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Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Protective Immunity Induced by Oral Immunization with a

Rotavirus DNA Vaccine Encapsulated in Microparticles

SHING C. CHEN,

1

DAVID H. JONES,

2

ELLEN F. FYNAN,

1,3

GRAHAM H. FARRAR,

2

J. CHRISTOPHER S. CLEGG,

2

HARRY B. GREENBERG,

4

AND

JOHN E. HERRMANN

1

*

Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester,

Massachusetts 01655

1

; Center for Applied Microbiology and Research, Salisbury SP4 OJG,

United Kingdom

2

; Department of Biology, Worcester State College, Worcester,

Massachusetts 01602

3

; and Division of Gastroenterology, Stanford

University School of Medicine, Stanford, California 94304

4

Received 23 December 1997/Accepted 26 March 1998

DNA vaccines are usually given by intramuscular injection or by gene gun delivery of DNA-coated particles

into the epidermis. Induction of mucosal immunity by targeting DNA vaccines to mucosal surfaces may offer

advantages, and an oral vaccine could be effective for controlling infections of the gut mucosa. In a murine

model, we obtained protective immune responses after oral immunization with a rotavirus VP6 DNA vaccine

encapsulated in poly(lactide-coglycolide) (PLG) microparticles. One dose of vaccine given to BALB/c mice

elicited both rotavirus-specific serum antibodies and intestinal immunoglobulin A (IgA). After challenge at 12

weeks postimmunization with homologous rotavirus, fecal rotavirus antigen was significantly reduced

com-pared with controls. Earlier and higher fecal rotavirus-specific IgA responses were noted during the peak

period of viral shedding, suggesting that protection was due to specific mucosal immune responses. The results

that we obtained with PLG-encapsulated rotavirus VP6 DNA are the first to demonstrate protection against an

infectious agent elicited after oral administration of a DNA vaccine.

Group A rotavirus infections cause an estimated 870,000

deaths each year in developing countries (12). They also cause

55,000 to 70,000 hospitalizations per year in the United States,

with an estimated cost of more than $1 billion (12). Because of

the widespread nature of rotavirus disease, development of

vaccines is considered key to their control (1, 12). Although

progress has been made in the development of live oral

rota-virus vaccines (32), improved vaccines are still needed,

partic-ularly in many developing countries where the need is the

greatest (1, 12, 22, 33) but where the live oral vaccines have

been less effective (25, 26). Development of killed rotavirus

vaccines and subunit vaccines may be possible (1), but these

types of vaccines do not provide endogenously synthesized

proteins and generally do not elicit cytotoxic T-lymphocyte

(CTL) responses (13) that may be important in controlling

rotavirus infection. The use of DNA encoding specific viral

proteins allows for the expression of immunizing proteins by

host cells that take up inoculated DNA. This results in the

presentation of normally processed proteins to the immune

system, which is important for raising immune responses

against the native forms of proteins (11, 36). Expression of

the immunogen in host cells also results in the immunogen

having access to class I major histocompatibility complex

presentation, which is necessary for eliciting CD8

1

CTL

responses.

Rotavirus virions have a three-layered protein capsid. The

protein-coated RNA core is coated by VP6, a protein that is

antigenically conserved among group A rotaviruses but does

not elicit antibodies that neutralize rotavirus in vitro. The two

outer capsid surface proteins, VP4 and VP7, elicit neutralizing

antibodies. In prior studies, we found that DNA vaccines

en-coding VP4, VP7, or VP6 were protective when administered

by gene gun delivery of the DNA to the epidermis (3, 15, 16).

Direct gene gun inoculation to the anal mucosa required

five-fold less DNA (0.5 rather than 2.5

m

g per mouse) to give the

same level of protection (17), suggesting that targeting

muco-sal tissue enhances the generation of protective immunity.

Both inoculation routes resulted in enhanced intestinal

immu-noglobulin A (IgA) responses after rotavirus challenge, but

neither induced detectable intestinal IgA prior to challenge.

Protective immune responses against rotavirus infections have

been correlated with production of rotavirus-specific fecal IgA

in vivo in human and porcine studies as well as in the murine

model (4, 10, 27, 34, 38). Thus, induction of intestinal IgA may

be an important correlate in the development of rotavirus

vaccines.

