I.
Introduction and Background
II.
Characteristics of Enteropathogenic Viruses
A.
Comparative Pathogenesis of Virulent and Attenuated Transmissible Gastroenteritis
Virus, Porcine Respiratory Coronavirus, and Rotavirus
III.
Mucosal Immunity to Enteropathogenic Viruses
A.
Studies of Active Immunity to Transmissible Gastroenteritis Virus and Porcine Respiratory
Coronavirus
B.
New Vaccine Approaches to Induce Immunity to Transmissible Gastroenteritis Virus
C.
Studies of Active Immunity to Group A Rotavirus
D.
New Vaccine Approaches to Induce Immunity to Rotavirus
Acknowledgments
References
I.
Introduction and Background
Enteropathogenic viruses, such as transmissible gastroenteritis virus (TGEV) and rotavirus,
replicate and induce lesions only in the gastrointestinal tract. The susceptible target
cell is the villous enterocyte (Saif, 1990). Thus active immunity against enteropathogenic
viral infections depends on stimulation of local immune responses within the intestine.
To date, only limited success has been achieved in the development of oral vaccines
to prevent neonatal viral diarrheas, and commercial vaccines show limited efficacy
in the field (Saif and Jackwood, 1990). Although use of live attenuated poliovirus
is often cited as a model for an effective oral vaccine, the mechanism of viral pathogenesis
and hence protective immunity differs from that needed to prevent viral diarrheas.
Poliovirus undergoes primary replication in Peyer's patches or intestinal lymphoid
cells (not epithelial cells), but the target cell for disease induction is the neuron
(Melnick, 1990). Thus stimulation of circulating antibodies using either live oral
or inactivated poliovirus vaccines can prevent the systemic spread of poliovirus to
the central nervous system and the paralytic disease.
Currently only oral vaccines containing live replicating organisms have been highly
effective in inducing mucosal immune responses, especially secretory, (S)IgA antibodies.
The oral administration of soluble or killed antigens generally induces immunity of
short duration or even systemic tolerance (reviewed in Mowat, 1994). Whether the problems
encountered with oral administration of soluble protein antigens can be overcome by
the use of improved mucosal adjuvants [muranyl dipeptide, immune stimulating complexes
(ISCOMs), cholera or Escherichia coli enterotoxins, avridine, proteosomes, cytokines,
etc.] or new and novel delivery systems (liposomes, live recombinant vectors, microspheres,
DNA plasmids, virus-like particles) requires further investigation, and specific examples
are given in this review.
Coronaviruses and rotaviruses are well-characterized enteropathogens that account
for a high percentage of the viral diarrheas in many animals (Saif and Wesley, 1992;
Saif et al., 1994a). In addition, rotaviruses are the leading cause of dehydrating
diarrhea in young children worldwide (Kapikian and Chanock, 1990). Thus these viruses
serve as important models to study mucosal immunity to enteric viruses. In this review,
the impact of the site of viral replication (intestine vs respiratory tract), vaccine
dose, and type (attenuated, inactivated) on the isotype, level, and distribution of
virus-specific antibody-secreting cells (ASCs) and protection against viral challenge
in pigs is summarized and discussed.
II.
Characteristics of Enteropathogenic Viruses
Enteropathogenic viruses belonging to at least five different families have been associated
with diarrhea in pigs (Table I
, Saif, 1990). Each of these viruses infects mainly the villous enterocytes of pigs
and, with the possible exception of astroviruses (Saif et al., 1980), induces villous
atrophy and a malabsorptive diarrhea (Saif, 1990). None of these viruses causes systemic
infections; hence, the localized nature of these intestinal viral infections is of
prime consideration for designing effective strategies to induce mucosal immunity.
A potential explanation for the localized nature of many enteric viral infections
was highlighted in a recent study (Rossen et al., 1996). The authors found that TGEV
enters and exits from polarized epithelial cells in vitro via the apical surface;
in contrast, another coronavirus, mouse hepatitis virus (MHV), enters the same cells
apically but exits basolaterally. The investigators speculated that similar differences
in the mode of release of coronaviruses from infected host cells could contribute
to the nature of the localized intestinal infections induced by TGEV (released apically
into the gut lumen) or the systemic infections associated with MHV (released basolaterally
into the blood and lymph).
