansen CM, Madsen ES, Strandbygaard B, Botner Aand Sorensen KJ (1998). Sequence analysis of porcinereproductive and respiratory syndrome virus of theAmerican type collected from Danish swine herds. Archivesof Virology 143: 1683–1700.

Mann JF, Acevedo R, Campo JD, Perez O and Ferro VA(2009). Delivery systems: a vaccine strategy for over-coming mucosal tolerance? Expert Review of Vaccines 8:103–112.

Martelli P, Gozio S, Ferrari L, Rosina S, De Angelis E, QuintavallaC, Bottarelli E and Borghetti P (2009). Efficacy of a modifiedlive porcine reproductive and respiratory syndrome virus(PRRSV) vaccine in pigs naturally exposed to a hetero-logous European (Italian cluster) field strain: Clinicalprotection and cell-mediated immunity. Vaccine 27:3788–3799.

Martin JH and Edwards SW (1993). Changes in mechanismsof monocyte/macrophage-mediated cytotoxicity duringculture. Reactive oxygen intermediates are involved inmonocyte-mediated cytotoxicity, whereas reactive nitrogenintermediates are employed by macrophages in tumor cellkilling. Journal of Immunology 150: 3478–3486.

Mateu E and Diaz I (2008). The challenge of PRRS immunology.Veterinary Journal 177: 345–351.

Mateu E, Diaz I, Darwich L, Casal J, Martin M and Pujols J (2006).Evolution of ORF5 of Spanish porcine reproductive andrespiratory syndrome virus strains from 1991 to 2005. VirusResearch 115: 198–206.

McNeil SE (2005). Nanotechnology for the biologist. Journal ofLeukocyte Biology 78: 585–594.

Meier WA, Galeota J, Osorio FA, Husmann RJ, Schnitzlein WMand Zuckermann FA (2003). Gradual development of

34 G. J. Renukaradhya et al.

the interferon-gamma response of swine to porcinereproductive and respiratory syndrome virus infection orvaccination. Virology 309: 18–31.

Meng XJ (2000). Heterogeneity of porcine reproductive andrespiratory syndrome virus: implications for current vaccineefficacy and future vaccine development. VeterinaryMicrobiology 74: 309–329.

Mengeling WL, Clouser DF, Vorwald AC and Lager KM (2002).The potential role of genetic recombination in the evolutionof new strains of porcine reproductive and respiratorysyndrome virus (PRRSV). Journal of Swine Health andProduction 10: 273–275.

Mengeling WL, Lager KM and Vorwald AC (1998). Clinicalconsequences of exposing pregnant gilts to strains ofporcine reproductive and respiratory syndrome (PRRS)virus isolated from field cases of “atypical” PRRS. AmericanJournal of Veterinary Research 59: 1540–1544.

Mengeling WL, Lager KM, Vorwald AC and Clouser DF (2003a).Comparative safety and efficacy of attenuated single-strain and multi-strain vaccines for porcine reproductiveand respiratory syndrome. Veterinary Microbiology 93:25–38.

Mengeling WL, Lager KM, Vorwald AC and Koehler KJ (2003b).Strain specificity of the immune response of pigs followingvaccination with various strains of porcine reproductive andrespiratory syndrome virus. Veterinary Microbiology 93:13–24.

Meulenberg JJ (2000). PRRSV, the virus. Veterinary Research 31:11–21.

Mowat AM (2003). Anatomical basis of tolerance and immunityto intestinal antigens. Nature Reviews Immunology 3:331–341.

Moyron-Quiroz J, Rangel-Moreno J, Carragher DM and RandallTD (2007). The function of local lymphoid tissues inpulmonary immune responses. Advances in ExperimentalMedicine and Biology 590: 55–68.

Murtaugh MP, Xiao Z and Zuckermann F (2002a). Immunolo-gical responses of swine to porcine reproductive andrespiratory syndrome virus infection. Viral Immunology 15:533–547.

