Immunology and Cell Biology

(2005)

83

, 257–262 doi:10.1111/j.1440-1711.2005.01335.x

© 2005 Australasian Society for Immunology Inc.

Special Feature

Oral hepatitis B vaccine candidates produced and delivered in plant material

S T E P H E N J S T R E A T F I E L D

Applied Biotechnology Institute, College Station, Texas, USA

Summary

Hepatitis B is a major global health problem; approximately two billion people are infected with thevirus worldwide, despite the fact that safe and efficacious vaccines have been developed and used for nearly20 years. Prohibitive costs for vaccine purchase and administration restrict uptake in many developing nations.Agencies such as the Global Alliance for Vaccination and Immunization are helping to make current vaccines moreavailable, but reduced costs would greatly aid this effort. Oral delivery is an option to reduce the expense ofadministering hepatitis B vaccines. It may also improve compliance, and orally delivered vaccines may be moreefficacious among poor responders to current vaccines. However, to induce protective efficacy, oral administrationmay require encapsulation of antigen and delivery of large doses. Plant-based expression systems offer an oraldelivery alternative with low production costs, and they also encapsulate the antigen. Some plant-based systems alsostabilize antigen and therefore reduce storage and distribution costs. The hepatitis B major surface antigen has beenexpressed in several plant systems. A variety of regulatory sequences and subcellular targets have been used toachieve expression suitable for early stage clinical trials. However, further increase in expression will be necessaryfor practical and efficacious products. Appropriate processing can yield palatable products with uniform antigenconcentration. The antigen expressed in plant systems shows extensive disulphide cross-linking and oligomerizationand forms virus-like particles. Oral delivery of the antigen in plant material can induce a serum antibody response,prime the immune system for a subsequent injection of antigen and give a boosted response to a prior injection.Small scale clinical trials in which the antigen has been delivered orally in edible plant material indicate safety andimmunogenicity.

Key words:

booster vaccine, edible vaccine, hepatitis B major surface antigen, hepatitis B vaccine, oral vaccine,plant-based vaccine.

Introduction

Vaccination programs greatly reduce mortality and morbidityrates. They are also a relatively inexpensive strategy forcombating infectious diseases. Set against the costs of diag-nosis and treatment, and additional costs incurred through lostproductivity of infected individuals, vaccination is clearlyhighly cost-effective. Despite this, many large at risk popula-tions are still not vaccinated against major infectious diseases.In some cases, such as HIV, an effective vaccine has not yetbeen developed, but in other cases, such as hepatitis B, aneffective and safe vaccine is available but costs of production,distribution and delivery have been prohibitive to widespreadadministration in many developing nations. Thus, cost con-straints result in preventable diseases not being properlytackled, and these diseases continue to inflict high levels ofmorbidity and mortality on large populations. Furthermore,incomplete market penetration of vaccines such as hepatitis Bcan act as a disincentive for developing new vaccines directedagainst infectious diseases affecting developing countries.Several governments and international agencies share the aimof ensuring much more widespread vaccination. For example,

the Global Alliance for Vaccines and Immunization (GAVI)helps to fund the purchase and administration of vaccines incountries where cost restricts their uptake.

The focus of this review is hepatitis B. Approximately twobillion people are infected with hepatitis B virus worldwide.Of these, approximately 350 million are chronically infected,and are thus at high risk of developing cirrhosis of the liverand liver cancer. These conditions lead to approximately onemillion deaths annually. Current vaccines comprise the viralmajor surface antigen produced in recombinant yeast. A courseof three injections confers more than 95% protection forinfants and more than 90% protection for adults. The vaccineshave an annual market in excess of US$1 billion. However,three factors limit their success, and hepatitis B looks set tocontinue as a major global health problem. First, the cost ofpurchasing and administering current vaccines is too high formany developing countries. Second, compliance is limited bythe inconvenience of needing trained personnel to administerinjections and the reluctance of many individuals to receiveinjections. Third, a small but significant proportion of individ-uals vaccinated are not protected by current vaccines. Thus,there are clear opportunities for new hepatitis B vaccines. Here,we discuss orally delivered plant-based hepatitis B vaccinecandidates developed to promote increased vaccination. Theoral delivery route may also serve to improve efficacy amongindividuals that respond relatively poorly to current vaccines.