Table I
Classification and Characteristics of Porcine Enteropathogenic Viruses
Discovery
Intestinal replication site
Family/virus
Size (nm)
Nucleic acid
Year
Investigator
Villous
Crypt
Enveloped
Coronaviridae/ transmissible gastroenteritis virus (TGEV)
60–220
ssRNA
1946
Doyle and Hutchings (Solf and Wesley, 1992)
+
–
Nonenveloped
Reoviridae/rotavirus (group A)
55–70
dsRNA
1976
Woode et al.
+
–
Rotavirus (Group B)
55–70
dsRNA
1980
Bridger
+
–
Rotavirus (Group C)
55–70
dsRNA
1980
Saif et al.
+
–
Rotavirus (Group E)
55–70
dsRNA
1986
Chasey et al.
?
?
Caliciviridae/calicivirus
30–40
ssRNA
1980
Bridger
+
–
1980
Saif et al.
Astroviridae/Astrovirus
28–30
ssRNA
1980
Bridger
+
–
1980
Saif et al.
Adenoviridae/Adenovirus
70–90
DNA
1981
Coussement et al.
+
±
A.
Comparative Pathogenesis of Virulent and Attenuated Transmissible Gastroenteritis,
Porcine Respiratory Coronavirus, and Rotavirus
Exposure of pigs to attenuated TGEV (TGEV-A), virulent TGEV (TGEV-V) or porcine respiratory
coronavirus (PRCV) results in distinct disease patterns related to differences in
virulence and tissue tropisms between the viruses (Table II
) (Pensaert and Cox, 1989;
Saif and Wesley, 1992; Saif et al., 1994b). Virulent TGEV replicates in villous epithelial
cells throughout the small intestine, inducing severe villous atrophy and a malabsorptive
diarrhea leading to nearly 100% mortality in seronegative, neonatal pigs. Attenuated
strains of TGEV replicate in scattered villous epithelial cells in the distal portion
of the small intestine of neonatal pigs and induce mild or no diarrhea (Frederick
et al., 1976). They also replicate more extensively in the respiratory tract compared
to virulent TGEV strains (Furuuchi et al., 1979). In contrast, PRCV strains replicate
in the upper and lower respiratory tract, with little or no replication in the intestine,
and generally cause subclinical infections or mild respiratory disease (Pensaert and
Cox, 1989). TGEV infections remain a leading cause of piglet diarrhea and mortality
in swine herds in North America, and commercial vaccines, even live attenuated oral
TGEV vaccines, are of limited efficacy in the field (Saif and Wesley, 1992). In previous
studies, PRCV induced partial protection against experimental challenge with TGEV
(Van Nieuwstadt et al., 1989; Cox et al., 1993), but the mechanisms involved were
not elucidated. These three antigenically related porcine coronaviruses with distinct
differences in virulence and tissue tropisms (enteric TGEV-A or TGEV-V or respiratory
PRCV) provided an ideal model to study interactions between bronchus-associated lymphoid
tissues (BALT) and gut-associated lymphoid tissues (GALT) in the induction of mucosal
immunity and protection against virulent TGEV challenge (Brim et al., 1995; Saif,
1996; VanCott et al., 1993, 1994).
Table II
Vertical and Longitudinal Sites of Replication and Villous Atrophy in the Intestine
for Porcine Coronaviruses and Group A Rotaviruses
Longitudinal
Vertical small intestine (site)
Villous atrophy small intestine
Respiratory tract
Virus
Diarrhea
Small intestine
Colon
Villous
Crypt
Site
Extent
Upper
Lower
Reference
Coronavirus
Virulent TGEV
Severe
D,J,I
–
+ Entire
–
D,J,I
Severe
±
±
Frederick et al. (1976)
Attenuated TGEV
Mild/none
J,I
–
+ Entire
–
J,I/none
Mild
+
±
Frederick et al. (1976)
Furuuchi et al. (1979)
PRCV
None
–
–
– NA
–
None
None
+
+
Pensaert and Cox (1989)
Rotavirus
Group A
Mild-severe
D,J,I
±
+ Entire
–
J,I
Moderate-severe
−
−
Theil et al. (1978)
By comparison, porcine group A rotaviruses also replicate throughout the small intestine
and occasionally the colon, inducing moderate to severe villous atrophy in the distal
small intestine (Table II, Theil et al., 1978). Similar to enzootic infections with
TGEV-V in seropositive herds, rotaviruses are a frequent cause of diarrhea in 2- to
3-week-old pigs with morbidity rates approaching 100%, but with lower mortality rates
(5–20%) (Paul and Stevenson, 1990; Saif et al., 1994a).