Murtaugh MP, Yuan S, Nelson EA and Faaberg KS (2002b).Genetic interaction between porcine reproductive andrespiratory syndrome virus (PRRSV) strains in cell cultureand in animals. Journal of Swine Health and Production 10:15–21.

Nardelli-Haefliger D, Wirthner D, Schiller JT, Lowy DR,Hildesheim A, Ponci F and De Grandi P (2003). Specificantibody levels at the cervix during the menstrual cycle ofwomen vaccinated with human papillomavirus 16 virus-likeparticles. Journal of the National Cancer Institute 95: 1128–1137.

Nayak B, Panda AK, Ray P and Ray AR (2009). Formulation,characterization and evaluation of rotavirus encapsulatedPLA and PLGA particles for oral vaccination. Journal ofMicroencapsulation 26: 154–165.

Nel A, Xia T, Madler L and Li N (2006). Toxic potential ofmaterials at the nanolevel. Science 311: 622–627.

Nelson EA, Christopher-Hennings J and Benfield DA (1994).Serum immune responses to the proteins of porcine repro-ductive and respiratory syndrome (PRRS) virus. Journal ofVeterinary Diagnostic Investigation 6: 410–415.

Neutra MR and Kozlowski PA (2006). Mucosal vaccines: thepromise and the challenge. Nature Reviews Immunology 6:148–158.

Neutra MR, Mantis NJ, Frey A and Giannasca PJ (1999). Thecomposition and function of M cell apical membranes:implications for microbial pathogenesis. Seminars inImmunology 11: 171–181.

Nielsen HS, Oleksiewicz MB, Forsberg R, Stadejek T, Botner Aand Storgaard T (2001). Reversion of a live porcinereproductive and respiratory syndrome virus vaccineinvestigated by parallel mutations. Journal of GeneralVirology 82: 1263–1272.

Nielsen J, Botner A, Bille-Hansen V, Oleksiewicz MB andStorgaard T (2002). Experimental inoculation of late termpregnant sows with a field isolate of porcine reproductiveand respiratory syndrome vaccine-derived virus. VeterinaryMicrobiology 84: 1–13.

Ogra PL (1984). Mucosal immune response to poliovirusvaccines in childhood. Review of Infectious Diseases 6(Suppl. 2): S361–368.

Opriessnig T, Halbur PG, Yoon KJ, Pogranichniy RM, HarmonKM, Evans R, Key KF, Pallares FJ, Thomas P and Meng XJ(2002). Comparison of molecular and biological character-istics of a modified live porcine reproductive and respira-tory syndrome virus (PRRSV) vaccine (ingelvac PRRS MLV),the parent strain of the vaccine (ATCC VR2332), ATCCVR2385, and two recent field isolates of PRRSV. Journal ofVirology 76: 11837–11844.

Osorio FA, Galeota JA, Nelson E, Brodersen B, Doster A, Wills R,Zuckermann F and Laegreid WW (2002). Passive transferof virus-specific antibodies confers protection againstreproductive failure induced by a virulent strain of porcinereproductive and respiratory syndrome virus and estab-lishes sterilizing immunity. Virology 302: 9–20.

Ostrowski M, Galeota JA, Jar AM, Platt KB, Osorio FA andLopez OJ (2002). Identification of neutralizing and non-neutralizing epitopes in the porcine reproductive andrespiratory syndrome virus GP5 ectodomain. Journal ofVirology 76: 4241–4250.

Otake S, Dee S, Corzo C, Oliveira S and Deen J (2010). Long-distance airborne transport of infectious PRRSV andMycoplasma hyopneumoniae from a swine populationinfected with multiple viral variants. Veterinary Microbiol-ogy 145: 198–208.

Pabst R and Binns RM (1989). Heterogeneity of lymphocytehoming physiology: several mechanisms operate in thecontrol of migration to lymphoid and non-lymphoid organsin vivo. Immunological Reviews 108: 83–109.