Correspondence: Stephen J Streatfield, Applied BiotechnologyInstitute, 101 Gateway Boulevard, Suite 100, College Station, TX77845, USA. Email: sstreatfield@appliedbiotech.org

Received 20 February 2005; accepted 22 February 2005.

258

SJ Streatfield

The oral delivery option

Current hepatitis B vaccines are delivered parenterally. Thecost of producing material considered safe for injection andadministering this material using needles keeps these vaccinesout of reach of many target populations. Oral delivery over-comes some of the administration expenses incurred byinjections. The equipment and trained personnel costs fordelivery are greatly reduced. Also, with oral delivery, thereare no safety concerns over the potential reuse of needles, andfear of injections becomes irrelevant. This could be importantin improving compliance where there is a reluctance toreceive injections, and is relevant even in countries wherevaccine expense is not a major factor. Oral delivery alsodirects the antigen to the mucosal lining of the intestine,which can result in priming of other mucosal surfaces. Thismay lead to improved efficacy against pathogens that invadevia these sites, and is relevant to combating the sexualtransmission of hepatitis B.

Despite these advantages, oral delivery of protein subunitvaccines, such as those directed against hepatitis B, raise someconcerns. One problem is that antigen will be subject todigestion in the gastrointestinal tract before it can induce animmune response. Also, only a fraction of remaining undi-gested or partially digested material is likely to be appropriatelypresented at the intestinal epithelium to induce an immuneresponse. Thus, achieving protective efficacy through oraldelivery will probably require administrating much moreantigen than is typically given by injection and/or protecting itfrom degradation. Encapsulation of antigen or expression in amodified live vector can guard against digestion. However,encapsulation adds to production costs, partly off-setting thecost advantages of avoiding injections, while live deliveryvehicles raise safety concerns. Clearly, the delivery of muchgreater antigen doses also adds to production costs.

The major concern with oral delivery is probably that evenif a substantial amount of antigen can be delivered to the gutepithelium, a sufficient or appropriate immune response maynot be induced. While oral delivery may be appropriate tovaccinate against pathogens that invade via the mucosallining of the intestine (e.g.

Vibrio cholerae

), it is not soclearly suited to other pathogens (e.g. hepatitis B virus). Thisconcern can only be assessed by experimentation withspecific disease agents, but as discussed below for plant-based vaccines, initial data are promising.

Another potential drawback to oral delivery is the possibil-ity of inducing oral tolerance whereby oral delivery of anantigen would prevent systemic responses to a subsequentexposure to the same antigen. In this case, individuals notprotected by an oral hepatitis B vaccine might also fail torespond to a subsequent parenteral vaccine, even though thelatter may have been effective if delivered first. Thus, thepotential for an oral hepatitis B vaccine candidate to induceoral tolerance will need to be carefully assessed by trials.

Plant-based oral vaccines

Plant-based vaccines allow oral delivery of an antigen whileovercoming the cost concerns described above. Production ofrecombinant proteins in plants is inexpensive compared toother expression systems.

1,2

Even under greenhouse condi-tions, the capital equipment requirements for growing and

harvesting plants are fairly modest and overhead costs arerelatively low. Field production further reduces expenses,although measures to ensure containment add to costs typicalfor food or feed crops. Thus, large quantities of antigen canbe produced relatively inexpensively in recombinant planttissues. Furthermore, the antigen is encapsulated in the pro-duction vehicle. Encapsulation facilitates oral delivery byprotecting the antigen from proteolytic digestion. Edibletransgenic plant tissue expressing the antigen can be proc-essed into a readily consumed form. This is achieved usingfood processing technologies that are very inexpensive com-pared to the protein purification schemes more typical forsubunit vaccines. Furthermore, provided an appropriate planttissue is chosen for antigen expression, the processed materialmay be stable at ambient temperatures for years. This obvi-ates the need to maintain a cold chain during storage anddistribution, so further reducing costs. Also, because no livevectors are involved, and the plant tissues do not harbouranimal pathogens, plant-based vehicles are an inherently safeform of delivery.