To date, commercial and experimental candidate vaccines have not been highly effective
in preventing enteric viral infections and gastroenteritis in humans or animals (reviewed
in Saif and Jackwood, 1990; Kapikian and Chanock, 1990). Poor efficacy has frequently
been encountered in the field using live oral or parenterally administered vaccines
to prevent coronavirus and rotavirus-induced diarrhea in swine (Saif and Jackwood,
1990). Likewise, clinical trials of candidate rotavirus vaccines in infants have often
failed in various aspects of safety, immunogenicity, or efficacy, especially when
tested in developing countries (Kapikian and Chanock, 1990). These results suggest
that more research is needed to optimize enteric vaccines to induce local mucosal
immune responses that more closely mimic ones elicited after exposure to the virulent
organism.
III.
Mucosal Immunity to Enteropathogenic Viruses
A unique mucosal immune system separate from the systemic immune system has evolved
to protect mucosal surfaces from pathogens and to exclude environmental antigens and
foreign proteins, thereby preventing them from evoking systemic-type inflammatory
immune responses (reviewed by Brandtzaeg, 1992; Husband, 1993; McGhee et al., 1992;
Mestecky, 1987). The mucosal immune system is characterized by a preponderance of
SIgA antibodies selectively secreted onto mucosal surfaces by an active transport
mechanism (polyimmunoglobulin receptor, PIgR). The SIgA antibodies play a major role
in preservation of mucosal integrity by down-regulation of systemic-type immune responses,
preventing invasion of pathogens from the mucosa by blocking of attachment or invasion,
neutralization (in the lumen or intracellularly), and “immune exclusion.” These functions
are in contrast to systemically induced IgG antibodies that mediate inflammatory reactions
leading to the killing and elimination of pathogens, thereby maintaining systemic
sterility.
Although in earlier studies SIgA was envisioned to act mainly at the luminal mucosal
surfaces, recent data suggest that dimeric IgA may bind antigens on the basolateral
side of intestinal epithelial cells (Kaetzel et al., 1992). These immune complexes
would then be transported across the epithelial cell via the PIgR and secreted back
into the intestinal lumen, thereby eliminating foreign antigens that have penetrated
the epithelium, Other recent reports suggest that SIgA may function intracellularly
in host defense by inhibiting viral replication or assembly in vitro (Armstrong and
Dimmock, 1992; Marzanec et al., 1992) and in vivo (Burns et al., 1996). If further
confirmed in vivo, such findings imply that SIgA can promote recovery from viral infections
as well as initial protection.
Another unique feature of the mucosal immune system compared to the systemic immune
system is the induction of antigen-specific B and T cells in IgA inductive organized
lymphoid tissue (GALT, BALT, etc.) and their distribution to remote mucosal effector
sites (i.e., lamina propria regions of the intestine, bronchi, genitourinary tract,
and secretory glands). This cellular distribution pathway linking distant mucosal
sites is referred to as the common mucosal immune system (Mestecky, 1987). Thus antigen
taken up (via M cells) and processed via GALT [Peyer's patches (PP) and aggregates
of lymphoid tissue in the lamina propria] induces activated T and B cells which migrate
from the PP through the MLN and via the thoracic duct into the systemic circulation,
subsequently repopulating distant mucosal tissues. Maturation of these B cells into
IgA plasma cells occurs within the mucosal effector sites in response to antigen,
T cells, and cytokines (Lebman and Coffman, 1994). Key studies in rabbits confirmed
that PP are an enriched source of IgA precursor cells which repopulate the lamina
propria of the intestine and distant mucosal sites (Craig and Cebra, 1971).
Among the first reports to document that antigenic stimulation at one mucosal site
(intestine) leads to SIgA antibody responses at a distinct mucosal site (mammary gland)
were the studies of lactogenic immunity to TGEV in swine by Bohl et al. (1972) and
Saif et al. (1972). The discovery and subsequent confirmation (Weisz-Carrington et
al., 1978) of the interrelationship between the SIgA system of the intestine and mammary
gland was an important tenet of the common mucosal immune system, and this system
was later confirmed in humans and other species (Mestecky, 1987; McGhee et al., 1992).