Targeting of rotaviruses to the gut-associated lymphoid

tis-sue by oral administration of an aqueous-based system of

mi-croencapsulated noninfectious rotaviruses generated serum

IgG and intestinal IgA antibody responses (24). This finding

suggests that mucosal targeting of DNAs expressing rotavirus

proteins might also generate immune responses. Recently, a

method for encapsulation of plasmid DNA which permits the

DNA to be orally administered has been developed. Plasmid

DNA encoding insect luciferase was encapsulated in

poly(lac-tide-coglycolide) (PLG) microparticles and oral administration

of these PLG microparticles stimulated serum IgG, IgM, and

IgA antibodies to luciferase (21). Luciferase-specific IgA was

also detected in stool samples, indicating a mucosal response.

In this study, we examined the ability of a PLG-encapsulated

rotavirus VP6 DNA vaccine to induce serum and mucosal

antibody responses and to protect against rotavirus infection

after challenge of adult mice.

* Corresponding author. Mailing address: Division of Infectious

Diseases, University of Massachusetts Medical School, 55 Lake Ave.

North, Worcester, MA 01655. Phone: (508) 2155. Fax: (508)

856-5981. E-mail: John.E.Herrmann@banyan.ummed.edu.

5757

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MATERIALS AND METHODS

Virus and mice.Epizootic diarrhea of infant mice (EDIM) rotavirus strain EW (P10[16], G3) was used for preparation of cDNA encoding VP6 and for virus challenge of mice. The virus challenge stock was prepared by passaging virus from intestinal homogenates of EDIM rotavirus-infected infant mice in adult mice. Virus for challenge was a stool sample diluted in saline. The 50% infective dose (ID50) of the stock virus was the 50% shedding dose as determined by detection of rotavirus antigen shed in feces of infected mice. The mice used for vaccine studies were obtained from rotavirus-free colonies (Charles River Lab-oratories, Portage, Mich.) at 6 to 8 weeks of age and were housed in plastic microisolater cages before and after immunization. The model developed by Ward et al. for BALB/c mice (35) was used to measure protective immunity. In this model, the endpoint is infection rather than illness, because illness is gen-erally limited to infant mice aged 15 days or younger. The adult mouse (6 weeks or older) becomes infected and sheds virus in feces for approximately 1 week postinfection. Protection after virus challenge was defined as significant reduc-tion in rotavirus antigen shedding in feces.

Encapsulated DNA vaccine.The plasmid encoding rotavirus VP6 DNA (Fig. 1) was prepared by insertion of murine rotavirus VP6 cDNA into the pCMV intron A TPA expression vector provided by J. Mullins, University of Washing-ton (plasmid JW4303) (37). This vector uses sequences from the cytomegalovirus (CMV) immediate-early promoter to drive transcription and sequences from bovine growth hormone genes to provide polyadenylation signals. To prepare VP6 DNA vaccine by cohesive end ligation, the TPA leader sequence was removed by treatment with restriction endonucleases HindIII and BamHI. The HindIII site was changed to a BamHI site, and the gene for VP6 (GenBank accession no. U36474) was inserted as a BamHI-BamHI fragment. The gene had been inserted in the BamHI site of plasmid Bluescript KS2and was released by BamHI digestion prior to insertion into plasmid JW4303. Newly constructed plasmids in the correct orientation were identified by restriction endonuclease digestion. Expression of rotavirus VP6 in transfected COS cells was confirmed by indirect immunofluorescent staining with monoclonal antibody to VP6. The monoclonal antibody had been prepared against a rotavirus SA-11 strain (5). The control DNA vaccine was the plasmid without the viral cDNA insert.