Ideally, in developing a plant-based expression system foran oral hepatitis B vaccine, four criteria should be met. First,the major surface antigen must be sufficiently concentrated toallow for the recommended dose to be delivered in an amountof material that is easily consumed. The standard dose forparenterally delivered hepatitis B vaccines varies from 5 to40

µ

g, depending on the specific product, the age of therecipient and whether the recipient is immunocompromised.It is anticipated that oral doses will need to be at leastequivalent to parenteral doses, but may need to be up to threeorders of magnitude greater in order to achieve comparableefficacy. Also, for convenience of administration, the amountof material delivered orally as a vaccine should not exceed afew tens of grams. Thus, for a potential oral dose of 10 mg ofhepatitis B major surface antigen to be delivered in 10 g ofplant material, the recombinant protein must constitute 0.1%of the material administered. Protein extraction procedures toconcentrate the antigen from plant expression hosts are bestavoided, because they add significant cost. However, for freshtissue, some degree of dehydration is necessary to avoid rapidprotein degradation following harvest, and this also servesto concentrate the antigen on a weight basis. Also, someinexpensive food processing technologies applied to planttissues yield protein-rich fractions, and can thus serve toconcentrate antigens up to 10-fold. Thus, depending on theavailable processing options for the plant tissue chosen forantigen production, expression level targets should be at least0.01 to 0.1% weight.

A second criteria for a plant-based hepatitis B vaccine isthat the antigen concentration should be uniform, allowing foreven dosing of subjects. Some degree of processing willlikely be required to achieve homogenization. Third, the plantmaterial should be palatable, encouraging uptake. This rulesout several plant expression systems such as tobacco and

Arabidopsis thaliana

used to show the feasibility of express-ing antigens in plants. Finally, the fourth criterion that shouldbe met is that the antigen should be stable during prolongedstorage of the vaccine at ambient temperatures, so avoidingthe expense of a cold chain. Grains are particularly suitablein this regard, although fresh tissue may be dehydrated toguard against antigen degradation.

Plant-based hepatitis B oral vaccines

259

Expression of hepatitis B antigens in plant systems

Several research groups have expressed the hepatitis B majorsurface antigen in plant tissues. The main difference betweenthe strategies pursued by these groups is the plant tissuechosen for expression. The protein was first expressed intobacco leaves to demonstrate that expression in plant tissuesis feasible.

3

However, this is not a practical expression systemfor oral delivery. Expression levels are low, with the majorsurface antigen constituting less than 0.01% of total solubleprotein, and the tissue is not well suited to inexpensiveprocessing to an edible form. Also, fresh leaves expressingthe antigen must be processed immediately. The majorsurface antigen has also been expressed in lettuce leaves,

4

which are more suitable for oral delivery. However, as fortobacco, expression of the antigen is poor, being less than0.000001% of fresh weight, and again, the leaves requireimmediate processing. Leaf expression has not been opti-mized by applying an extensive array of molecular tools.However, the expression levels achieved in the above studiesare so low that leaf tissues are unlikely to be suitable forlarge-scale production and oral delivery of hepatitis B majorsurface antigen.

The major surface antigen has also been expressed intissues more practical for oral delivery. Potato tubers havereceived considerable attention. The hepatitis B major surfaceantigen has been expressed in potato tubers at levelsapproaching 0.002% of fresh weight.