Thus antigen-specific B and T cells induced in IgA inductive lymphoid tissues (GALT,
BALT, etc.) are disseminated to remote mucosal effector sites (i.e., lamina propria
of the gut, mammary gland, bronchi, genitourinary tract, etc.).
Recent studies, including further studies of immunity to porcine coronaviruses (Van
Cott et al., 1993, 1994; Saif et al., 1994b; Saif, 1996), have suggested that functional
compartmentalization and limited reciprocity may exist within some components of the
common mucosal immune system. For example, migration of cells from BALT is more limited
than from GALT (Sminia et al., 1989) and BALT exposure often leads to dissemination
of non-IgA committed secondary B cells (Cebra et al., 1984). In addition, IgA precursor
cells derived from GALT more readily repopulate the gut lamina propria than distant
mucosal sites (Cebra et al., 1984; Brandtzaeg, 1992). Such observations have important
implications for the design of effective mucosal vaccines, but information on effective
and practical procedures to induce protective immunity at mucosal surfaces is lacking.
In the following sections, our studies of the induction of mucosal immunity and protection
using the antigenically related porcine coronaviruses, TGEV and PRCV, are reviewed
as are results of studies comparing different types of rotavirus vaccines.
A.
Studies of Active Immunity to Transmissible Gastroenteritis Virus and Porcine Respiratory
Coronavirus
To analyze the interrelationships between BALT and GALT related to protective immunity,
we used as a model the three antigenically related porcine coronaviruses. Virulent
TGEV replicates primarily in the intestine and induces diarrhea; attenuated TGEV replicates
in the intestine and the upper respiratory tract but induces no diarrhea; and PRCV
replicates in the upper and lower respiratory tract, but induces only a subclinical
infection (Table II; Frederick et al., 1976; Pensaert and Cox, 1989; Saif and Wesley,
1992). These questions were addressed: Is PRCV a more effective candidate vaccine
for TGEV than attenuated TGEV? Does a high dose of attenuated TGEV administered orally
induce greater ASC responses in GALT than a lower dose (comparable or higher virus
titer than commercial TGEV vaccines)? What are the comparative IgA and IgG ASC responses
induced in GALT and BALT and the level of protection after inoculation with PRCV,
TGEV-A, or TGEV-V and challenge with TGEV-V? In pigs recovered from infection with
TGEV-V and reexposed to TGEV-V, what are the correlates of protective immunity?
We first investigated the comparative immune responses to live PRCV versus TGEV-V,
the degree of protection induced against TGEV-V challenge, and potential correlates
of protection. Three groups of 11-day-old TGEV seronegative pigs were oronasally inoculated
with virulent TGEV, PRCV, or mock-infected cell-culture fluids, respectively, and
challenged 24 days later with virulent TGEV (Brim et al., 1995; Saif et al., 1994b;
Saif, 1996; VanCott et al., 1993, 1994). Immune responses in intestinal (gut lamina
propria and mesenteric lymph nodes (MLNs) and respiratory (bronchial lymph nodes,
BLN) lymphoid tissues were assessed at challenge and postchallenge day (PCD) 4 by
enumeration of IgA and IgG TGEV-specific ASC by ELISPOT and by lymphoproliferative
assays (LPAs) using inactivated TGEV as antigen. The major ASC responses and percent
of pigs protected are summarized in Table III
. All pigs inoculated with TGEV-V developed diarrhea, shed TGEV in feces, and recovered.
The presence of high numbers of IgA-ASC in the gut lamina propria (LP) and high LPA
responses in the MLN at challenge (PCD 0) was associated with 100% protection against
diarrhea after TGEV challenge. No significant increases were observed in numbers of
ASC or LPA responses in the gut LP or MLN, respectively, after TGEV challenge (PCD
4), reflecting the lack of viral replication associated with complete protection.