Plasmid DNA was encapsulated in PLG microparticles by the solvent extrac-tion technique as previously described (20, 21). In brief, the DNA was emulsified with PLG dissolved in dichloromethane, and this water-in-oil emulsion was emulsified with aqueous polyvinyl alcohol (an emulsion stabilizer) to form a (water-in-oil)-in-water double emulsion. This double emulsion was added to a large quantity of water to dissipate the dichloromethane, which resulted in the microdroplets hardening to form microparticles. These were harvested by cen-trifugation, washed several times to remove the polyvinyl alcohol and residual solvent, and finally lyophilized. The microparticles containing DNA had a mean diameter of 0.5mm. To test for DNA content, the microparticles were dissolved in 0.1 M NaOH at 100°C for 10 min. The A260was measured, and DNA was calculated from a standard curve. Incorporation of DNA into microparticles was 1.76 to 2.7mg of DNA per mg of PLG for the VP6 DNA vaccine and 1.75 to 3.61

mg per mg of PLG for the plasmid control.

Immunization of mice.Three groups of BALB/c mice were inoculated orally (by gavage) with PLG-encapsulated plasmid DNA encoding murine rotavirus VP6 (n513 mice total) or control plasmid DNA (n510 mice total). The microparticles were suspended in a solution of 0.1 M sodium bicarbonate in distilled water (pH 8.5) and given at 0.5 ml/mouse. The DNA dose administered was approximately 50mg per mouse.

Antigen and antibody testing.For monitoring viral antigen shedding in mouse feces, we used a monoclonal antibody-based enzyme-linked immunosorbent as-say (ELISA) in microtiter plates as previously described (14). For evaluating serum antibody responses, an indirect ELISA for total antibody (IgG, IgM, and

IgA) (3, 10, 15, 16) was used with EDIM rotavirus-coated wells. Intestinal IgA antibodies to EDIM virus were determined by use of IgA-specific peroxidase-labeled antiglobulin in an indirect ELISA (3, 10, 15, 16). Five percent (wt/vol) stool suspensions in 0.01 M phosphate-buffered saline (PBS; pH 7.1) were fur-ther diluted 1:4 (final dilution of 1:80) and used for assays of fecal IgA.

Statistical analyses.Statistical analyses were performed using a nonparamet-ric Wilcoxon two-sample test for ranked data and analysis of variance and the Student-Newman-Keuls test for multiple comparison of the differences among experimental groups.

RESULTS

Serum antibodies.

Inoculated mice were examined for

se-rum antibodies (IgG, IgM, and IgA) every 2 weeks for 12

weeks. A single immunization was sufficient to elicit a serum

antibody response by 4 weeks after inoculation, with peak titers

being reached by 6 weeks (Fig. 2). Twofold dilutions were

tested starting at a 1:100 dilution.

Protection against rotavirus challenge.

Mice were

chal-lenged with 100 ID

50

of EDIM rotavirus at 12 weeks

postim-munization to determine if the impostim-munizations had provided

protection. The challenge virus used was given by oral gavage.

Protection was assessed by testing for the reduction of

rotavi-rus antigen shedding in stools. Significant reductions (P

,

0.0002) in virus antigen shed were noted on days 2, 3, and 4

(Fig. 3).

Rotavirus-specific IgA in stools.

Immunized mice were

ex-amined for intestinal rotavirus-specific IgA before virus

chal-lenge at 4, 6, 8, 10, and 12 weeks postimmunization. The results

of the tests for detection of rotavirus-specific IgA in stools are

shown in Fig. 4. Significant production of fecal IgA (P

,

0.003)

was detected in the PLG-encapsulated VP6 DNA-vaccinated

animals at 6, 8, 10, and 12 weeks, suggesting that rotavirus

antigen was expressed and a mucosal antibody response had

been induced. In previous studies using gene gun delivery, we

did not detect rotavirus-specific IgA in the stools of

DNA-immunized animals until after they were challenged with live

rotavirus (3, 15, 16).

[image:2.612.121.218.70.199.2]

To determine if oral immunization also enhanced IgA

re-sponses after virus challenge, intestinal IgA was measured by

an IgA rotavirus-specific ELISA. The mice orally inoculated

with the PLG-VP6 DNA vaccine gave higher and earlier A

492

values (P

,

0.01) at 0, 1, 3, and 5 days postchallenge than mice

FIG. 1. Diagram of the pCMVIA vector and the virus cDNA insert. SV 40

Ori, simian virus 40 origin of replication; CMV Pro, CMV immediate-early promoter, intron A (largest CMV intron); BGH, bovine growth hormone gene (provides polyadenylation [pA] signals).