Panyam J and Labhasetwar V (2003). Biodegradable nanoparti-cles for drug and gene delivery to cells and tissue.Advanced Drug Delivery Reviews 55: 329–347.

Peng G, Li S, Wu W, Sun Z, Chen Y and Chen Z(2008). Circulating CD4+ CD25+ regulatory T cells cor-relate with chronic hepatitis B infection. Immunology 123:57–65.

Petrovsky N and Aguilar JC (2004). Vaccine adjuvants: currentstate and future trends. Immunology and Cell Biology 82:488–496.

Pitkin A, Deen J and Dee S (2009). Further assessment of fomitesand personnel as vehicles for the mechanical transportand transmission of porcine reproductive and respiratorysyndrome virus. Canadian Journal of Veterinary Research73: 298–302.

Plotkin SA (2005). Vaccines: past, present and future. NatureMedicine 11: S5–11.

Quinn MT and Gauss KA (2004). Structure and regulation of theneutrophil respiratory burst oxidase: comparison withnonphagocyte oxidases. Journal of Leukocyte Biology 76:760–781.

Randall TD (2010). Bronchus-associated lymphoid tissue (BALT)structure and function. Advances in Immunology 107:187–241.

Read S, Malmstrom V and Powrie F (2000). Cytotoxic Tlymphocyte-associated antigen 4 plays an essential role inthe function of CD25(+)CD4(+) regulatory cells that control

Mucosal vaccines to prevent porcine reproductive and respiratory syndrome 35

intestinal inflammation. Journal of Experimental Medicine192: 295–302.

Renukaradhya GJ, Alekseev K, Jung K, Fang Y and Saif LJ (2010).Porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatoryresponse to porcine respiratory coronavirus in pigs. ViralImmunology 23: 457–466.

Rimmelzwaan GF, Baars MM, De Lijster P, Fouchier RA andOsterhaus AD (1999). Inhibition of influenza virus replica-tion by nitric oxide. Journal of Virology 73: 8880–8883.

Roberts P, Jeffery PK, Mitchell TJ, Andrew PW, Boulnois GJ,Feldman C, Cole PJ and Wilson R (1992). Effect ofimmunization with Freund’s adjuvant and pneumolysin onhistologic features of pneumococcal infection in the ratlung in vivo. Infection and Immunity 60: 4969–4972.

Rodriguez A, Troye-Blomberg M, Lindroth K, Ivanyi J, Singh Mand Fernandez C (2003). B- and T-cell responses to themycobacterium surface antigen PstS-1 in the respiratorytract and adjacent tissues. Role of adjuvants and routesof immunization. Vaccine 21: 458–467.

Rossow KD, Bautista EM, Goyal SM, Molitor TW, Murtaugh MP,Morrison RB, Benfield DA and Collins JE (1994). Experi-mental porcine reproductive and respiratory syndromevirus infection in one-, four-, and 10-week-old pigs. Journalof Veterinary Diagnostic Investigations 6: 3–12.

Rossow KD, Benfield DA, Goyal SM, Nelson EA, Christopher-Hennings J and Collins JE (1996). Chronological immuno-histochemical detection and localization of porcinereproductive and respiratory syndrome virus in gnotobioticpigs. Veterinary Pathology 33: 551–556.

Rowland RR (2007). The stealthy nature of PRRSV infection: thedangers posed by that ever-changing mystery swinedisease. Veterinary Journal 174: 451.

Royaee AR, Husmann RJ, Dawson HD, Calzada-Nova G,Schnitzlein WM, Zuckermann FA and Lunney JK (2004).Deciphering the involvement of innate immune factors inthe development of the host response to PRRSV vaccina-tion. Veterinary Immunology and Immunopathology 102:199–216.

Sakaguchi S, Wing K, Onishi Y, Prieto-Martin P and Yamaguchi T(2009). Regulatory T cells: how do they suppress immuneresponses? International Immunology 21: 1105–1111.