In contrast, pigs inoculated with PRCV had no clinical disease and shed virus in nasal
secretions
but not feces. At challenge (PCD 0), the PRCV-exposed pigs had mainly IgG ASC and
high LPA responses in the BLN, but low ASC numbers and LPA responses in the intestine
(gut LP or MLN, respectively). About 58% of the pigs were protected against diarrhea
(compared to 10% of controls) and only 17% were protected against fecal TGEV shedding
(comparable to controls). After TGEV challenge (PCD 4), the numbers of IgG-ASC and
to a lesser extent IgA-ASC increased rapidly in the intestinal lamina propria of the
PRCV-exposed pigs, suggesting that virus-specific IgG-ASC precursors derived in BALT
or systemic lymphoid tissues of the PRCV-exposed pigs may migrate to the intestine
in response to TGEV challenge and contribute to the partial protection observed. The
higher numbers of IgA-ASC in BALT of TGEV-exposed pigs compared to PRCV-exposed pigs
at PCD 4 probably reflects TGEV replication and restimulation in the gut resulting
in trafficking of IgA precursor cells from GALT to BALT (Husband, 1994; Mestecky,
1987; McGhee et al., 1992). Thus TGEV infections or vaccines that induce immunity
via GALT and secondarily via BALT may prevent PRCV infections. Whether the more frequent
use of live attenuated TGEV vaccines in the United States (which induce IgG ASC in
BLN, Table IV
) compared to Europe has had an impact on limiting the spread of PRCV infections in
the United States is unknown, but at present PRCV infections appear to be less widespread
among swine in the United States than in Europe. Thus our major conclusions were that
functional compartmentalization exists in the BALT and GALT responses: immunization
via BALT (PRCV infection) induced a systemic type of response (IgG-ASC) with low numbers
of ASC and LPA responses in the gut and provided incomplete protection against TGEV-V.
Immunization via GALT (TGEV-V infection) induced high numbers of IgA-ASC and high
LPA responses in the gut and provided complete protection against TGEV-V induced diarrhea.
Further studies on the induction and immune regulation of responses to TGEV and PRCV
that affect the distribution of ASC and T lymphocytes should provide important insights
to optimize oral vaccine regimens to elicit protective mucosal immune responses against
enteric pathogens.
Table III
Comparison of Intestinal and Respiratory ASC Responses and Protective Immunity Induced
by TGEV and PRCV Strains in Neonatal Pigs at Postchallenge Day (PCD) 0 and 4a
Mean No. ASC/5 ⊠ 105 MNC at PCD 0
Mean No. ASC/5 ⊠ 105 MNC at PCD 4
Percent protection against TGEV challenge
Virus inoculum group
Intestinal lamina propria
Bronchial lymph node
Intestinal lamina propria
Bronchial lymph node
Diarrhea (%)
Shedding (%)
IgG
IgA
G/Ab
IgG
IgA
G/A
IgG
IgA
G/A
IgG
IgA
G/A
Virulent TGEV
109
620
0.18
25
1
25
15
109
0.14
300
94
3.2
100
80
PRCV
<1
1
UD
223
1
223
150
4
38
320
7
46
58
17
Controls
<1
<1
—
<1
<1
—
<1
<1
—
<1
<1
—
10
22
a
Data summarized from VanCott et al. (1994).
b
G/A, ratio of IgG to IgA ASCs; UD, undetermined because numerator <1.
Table IV
COMPARISON OF INTESTINAL AND RESPIRATORY PRIMARY AND MEMORY ASC RESPONSES INDUCED
BY VIRULENT TGEV AND LOW VERSUS HIGH DOSES OF ATTENUATED TGEV IN NEONATAL PIGSa
Mean No. ASC/5 × 105 MNC
Virus inoculum group/responseb
Mesenteric lymph node
Bronchial lymph node
IgG
IgA
G/Ac
IgG
IgA
G/Ac
Virulent TGEV
Primary
48
9
5
7
1
7
Memory
5295
1159
5
2989
327
9
Attenuated TGEV
Low dose (106 pfu)
Primary
2
<1
UD
16
1
16
Memory
60
<10
UD
866
34
25
High dose (108 pfu)
Primary
9
1
9
28
1
28
Memory
1133
79
14
4475
159
28
a
Data summarized from VanCott et al. (1993).
b
Primary immune responses were assayed by ELISPOT directly on mononuclear cells (MNCs)
obtained from pigs at PID 12 and 24 and the mean numbers of ASC per 5 × 105 MNC are
shown. Memory or secondary immune responses were assayed by ELISPOT after in vitro
TGEV stimulation (5 days) of MNC obtained from pigs at PID 24 and 40 and the mean
numbers of ASC per 5 × 105 MNC are shown.
c
G/A, ratio of IgG to IgA ASCs; UD, undetermined.