FIG. 2. Rotavirus-specific serum ELISA antibodies mice from BALB/c mice that had been orally inoculated (by gavage) with PLG-encapsulated VP6 DNA vaccine (n513) or with PLG-encapsulated control plasmid DNA (n510). Serum was collected at the times indicated and tested by an ELISA for total antibody (IgG, IgM, and IgA) every 2 weeks for 12 weeks. Results are expressed as geometric mean titers6standard error.

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that had been orally inoculated with PLG-control plasmid

DNA (Fig. 5).

DISCUSSION

Our studies with PLG-encapsulated DNA vaccines

pre-sented here used VP6 DNA. We selected the VP6 DNA

vac-cine for these studies because of the broad implications a

VP6-based vaccine has for the prevention of rotavirus

infec-tions. VP6 is an antigenically conserved protein among all

group A rotaviruses of both animal and human origin. Thus,

serotype specificity may not be a problem with VP6-based

vaccines. The protection was not as complete as that we

re-ported previously with gene gun immunizations, where

rotavi-rus antigen was not detected in the stools of mice immunized

with VP6 DNA vaccine (3, 15, 16). This could be related to

vaccine dose. The dose of 50

m

g per mouse used was based on

doses used for expression of other antigens and may not be

optimal for our system, and lower doses of VP6 DNA vaccine

given by gene gun also resulted in partial protection (17).

Dose-response studies will determine the optimal vaccine

dose. Compared with gene gun delivery used in our previous

studies (3, 15, 16), more DNA is required when encapsulated

and given orally (50

m

g, compared with two doses of 2.5

m

g

given by gene gun). Although it may also be possible to induce

immune responses by oral administration of naked DNA in a

saline solution, encapsulated material protects the DNA from

degradation by nucleases. Oral administration of naked DNA

encoding luciferase did induce immune responses, but PLG

encapsulation enhanced the responses (22). We expect to

com-pare naked DNA with encapsulated DNA in future

experi-ments.

Protective immunity was measured by reduction of rotavirus

antigen shed after challenge, because adult mice (mice older

than 2 weeks) do not develop diarrhea following rotavirus

infection. However, as pointed out by others, protection from

rotavirus infection may be a more stringent measure of

pro-tection than propro-tection from disease, because infection can

occur in the absence of disease (31). In studies with murine

rotaviruses given orally to mice, protection against rotavirus

challenge is associated with rotavirus-specific fecal IgA (10,

35). We have also found that fecal IgA antibodies were rapidly

induced in mice immunized with rotavirus DNA vaccines, but

only after they were virus challenged (3, 15, 16). We had

previously shown that VP6 DNA vaccine induced both serum

antibodies and CTL responses after gene gun immunization (3,

15, 16). The serum antibodies did not neutralize rotavirus in

vitro; thus, it is unlikely that traditional virus neutralization is

involved in the protection found.

The mechanism of protection seen with the VP6 DNA

vac-cine and also with VP6-based virus-like particles (31) are not

known. Among potential mechanisms of protection are

cell-mediated immunity and IgA-cell-mediated intracellular

neutraliza-tion of virus that is undergoing assembly. In studies with

rota-virus VP 2, 6 rota-virus-like particles (31), protective immunity was

obtained by coadministration of cholera toxin, which is known

FIG. 3. Protection against EDIM rotavirus challenge in BALB/c mice. Mice [image:3.612.82.257.68.231.2]

were challenged with 100 ID50 of virus per mouse 12 weeks after receiving PLG-encapsulated VP6 DNA vaccine (n513) or PLG-encapsulated control plasmid DNA (n510) by oral gavage. Virus shedding in feces, determined by an ELISA for detecting rotavirus antigen, is given as A4926standard deviation. A positive test is one in which the A492is$0.1. There were significant differences (P,0.0002) in viral shedding between the mice receiving the plasmid encoding VP6 and the plasmid control on the days indicated by an asterisk.