Schmidt AC, Wenzke DR, Mcauliffe JM, St Claire M, Elkins WR,Murphy BR and Collins PL (2002). Mucosal immunizationof rhesus monkeys against respiratory syncytial virussubgroups A and B and human parainfluenza virus type 3by using a live cDNA-derived vaccine based on a hostrange-attenuated bovine parainfluenza virus type 3 vectorbackbone. Journal of Virology 76: 1089–1099.

Semete B, Booysen L, Lemmer Y, Kalombo L, Katata L,Verschoor J and Swai HS (2010). In vivo evaluation of thebiodistribution and safety of PLGA nanoparticles as drugdelivery systems. Nanomedicine 6: 662–671.

Shephard MJ, Todd D, Adair BM, Po AL, Mackie DP and Scott EM(2003). Immunogenicity of bovine parainfluenza type 3virus proteins encapsulated in nanoparticle vaccines,following intranasal administration to mice. Research inVeterinary Science 74: 187–190.

Silva-Campa E, Cordoba L, Fraile L, Flores-Mendoza L, MontoyaM and Hernandez J (2009a). European genotype of porcinereproductive and respiratory syndrome (PRRSV) infectsmonocyte-derived dendritic cells but does not induce Tregcells. Virology 396: 264–271.

Silva-Campa E, Flores-Mendoza L, Resendiz M, Pinelli-SaavedraA, Mata-Haro V, Mwangi W and Hernandez J (2009b).Induction of T helper 3 regulatory cells by dendritic cellsinfected with porcine reproductive and respiratorysyndrome virus. Virology 387: 373–379.

Singh M, Briones M and O’hagan DT (2001). A novelbioadhesive intranasal delivery system for inactivatedinfluenza vaccines. Journal of Controlled Release 70:267–276.

Sirinarumitr T, Zhang Y, Kluge JP, Halbur PG and Paul PS (1998).A pneumo-virulent United States isolate of porcine repro-ductive and respiratory syndrome virus induces apoptosisin bystander cells both in vitro and in vivo. Journal ofGeneral Virology 79 (Pt 12): 2989–2995.

Sminia T, Van Der Brugge-Gamelkoorn GJ and Jeurissen SH(1989). Structure and function of bronchus-associatedlymphoid tissue (BALT). Critical Reviews in Immunology9: 119–150.

Srivastava P (2002). Interaction of heat shock proteins withpeptides and antigen presenting cells: chaperoning of theinnate and adaptive immune responses. Annual Reviews inImmunology 20: 395–425.

Sun J, Dodd H, Moser EK, Sharma R and Braciale TJ (2011).CD4+ T cell help and innate-derived IL-27 induce Blimp-1-dependent IL-10 production by antiviral CTLs. NatureImmunology 12: 327–334.

Suradhat S and Thanawongnuwech R (2003). Upregulationof interleukin-10 gene expression in the leukocytes ofpigs infected with porcine reproductive and respiratorysyndrome virus. Journal of General Virology 84: 2755–2760.

Suradhat S, Thanawongnuwech R and Poovorawan Y (2003).Upregulation of IL-10 gene expression in porcine periph-eral blood mononuclear cells by porcine reproductive andrespiratory syndrome virus. Journal of General Virology 84:453–459.

Suvas S, Azkur AK, Kim BS, Kumaraguru U and Rouse BT (2004).CD4+CD25+ regulatory T cells control the severity of viralimmunoinflammatory lesions. Journal of Immunology 172:4123–4132.

Suzuki H, Sekine S, Kataoka K, Pascual DW, Maddaloni M,Kobayashi R, Fujihashi K, Kozono H and McGhee JR (2008).Ovalbumin-protein sigma 1 M-cell targeting facilitates oraltolerance with reduction of antigen-specific CD4+ T cells.Gastroenterology 135: 917–925.