In a similar series of experiments, we also examined the effect of the dose (106 versus
108 pfu) of live TGEV-A administered oronasally to 11-day-old TGEV seronegative pigs,
on primary and memory ASC responses in the MLN and BLN (Saif et al., 1994b; Saif,
1996; VanCott et al., 1993). Our findings (summarized in Table IV) revealed that the
high dose of TGEV-A (108 pfu) induced 2–4 times more primary IgG ASC and about 5–20
times more memory IgG ASC in the MLN and BLN than the lower dose. Only the high dose
of TGEV-A elicited low numbers of primary or memory IgA ASC in the MLN, but numbers
were 9–15 times lower than after inoculation with TGEV-V. Of interest were the two-
to fourfold higher numbers of primary and memory IgG ASC induced in BALT by the high-dose
TGEV-A compared to TGEV-V consistent with reports that attenuated strains of TGEV
replicate more extensively in the respiratory tract compared to virulent TGEV strains
(Furuuchi et al., 1979). Thus the high degree of attenuation of TGEV vaccines leading
to reduced viral replication in the intestine of the sow (Saif and Jackwood, 1990;
Saif and Wesley, 1992) and the use of low doses (≤106 pfu/ml) of live attenuated TGEV
vaccines orally in piglets (Saif et al., 1994b; VanCott et al., 1993) were major determinants
in their failure to induce SIgA antibodies in sow's milk or IgA ASC in the piglets’
intestines, respectively. Such factors presumably contribute to the corresponding
lack of efficacy of TGEV vaccines in the field.
B.
New Vaccine Approaches to Induce Active Immunity to TGEV
Several new potential vaccine approaches to induce immunity to TGEV have recently
been reported based on delivery of antigenic peptides of the TGEV S protein in orally
administered live bacterial vectors (Der Vartanian et al., 1997; Smerdou et al., 1996).
In studies by Der Vartanian et al., (1997), two antigenic peptides of the TGEV S protein,
TGEV S
A
and S
C
, were tandemly inserted (25 amino acids) into the major CIpG subunit of the CS 31A
fibrillae of Escherichia coli K-12 strain. The responses of mice to these constructs
were as follows: (1) The two TGEV epitopes were immunogenic when injected intraperitoneally
(IP) into mice as hybrid CIpG subunits, chimeric CS31A polymers, or recombinant bacteria;
(2) the chimeric CS31A fibrillae elicited TGEV antibodies in the serum of mice reactive
with TGEV peptides and native TGEV; and (3) mice inoculated orally with the recombinant
bacteria produced IgA intestinal antibodies reactive against the CS31A fibrillae and
TGEV S
C
peptide.
In another approach, a recombinant live attenuated Salmonella typhimurium was used
for oral delivery of a TGEV peptide vaccine in rabbits (Smerdou et al., 1996). An
antigenic peptide of the TGEV S protein (S
D
, amino acids 378–395) was expressed as a fusion protein with E. coli LT-B in the
Salmonella. The rationale for fusion with LT-B was to enhance the immunogenicity of
the bivalent vaccine since LT-B also functions as an oral adjuvant. Studies of immune
responses of rabbits inoculated with purified LT-B/S
D
fusion products expressed from Salmonella or the recombinant Salmonella revealed that
neutralizing antibodies to TGEV were induced by the purified LT-B/S
D
and TGEV antibodies were elicited in serum and intestinal secretions after oral inoculation
with the recombinant Salmonella. Thus, if similar TGEV neutralizing IgA antibody responses
can be induced in the intestines of pigs by the recombinant bacterial vaccines, such
vaccines warrant further study to access their ability to induce protective immunity
to TGEV in pigs.
In our laboratory, we are currently exploring optimal oral adjuvants and delivery
systems for recombinant TGEV S and M protein vaccines. Preliminary data indicate S
and M protein vaccines administered IP with incomplete Freund's adjuvant (IFA) induced
higher numbers of memory ASCs in GALT than an inactivated TGEV vaccine administered
IP (Sestak et al., 1997).
C.