5

To achieve this level ofexpression, a variety of untranslated leader sequences, poly-adenylation signals and organellar targeting sequences weretested. Varying the polyadenylation and targeting sequenceshad the greatest effect on expression. The native hepatitis Bviral signal sequence and a plant sequence directing theprotein to the cell surface both directed relatively high levelsof expression, as did the standard endoplasmic reticulumretention sequence and a plant vacuolar targeting sequence.However, a plant plastid target was unsuccessful in directingantigen accumulation. Although selected strategies raisedexpression, the highest expressing line, which expressedantigen at twofold to threefold the level of the next line, hadfour insertions of the expression vector. Lines with multipleinsertions are subject to silencing in later generations, andincrease the regulatory burden for characterization of pro-duction lines. Depending on the species and the means ofpropagation, they may also complicate plant breeding for linedevelopment. Furthermore, potato tubers cannot be inexpen-sively processed to give a protein rich fraction. Thus, expres-sion levels achieved in potatoes are still approximately twoorders of magnitude less than estimates of those required forpractical oral delivery. In addition, when assessed withanother recombinant protein, expression levels vary morethan fourfold between tubers.

6

Thus, before delivery, a formof processing must be used that will thoroughly mix thetransgenic material to give a homogeneous level of antigen.

The major surface antigen has also been expressed in fruittissues. Expression in cherry tomatillos has been reportedat 0.000001% of fresh weight

7

and low levels of expressionhave also been achieved in tomatoes. As with fresh leaves,an array of expression technologies have not yet been applied,but the starting level for expression is so low as to makethese tissues impractical for oral vaccines. Dehydration will

concentrate the antigen, but the fruits cannot be inexpensivelyprocessed to enrich it. Fresh fruits also have the disadvantagethat they must be processed immediately upon harvest toguard against antigen degradation. Furthermore, as with vege-table tubers, expression levels vary between fruits, soappropriate processing must be applied to ensure an evenconcentration of antigen.

Grains are a further option for the expression of candidatevaccine antigens. Corn in particular has been explored with aview to achieving expression levels of foreign proteins suit-able for commercial purposes. Some recombinant proteinsproduced in corn have entered the marketplace. A recentexample with the potential for large-scale industrial pro-duction is trypsin, which offers a nonanimal source alter-native for pharmaceutical processing.

8

Recombinant proteinhas been expressed in grain well in excess of 0.01% of seedweight,

9

and expression is fairly uniform among ears ofdeveloped production lines. Seed can be inexpensively proc-essed into a palatable form,

10

and processing can also serve toconcentrate antigen in the protein rich germ fraction.

11

Also,dry seed can stably store recombinant protein for at least ayear at ambient temperatures.

11

Thus, corn meets the fourcriteria given above for a successful expression system: highexpression, uniform expression, palatable product and stablerecombinant protein.

The hepatitis B major surface antigen has been expressedin corn seed by the research group at ProdiGene (CollegeStation, TX, USA) (Lamphear

et al.

, unpubl. data). Severalapproaches were used to raise expression, including the use ofa maize codon optimized gene, strong plant promoters, sub-cellular targeting sequences and selective breeding strategies.High expressing lines were identified with a single copy ofthe transgene integrated into the maize genome. Expressionlevels in single seeds harvested from first generation trans-genic plants exceeded 0.2% of total soluble protein. The antigenis largely concentrated in the germ fraction of the seed, whichis almost 20% protein. By milling seed to fractionate germ,the concentration of the major surface antigen has beenincreased fivefold. Bulk grain expressing the antigen hasbeen processed to yield germ with an antigen concentration ofapproximately 0.003% of weight. Thus, antigen levels havealready been achieved that are within approximately 30-foldof the higher estimates of what will be required for a practicaloral vaccine. Further line development and improved expres-sion vectors should close this gap to yield a very promisingvaccine candidate. In addition, the antigen is most concen-trated in the germ fraction. This fraction is palatable withoutthe need for further processing steps involving heat or pres-sure that might denature the recombinant protein. Also,preparation of germ from transgenic corn expressing a targetantigen has previously been shown to yield a product with auniform antigen concentration.