Thanawongnuwech R, Young TF, Thacker BJ and Thacker EL(2001). Differential production of proinflammatory cyto-kines: in vitro PRRSV and Mycoplasma hyopneumoniaeco-infection model. Veterinary Immunology and Immuno-pathology 79: 115–127.

Thiel V and Weber F (2008). Interferon and cytokine responsesto SARS-coronavirus infection. Cytokine Growth FactorReviews 19: 121–132.

Thomas C, Gupta V and Ahsan F (2009). Influence of surfacecharge of PLGA particles of recombinant hepatitis B surfaceantigen in enhancing systemic and mucosal immuneresponses. International Journal of Pharmacology 379:41–50.

Trandem K, Jin Q, Weiss KA, James BR, Zhao J and Perlman S(2011a). Virally expressed IL-10 ameliorates acute encepha-lomyelitis and chronic demyelination in coronavirus-infected mice. Journal of Virology 85: 6822–6831.

Trandem K, Zhao J, Fleming E and Perlman S (2011b). Highlyactivated cytotoxic CD8 T cells express protective IL-10 atthe peak of coronavirus-induced encephalitis. Journal ofImmunology 186: 3642–3652.

Tschernig T and Pabst R (2000). Bronchus-associated lymphoidtissue (BALT) is not present in the normal adult lung but indifferent diseases. Pathobiology 68: 1–8.

Vahlenkamp TW, Tompkins MB and Tompkins WA (2005). Therole of CD4+CD25+ regulatory T cells in viral infections.Veterinary Immunology and Immunopathology 108:219–225.

36 G. J. Renukaradhya et al.

Van Der Poel WH, Kramps JA, Quak J, Brand A and VanOirschot JT (1995). Persistence of bovine herpesvirus-1-specific antibodies in cattle after intranasal vacci-nation with a live virus vaccine. Veterinary Record 137:347–348.

Van Oirschot JT, Rziha HJ, Moonen PJ, Pol JM and Van Zaane D(1986). Differentiation of serum antibodies from pigsvaccinated or infected with Aujeszky’s disease virus by acompetitive enzyme immunoassay. Journal of GeneralVirology 67 (Pt 6): 1179–1182.

Van Reeth K, Labarque G, Nauwynck H and Pensaert M (1999).Differential production of proinflammatory cytokines in thepig lung during different respiratory virus infections:correlations with pathogenicity. Research in VeterinaryScience 67: 47–52.

Van Reeth K, Van Gucht S and Pensaert M (2002). In vivo studieson cytokine involvement during acute viral respiratorydisease of swine: troublesome but rewarding. VeterinaryImmunology and Immunopathology 87: 161–168.

Vaughan M (1997). Oxidative modification of macromolecules,Minireviews Series. Journal of Biological Chemistry 272:18513.

Vanhee M, Delputte PL, Delrue I, Geldhof MF and Nauwynck HJ(2009). Development of an experimental inactivatedPRRSV vaccine that induces virus-neutralizing antibodies.Veterinary Research 40: 63–77.

Voicu IL, Silim A, Morin M and Elazhary MA (1994). Interactionof porcine reproductive and respiratory syndromevirus with swine monocytes. Veterinary Record 134:422–423.

Vu HL, Kwon B, Yoon KJ, Laegreid WW, Pattnaik AK and OsorioFA (2011). Immune evasion of porcine reproductive andrespiratory syndrome virus through glycan shieldinginvolves both glycoprotein 5 as well as glycoprotein 3.Journal of Virology 85: 5555–5564.

Wagstrom EA, Chang CC, Yoon KJ and Zimmerman JJ (2001).Shedding of porcine reproductive and respiratorysyndrome virus in mammary gland secretions ofsows. American Journal of Veterinary Research 62:1876–1880.