FIG. 4. ELISA for rotavirus-specific IgA in stool suspensions from mice that had been orally inoculated (by gavage) with PLG-encapsulated VP6 DNA vac-cine (n513) or with PLG-encapsulated control plasmid DNA (n510). The stools were diluted 1:80 (wt/vol) in PBS. Results are expressed as A4926 stan-dard deviation. Values of.0.1 are considered positive for IgA. There were significant differences (P,0.004) in fecal IgA values between the mice receiving the plasmid encoding VP6 and the plasmid control on the days indicated by an asterisk.

FIG. 5. ELISA for rotavirus-specific IgA in stool suspensions from mice that had been orally inoculated (by gavage) with PLG-encapsulated VP6 DNA vac-cine (n513) or with PLG-encapsulated control plasmid DNA (n510) and challenged with EDIM rotavirus. The stools were diluted 1:80 (wt/vol) in PBS. Results are expressed as A4926standard deviation. An A492of.0.1 is consid-ered positive for IgA. There were significant differences (P,0.01) in fecal IgA values between the mice receiving the plasmid encoding VP6 and the plasmid control on the days indicated by an asterisk.

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to enhance both mucosal antibody responses and CTL

re-sponses, and it is possible that either or both types of immune

responses are involved. IgA-mediated intracellular virus

neu-tralization has been shown for Sendai virus and influenza virus

(28, 29), and studies with IgA monoclonal antibodies to VP6

suggest that IgA-mediated intracellular neutralization may

also occur with rotaviruses (2). Based on these findings and our

demonstration of enhanced IgA responses in VP6

DNA-vac-cinated mice both before and after virus challenge,

IgA-medi-ated intracellular neutralization in the intestinal mucosa may

be a factor in the protective immunity that we have obtained.

We expect to test DNA vaccines in immunodeficient mice to

help determine the relative importance of CTL responses and

intestinal IgA in the protection obtained with VP6 DNA

vac-cines.

Determination of the cell or cells targeted by the

encapsu-lated DNA and the ultimate fate of the DNA was beyond the

scope of this study, but it is likely that the cells involved are

similar to those that have been shown to be involved in the

uptake of PLG microparticles. Following oral administration

to mice, PLG microparticles 1 to 10

m

m in diameter were taken

up into the Peyer’s patches of the gut-associated lymphoid

tissue. Those particles

$

5

m

m that were taken up remained

localized for up to 35 days, whereas those particles

,

5

m

m

were disseminated within macrophages, mesenteric lymph

nodes, blood circulation, and spleen (8, 9). PLG microparticles

are not selectively targeted to M cells, but nonspecific binding

to M cells and subsequent transcytosis has been shown in

rabbits (18, 19). PLG microparticles

,

5

m

m have also been

shown to cross the intestinal mucosa at the site of Peyer’s

patches in rats (6). The DNA-containing PLG microparticles

used in our study had a mean diameter of 0.5

m

m. It has been

presumed that PLG microparticles containing antigen bind to

and are transported by M cells in a manner similar to that

found with empty PLG microparticles (30). Supporting this

assumption, uptake of bovine serum albumin encapsulated in

PLG microparticles by Peyer’s patches has been shown in a rat

model (7).

The use of DNA vaccines is a new approach to

immuniza-tion that may provide more effective rotavirus vaccines. It has

been suggested that this approach and the virus-like particle

approach may make a third generation of rotavirus vaccines

(33). DNA vaccines encapsulated in PLG microparticles

com-bine the advantages of DNA-based vaccination with the ease of

administration by the oral route and concomitant induction of

mucosal immune responses. The results that we obtained with

PLG-encapsulated rotavirus VP6 DNA are the first to

demon-strate protection against an infectious agent elicited after oral

administration of a DNA vaccine.

ACKNOWLEDGMENTS

This study was supported by a grant from the World Health

Orga-nization, by grants R01 AI39637 and R41 AI40449 from the National

Institutes of Health to J.E.H., and by a VA Merit Review Grant to

H.B.G.

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on November 9, 2019 by guest

Figure

FIG. 1. Diagram of the pCMVIA vector and the virus cDNA insert. SV 40Ori, simian virus 40 origin of replication; CMV Pro, CMV immediate-early
FIG. 5. ELISA for rotavirus-specific IgA in stool suspensions from mice thathad been orally inoculated (by gavage) with PLG-encapsulated VP6 DNA vac-

References

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