12

The expressed antigen wasstable at ambient temperatures for more than a year.

11

The major surface antigen of hepatitis B virus has alsobeen expressed in plant cell culture systems. The emphasishas been on using tissue cultures to analyze oligomerizationof the antigen rather than to achieve high levels of expression.Indeed, the cost advantages of plant-based vaccines for oraldelivery do not apply to tissue culture systems. However,plant tissue cultures could be used as an alternative to yeast toproduce antigen for purification. Expression levels have

260

SJ Streatfield

approached 0.007% of fresh weight in soybean culture, butonly approximately one-tenth of this in tobacco culture.

13

Higher expression in soybean culture probably reflects multi-ple copies of the transgene. In addition, expression in tobaccoculture declined through rounds of subculturing. However,tobacco cell suspension culture has been used to secrete themajor surface antigen into the culture medium.

14

The antigenaccumulated to 10

µ

g/L. Improvements of several orders ofmagnitude will be necessary for plant cell culture systemsto be competitive, particularly given the slow growth rates ofplant cells compared to yeast.

Current hepatitis B vaccines contain the major surfaceantigen, and therefore expression of this protein has been thefocus of most efforts with plants. However, inclusion offurther viral envelope proteins in a vaccine may serve toimprove efficacy among current poor responders. With this inmind, expression cassettes containing pre-S2/S gene chimerashave been used in potato to express part of the viral middleprotein along with the major surface antigen.

15,16

However,expression of the chimeric gene was from threefold to 10-foldlower than that of the major surface antigen.

Characterization of the major surface antigen in plant tissues

When isolated from human serum, the hepatitis B majorsurface antigen is assembled into 22 nm spherical particleswith the protein embedded into a phospholipid bilayer.Similar sized particles are formed when the antigen isexpressed in yeast, and such particles have also been observedwith whole plant expression.

3

The most detailed characteriza-tion studies in plants have been performed on the majorsurface antigen expressed in tissue cultures, where the pre-dominant form of the antigen is a dimer with disulphidecross-links.

13

The antigen accumulates in the endoplasmicreticulum in particles with a much wider size distribution thanthat seen in human serum. These particles have a tubularstructure

17

comparable to that seen when the major surfaceantigen is expressed in Chinese hamster ovary cells. Similartubular structures have been observed in potato tubersexpressing the antigen.

17

In both plant cell culture and potatotubers the antigen appears to be partially glycosylated.

17

Afurther expression cassette for production of the major surfaceantigen in plant tissue culture included sequence encoding aplant signal sequence. This signal was not cleaved from themajor surface antigen, and the fusion protein accumulated inthe endoplasmic reticulum.

18

The extent of disulphide bondformation and consequent oligomerization was increased overthat seen without the plant signal sequence. Extensive disul-phide cross-linking and oligomerization are important informing virus-like particles and in promoting immunogenicity.Indeed, the plant signal sequence–major surface antigen fusionprotein proved more immunogenic than the major surfaceantigen when the two plant cell culture-expressed proteins wereinjected into mice.

18

Thus, the form of the antigen observed inplant tissues holds promise for an effective vaccine.

Efficacy of plant-based hepatitis B vaccines

When extracted from transgenic tobacco leaves and injectedinto mice, the hepatitis B major surface antigen showed

comparable B- and T-cell responses to those observed withthe recombinant yeast protein.

19

These promising results withparenteral delivery prompted a series of further immuno-genicity studies with the major surface antigen expressed inplant tissues suitable for oral delivery. For hepatitis B vaccinecandidates, a serum antibody response to the major surfaceantigen exceeding 10 mIU/mL is indicative of protection inhumans. Feeding mice with three doses of approximately5

µ

g each of the major surface antigen expressed in potatotuber prompted a primary serum response peaking at approx-imately 70 mIU/mL approximately 3 weeks after the lastdose.