Studies of Active Immunity to Group A Rotavirus
The gnotobiotic piglet model of porcine and human rotavirus-induced diarrhea has been
used to further evaluate the influence of vaccine type (attenuated or binary-ethyleneimine
inactivated) compared to wild-type virus infection on induction of intestinal ASC
responses and protective immunity (Chen et al., 1995; Saif et al., 1996; Yuan et al.,
1996, 1998). Results of oral or IM inoculation of 3- to 5-day-old pigs with Wa human
rotaviruses (G1, P1A) and homologous virulent rotavirus oral challenge at postinoculation
day (PID) 21 are summarized in Table V
. B-cell responses (ASC) were measured by ELISPOT for intestinal (lamina propria)
and systemic (peripheral blood lymphocytes, PBL) lymphoid tissues at challenge (PID
21). The major findings were that the numbers of IgA ASCs in the intestinal lamina
propria and PBL were significantly greater in virulent-rotavirus inoculated pigs (mimic
natural infection) than in the other groups (attenuated, inactivated, controls) and
were correlated (r = 0.9) with the high degree of protection against diarrhea (89%
of piglets protected). The transient appearance of IgA ASC in the blood mirrored the
IgA ASC responses in the gut and could serve as an indicator for IgA ASC intestinal
responses after rotavirus infection. Piglets inoculated with attenuated rotavirus
had partial protection against diarrhea (44% protected) and the second highest numbers
of IgA and IgG ASC in the intestinal lamina propria. Interestingly pigs inoculated
IM or perorally (PO) with inactivated rotavirus in IFA had a very high number of IgG
ASCs in PBL, but few IgG or IgA ASCs in the intestinal lamina propria and, like pigs
given inactivated virus PO, minimal protection (0–17%) against diarrhea. Thus, the
vaccine type influenced the site, isotype, and level of the ASC response and, similar
to the results of the TGEV studies, the degree of protection was correlated with the
numbers of IgA ASCs induced in the intestine.
Table V
COMPARISON OF MUCOSAL AND SYSTEMIC ASC RESPONSES AND PROTECTIVE IMMUNITY INDUCED BY
VIRULENT, ATTENUATED AND INACTIVATED ROTAVIRUS VACCINES IN NEONATAL PIGS AT PCD 0
(PID 21)a
Mean No. ASC/5 ⊠ 105 MNC
Percent protection against rotavirus challenge
Virus inoculum group
Intestinal lamina propria
Peripheral blood lymphocytes
Diarrhea
Shedding
IgG
IgA
G/Ab
IgG
IgA
G/Ab
Live
Virulent rotavirus (PO)
64
53
1.2
2
6
0.3
89%
100%
Attenuated rotavirus (PO)
41
6
6.8
2
1
2
44%
19%
Inactivated
Rotavirus (PO)
0.7
5
0.14
88
1
88
0%
0%
Rotavirus (IM)
4
3
1.3
237
2
119
17%
0%
Controls
<1
<1
<1
<1
14%
0%
a
Data summarized from Yuan et al., (1996, 1998); Saif et al., (1996, p. 199).
b
G/A, ratio of IgG to IgA ASCs.
Similarly, in studies of natural rotavirus infections in children, higher fecal IgA
antibody titers to rotavirus were associated with protection against infection and
illness (Matson et al., 1993). Mouse studies of rotavirus-induced infection revealed
similar findings: induction of intestinal IgA antibody responses were positively associated
with protection against rotavirus shedding (Feng et al., 1994).
D.
New Vaccine Approaches to Induce Immunity to Rotavirus
Although not yet evaluated in swine, a new strategy for rotavirus vaccines is the
creation of recombinant virus-like particles (VLPs) produced by the coexpression of
the four rotavirus capsid genes (VP2/4/6/7) in a baculovirus expression system (Crawford
et al., 1994). The VLP vaccines administered with IFA have been tested in rotavirus
seronegative mice and rabbits (Conner et al., 1996) and as a maternal vaccine to enhance
passive immunity in rotavirus seropositive cows (Fernandez et al., 1996). The VLP
vaccines were shown to be noninfectious (no RNA), stable, antigenically authentic,
and highly immunogenic in the above species. They induced protective immunity against
rotavirus shedding in vaccinated mice and rabbits (Conner et al., 1996) and passive
immunity against rotavirus diarrhea in calves fed colostrum from the VLP-vaccinated
cows (Fernandez et al., 1998). Thus VLP vaccines show promise as novel vaccines designed
to induce mucosal immunity against rotavirus. Further research is needed to identify
the optimal delivery systems and mucosal adjuvants for use with the VLP vaccines to
most effectively stimulate mucosal immunity.