Wensvoort G, Terpstra C, Pol JM, Ter Laak EA, Bloemraad M,De Kluyver EP, Kragten C, Van Buiten L, Den Besten A,Wagenaar F, Broekhuijsen JM, Moonen PLJM, Zetstra T, deBoer EA, Tibben HJ, de Jong MF, Van’t Veld P, GroenlandGJR, Van Gennep JA, Voets MT, Verheijden JHMand Braamskamp J (1991). Mystery swine disease inThe Netherlands: the isolation of Lelystad virus. VeterinaryQuarterly 13: 121–130.

Werner GH, Maral R, Floch F, Migliore-Samour D and Jolles P(1975). Adjuvant and immunostimulating activities of water-soluble substances extracted from Mycobacterium tubercu-losis (var. hominis). Biomedicine 22: 440–452.

White RG, Coons AH and Connolly JM (1955). Studies onantibody production. IV. The role of a wax fraction ofMycobacterium tuberculosis in adjuvant emulsions on the

production of antibody to egg albumin. Journal ofExperimental Medicine 102: 83–104.

Wongyanin P, Buranapraditkun S, Chokeshai-Usaha K, Thana-wonguwech R and Suradhat S (2010). Induction ofinducible CD4+CD25+Foxp3+ regulatory T lymphocytesby porcine reproductive and respiratory syndrome virus(PRRSV). Veterinary Immunology and Immunopathology133: 170–182.

Wongyanin P, Buranapraditkun S, Thanawonguwech R, Roth JAand Suradhat S (2009). Effect of nucleocapsid protein ofPRRSV on the induction of interleuklin-10 producinglymphocytes. International PRRS Symposium, December4–5, 2009, Chicago, IL, Abstract p. 95.

Woodland DL and Randall TD (2004). Anatomical features ofanti-viral immunity in the respiratory tract. Seminars inImmunology 16: 163–170.

Yang H and Parkhouse RM (1996). Phenotypic classification ofporcine lymphocyte subpopulations in blood and lymphoidtissues. Immunology 89: 76–83.

Yang H and Parkhouse RM (1997). Differential expression ofCD8 epitopes amongst porcine CD8-positive functionallymphocyte subsets. Immunology 92: 45–52.

Yoo D, Song C, Sun Y, Du Y, Kim O and Liu HC (2010).Modulation of host cell responses and evasion strategies forporcine reproductive and respiratory syndrome virus. VirusResearch 154: 48–60.

Yoon KJ, Zimmerman JJ, Swenson SL, McGinley MJ, Eernisse KA,Brevik A, Rhinehart LL, Frey ML, Hill HT and Platt KB(1995). Characterization of the humoral immune responseto porcine reproductive and respiratory syndrome (PRRS)virus infection. Journal of Veterinary Diagnostic Investiga-tion 7: 305–312.

Zhang L, Tian X and Zhou F (2006). In vivo effects ofoligodeoxynucleotides containing synthetic immunostimu-latory motifs in weaned piglets. International Immuno-pharmacology 6: 1623–1631.

Zhang L, Tian X and Zhou F (2007). Intranasal administrationof CpG oligonucleotides induces mucosal and systemicType 1 immune responses and adjuvant activity to porcinereproductive and respiratory syndrome killed virus vaccinein piglets in vivo. International Immunopharmacology 7:1732–1740.

Zimmerman J (2003). PRRS virus – what happens after apig becomes infected with PRRS virus? In 2003 PRRSCompendium Producer Edition, Chapter V, Des Moines,Iowa: National Pork Board, pp. 36–43.

Zimmerman J, Benfield DA, Murtaugh MPEA, Straw BE,Zimmerman JJ, D’Allaire S and Taylor DJ (2006). Porcinereproductive and respiratory syndrome virus (porcinearterivirus). In: Straw BE, Zimmerman J, D’Allaire S et al.(eds) Diseases of Swine. 9th edn. Ames, Iowa: BlackwellPublishing, pp. 387–418.

Zuckermann FA (1999). Extrathymic CD4/CD8 double positiveT cells. Veterinary Immunology and Immunopathology 72:55–66.

Mucosal vaccines to prevent porcine reproductive and respiratory syndrome 37