5

Subsequent injection with yeast-derived antigenresulted in an immediate high and prolonged boost in anti-body titre. In a further study, mice were injected with asubimmunogenic dose of yeast-derived antigen and weresubsequently fed three approximately 40

µ

g oral doses ofantigen expressed in potato tuber.

20

This also resulted in ahigh prolonged boost in antibody titre. However, all thesefeedings had 10

µ

g of cholera toxin adjuvant added to thepotato material, and no commercial vaccine will include thisadjuvant. In the absence of adjuvant, primary immunizationwith three approximately 40

µ

g oral doses of potato-expressedantigen did not induce a response and a subsequent injectionwith yeast-derived antigen gave a much more modest boostin antibody titre.

20

This emphasizes the fact that much higheroral doses of the major surface antigen are likely to benecessary for efficacious vaccines.

The effect on immunogenicity of including plant-expressedmiddle protein sequences has also been assessed. Mice werefed three doses of potato tubers expressing either middleprotein sequences plus major surface antigen, or majorsurface antigen alone. Again, cholera toxin was included asan adjuvant. They were subsequently injected with yeast-derived major surface antigen. Including middle proteinsequences resulted in a significantly higher antibody titrefollowing the booster injection.

16

Ultimately, includingsequences in addition to the major surface antigen may beuseful in producing a more efficacious oral vaccine.

Clinical trials with plant-based hepatitis B vaccines

Two plant expression systems have taken an oral hepatitis Bvaccine candidate into small-scale clinical trials without theuse of adjuvant. Lettuce leaves expressing the major surfaceantigen were administered in two oral doses to three humanvolunteers.

4

These individuals had no history of vaccinationagainst hepatitis B or hepatitis B infection. Oral dosescontained no more than 1

µ

g of antigen. All volunteersresponded, with two of them having serum responses inexcess of the benchmark level of 10 mIU/mL. This is surpris-ing, given the low dose level. However, antibody levelsdeclined rapidly. No ill effects were recorded.

In a second larger and recently published study, 33 humanvolunteers who had been previously vaccinated against hepa-titis B and had shown responses in excess of 10 mIU/mL werefed two or three doses of approximately 100 g of raw potatotubers containing approximately 1 mg of the major surfaceantigen per dose.

21

More than half of the subjects showedincreased antibody titres. The transgenic potato tubers weresimilarly tolerated to non-transgenic control tubers.

Plant-based hepatitis B oral vaccines

261

Towards a practical plant-based vaccine

Progress towards a practical plant-based hepatitis B vaccinecan be judged against the four criteria laid out above. Theseare to achieve high expression, develop a product withuniform antigen concentration, prepare a palatable formula-tion for delivery, and have a product that can be stored atambient temperatures for extended periods. Clearly, theproduct must also be efficacious and safe.

With regard to expression levels, two approaches showpromise: vegetable tubers and grains. Effort has been placedon raising expression in these systems, and they are now suchas to allow larger scale clinical trials. However, animalstudies and early clinical trials indicate that at current expres-sion levels, an adjuvant may need to be administered alongwith the vaccine candidate to achieve widespread protectiveefficacy. Thus, significant further increases in expression arelikely to be necessary for a practical vaccine. Current expres-sion levels in corn seed exceed those in potato tubers and cornis more amenable to further increases in expression; forexample, by breeding into lines with high protein content.

An alternative strategy that has not yet been reported for themajor surface antigen is to express the protein in leaf tissuesfrom the chloroplast genome. High levels of recombinantprotein expression have been achieved in tobacco chloro-plasts,

22

but for a practical oral vaccine, the major surfaceantigen should be expressed in edible leaves. At present,species with edible leaves are not amenable to chloroplasttransformation. A further option for leaf expression is to use aplant viral expression system in which leaf tissues are infectedwith a recombinant plant virus encoding the foreign protein.Although mostly applied to tobacco, this approach has alsobeen used with species that have edible leaves, includingspinach.

23

However, plant viral systems have not yet givenparticularly high levels of expression and are most suitable forthe expression of peptides and small proteins.

In general, fresh tissues do not make practical oral deliveryvehicles, because the expressed antigen is neither evenlyexpressed nor stable for extended periods. However, appro-priately processed foodstuffs have a uniform and stableantigen concentration. Processing of potato tubers to a palata-ble form requires the application of heat, but boiling of tubersexpressing the major surface antigen leads to a loss ofantigenic activity

24

and greatly reduces immunogenicity.

20

In contrast, milling of corn grain expressing the major surfaceantigen to yield the palatable germ fraction concentrates theantigen. Alternatively, as shown with another heat sensitiveantigen, corn grain can stabilize a recombinant protein suchthat heat treatments can be applied to prepare a palatableproduct without denaturing the expressed protein.

10

Thus, forreasons of the expression level achieved, available approachesto further improve expression, and the potential for process-ing technologies to yield palatable products with an even andstable concentration of antigen, corn appears to be a preferredsystem for production of an oral hepatitis B vaccine. Grainsare also relatively inexpensive to produce and process topalatable forms.

In developing nations, the cost of the parenterally admin-istered vaccine has recently reduced from approximatelyUS$20 per paediatric dose to between US$0.25 and US$0.50.This reflects the efforts of organizations such as GAVI to

ensure wider vaccination. However, a plant-based hepatitis Boral vaccine would still be very cost-effective. For example,in the case of corn, with a current expression level in grain of0.0006% of seed weight, a standard 10

µ

g paediatric dosecould be delivered in approximately 1.5 g of corn materialand could cost 0.1 cents. This assumes growing costs ofUS$1000 per acre, a yield of 3000 kg per acre, and food-based processing costs equivalent to raw material costs. Anoral dose may need to be up to 1000-fold greater than this, butline development through breeding and improved approachesto expression should increase the level of the antigen in grainat least an order of magnitude. Also, preparation of a germfraction boosts antigen concentration fivefold. Thus, a 10 mgoral dose could still be delivered in approximately 30 g ofprocessed plant material for approximately 10 cents.

The standard parenterally delivered hepatitis B vaccine istypically administered to children in a series of three injections.Increasingly, the hepatitis B vaccine is one component in amultivalent vaccine. Thus, it may be impractical to initiallypursue a stand alone alternative for widespread administrationof a hepatitis B vaccine. However, booster treatments toparticular target groups are typically administered separately.A booster vaccine could initially target poor responders or atrisk groups, such as homosexual men or health workers.Successful application in this context may ultimately lead tooral administration replacing primary injection.

References

1 Daniell H, Streatfield SJ, Wycoff K. Medical molecular farming:production of antibodies, biopharmaceuticals and edible vac-cines in plants.

Trends Plant Sci.

2001;

6

: 219–26.2 Hood EE, Woodard SL, Horn ME. Monoclonal antibody manu-

facturing in transgenic plants – myths and realities.

Curr. Opin.Biotechnol.

2002;

13

: 630–35.3 Mason HS, Lam DM, Arntzen CJ. Expression of hepatitis B

surface antigen in transgenic plants.

Proc. Natl Acad. Sci. USA

1992;

89

: 11 745–9.4 Kapusta J, Modelska A, Figlerowicz M

et al.

A plant-derivededible vaccine against hepatitis B virus.

FASEB J.

1999;

13

:1796–9.

5 Richter LJ, Thanavala Y, Arntzen CJ, Mason HS. Production ofhepatitis B surface antigen in transgenic plants for oral immuni-zation.

Nat. Biotechnol.

2000;

18

: 1167–71.6 Tacket CO, Mason HS, Losonsky G, Clements JD, Levine MM,

Arntzen CJ. Immunogenicity in humans of a recombinant bac-terial antigen delivered in a transgenic potato.

Nat. Med.

1998;

4

:607–609.

7 Gao Y, Ma Y, Li M

et al.

Oral immunization of animals withtransgenic cherry tomatillo expressing HBsAg.

World J. Gastro-enterol.

2003;

9

: 996–1002.8 Woodard SL, Mayor JM, Bailey MR

et al.

Maize (

Zea mays

)-derived bovine trypsin: characterization of the first large-scale,commercial protein product from transgenic plants.

Biotechnol.Appl. Biochem.

2003;

38

: 123–30.9 Hood EE, Witcher DR, Maddock S

et al.

Commercial productionof avidin from transgenic maize: characterization of transform-ant, production, processing, extraction and purification.

Mol.Breed.

1997;

3

: 291–306.10 Streatfield SJ, Mayor JM, Barker DK

et al.

2000 congresssymposium on molecular farming: development of an ediblesubunit vaccine in corn against enterotoxigenic strains of

Escherichia coli

.

In Vitro Cell. Dev. Biol. Plant

2002;

38

: 11–17.

262

SJ Streatfield

11 Lamphear BJ, Streatfield SJ, Jilka JM

et al.

Delivery of subunitvaccines in maize seed.

J. Control. Release

2002;

85

: 169–80.12 Streatfield SJ, Lane JR, Brooks CA

et al.

Corn as a productionsystem for human and animal vaccines.

Vaccine

2003;

21

:812–15.

13 Smith ML, Mason HS, Shuler ML. Hepatitis B surface antigen(HBsAg) expression in plant cell culture: kinetics of antigenaccumulation in batch culture and its intracellular form.

Biotech-nol. Bioeng.

2002;

80

: 812–22.14 Kumar GBS, Ganapathi TR, Revathi CJ, Prasad KSN, Bapat VA.

Expression of hepatitis B surface antigen in tobacco cell suspen-sion cultures.

Protein Expr. Purif.

2003;

32

: 10–17.15 Ehsani P, Khabiri A, Domansky NN. Polypeptides of hepatitis B

surface antigen produced in transgenic potato.

Gene

1997;

190

:107–11.

16 Joung YH, Youm JW, Jeon JH

et al.

Expression of the hepatitisB surface S and preS2 antigens in tubers of

Solanum tuberosum

.

Plant Cell Report

2004;

22

: 925–30.17 Smith ML, Richter L, Arntzen CJ, Shuler ML, Mason HS.

Structural characterization of plant-derived hepatitis B surfaceantigen employed in oral immunization studies.

Vaccine

2003;

21

: 4011–21.18 Sojikul P, Buehner N, Mason HS. A plant signal peptide-

hepatitis B surface antigen fusion protein with enhanced stability

and immunogenicity expressed in plant cells.

Proc. Natl Acad.Sci. USA

2003;

100

: 2209–14.19 Thanavala Y, Yang Y-F, Lyons P, Mason HS, Arntzen C. Immuno-

genicity of transgenic plant-derived hepatitis B surface antigen.

Proc. Natl Acad. Sci. USA

1995;

92

: 3358–61.20 Kong Q, Richter L, Yang YF, Arntzen CJ, Mason HS, Thanavala Y.

Oral immunization with hepatitis B surface antigen expressed intransgenic plants.

Proc. Natl Acad. Sci. USA

2001;

98

: 11539–44.

21 Thanavala Y, Mahoney M, Pal S

et al.

Immunogenicity inhumans of an edible vaccine for hepatitis B.

Proc. Natl Acad.Sci. USA

2005;

102

: 3378–82.22 Daniell H, Lee S-B, Panchal T, Wiebe PO. Expression of the

native cholera toxin B subunit gene and assembly of functionaloligomers in transgenic tobacco chloroplasts.

J. Mol. Biol.

2001;

311

: 1001–1009.23 Yusibov V, Hooper DC, Spitsin SV

et al.

Expression in plantsand immunogenicity of plant virus-based experimental rabiesvaccine.

Vaccine

2002;

20

: 3155–64.24 Dogan B, Mason HS, Richter L, Hunter JB, Shuler ML. Process

options in hepatitis B surface antigen extraction from transgenicpotato.

Biotechnol. Prog.

2000;

16

: 435–41.