US scientists have developed an experimental

vaccine against the killer bug Escherichia coliO157. Although further clinical trials will be re-

quired before the vaccine can be declared pro-

tective in humans, the scientists are also look-

ing at its potential for mass vaccination of

cattle to eliminate the pathogen at source.

Food poisoning by this bacterium has been the

major international ‘food scare’ story in recent

months, and there have been several instances.

• The worst single outbreak occurred in

Scotland where, by January 1998, 18 elderly

people had died and approximately 500

others had been taken ill

• In the summer of 1996, 10,000 Japanese

were affected and there were 12 deaths

• There have been an estimated 20,000 cases

in the USA, with 250 associated deaths

E. coli O157 particularly affects the young

and the elderly. The most critical effects are

haemorrhagic colitis (resulting in bloody diar-

rhoea) and haemolytic uraemic syndrome,

which can result in kidney damage and, poss-

ibly, kidney failure. The bacterium lives in cattle

and other domesticated species without caus-

ing symptoms; contamination of meat during

butchering is considered the primary source of

the pathogen, although cross contamination

from animal faeces is also possible.

Conjugation processTo make an effective vaccine, scientists from

the National Institutes of Health (NIH), National

Institute of Child Health and Human

Development (Bethesda, MD, USA) chemically

linked (conjugated) a bacterial-surface-polysac-

charide antigen (the zero-specific polysacchar-

ide of lipopolysaccharide) from the bacterium

to the genetically inactivated protein toxin from

another bacterium, Pseudomonas aeruginosa.

This conjugation process boosts the efficacy of

the polysaccharide as a vaccine, and the same

group of scientists applied the principle to

eradicating Haemophilus influenzae type b,

which causes meningitis in children.

Immunological responseDr Szu Shousun and colleagues at the NIH and

the Carolinas Medical Center (NC, USA) found

that volunteers vaccinated in a clinical trial were

able to produce enough antibody to E. coli O157

to kill the microorganism in vitro. More than 80%

of 87 volunteers responded with a fourfold in-

crease in immunoglobulin G against this antigen

one week after vaccination. Significantly, all vol-

unteers had responded with the same increase

after four weeks and the team found that the im-

munological response was sustained for at least

six months. The only side effect was a mild skin

reaction (erythema) at the injection site in some

volunteers. The researchers report their results in

more detail in the February issue of the Journal ofInfectious Diseases1.

The researchers point out that zero-specific

polysaccharide cannot induce an antibody re-

sponse in humans. Proteins, on the other hand,

are effective antigens at all ages, which is why,

the team says, conjugating polysaccharides to

proteins is such an important advance in mak-

ing vaccines. The immune system of recipients

of the conjugate vaccine makes antibodies

against the bacterial-surface polysaccharide as

if it were a protein.

Supplanting antibiotic therapyE. coli O157 has been found to be a very persis-

tent microorganism, surviving in animal faeces

and even on dry surfaces for periods of months.

Patients with haemolytic uraemic syndrome do

not respond well to antibiotic treatment, so

vaccination could be very important for those

at risk and in the control of an outbreak. Indeed,

the researchers point out that antibiotics may

actually increase symptoms by releasing toxins

as the bacteria are lysed.

The researchers add that they are soon to

move on from this Phase I study to Phase II in

children.

Reference01 Shouson, S. et al. (1998) J. Infect. Dis. 177,

383–387

PSTT Vol. 1, No. 2 May 1998 update news

47

Sweet vaccine against E. coli O157David Bradley, tel/fax: +44 1954 202218, Web: http://www.camsoft.com/elemental/

Axis Genetics recently announced details of li-

censing agreements signed with US agribusi-

ness and biotechnology company Mycogen

Corporation, for 11 families of patents. The

agreements grant Axis a nonexclusive licence

for human health applications of technology to

alter plants genetically to produce vaccines for

oral delivery. Axis will pay annual licence fees

and royalties upon commercialization of edible

vaccine products.

Eight out of the 11 patents cover the use of

viral subgenomic promoters, transfer vectors,

hybrid RNA viruses and methods of inserting

viral DNA into plant material, and the viral

engineering patents allow Axis to proceed in

the development of vaccines for cancers, auto-

immunity, allergies and infectious diseases for

either human or animal use.

Oral vaccines from plantsAdrian Smith, Pharmaceutical Science & Technology Today, tel: +44 1223 315961, fax: +44 1223 464430, e-mail: pstt@elsevier.co.uk

Copyright ©1998 Elsevier Science Ltd. All rights reserved. 1461-5347/98/$19.00

Focus on oral routeAxis Genetics, based in Cambridge, UK, focuses

on the research, development and production

of a new generation of recombinant vaccines

from plants, the company’s stated aim being to

‘develop and commercialize a range of innova-

tive efficacious vaccines from plants for human

use’, based on a broad level of patent coverage.

The company has identified the oral route to be

highly appropriate in the prevention of some

enteric diseases. The company claims that this

recent agreement will facilitate its efforts to

develop a new generation of highly effective,

orally administered plant-based vaccines, which

will stimulate the immune system via the

mucosal route more conveniently and effec-

tively than by the more conventional method of

injected vaccine delivery.

‘One of the things that the vaccine industry

hasn’t been very good at historically,' says Dr Iain

Cubitt, Axis’ Chief Executive Officer, ‘is that

they’ve got most vaccines in injectable forms so

they miss a lot of the body’s immune system –

the mucosal immune system, which of course is

the normal entry route of pathogens into the

body, through the mouth, the lungs, genital

tract and gums, etc. So, if you deliver nasally,

you stimulate mucosal immunity. Companies are

now producing nasally-applied influenza vac-

cines and that, to me, is extremely logical. Why

inject something if you want a mucosal re-

sponse? Oral delivery can, if you use the right

proteins, stimulate a mucosal response.'

Core technologiesThe company takes three approaches to the de-

velopment of recombinant vaccines in plants.

• Genetic modification of plant viruses to pro-

duce chimaeric virus particles (CVPs) which

present biologically-active polypeptides on

their surface. According to Axis, such CVPs

can be produced in growing plants, and then

extracted and administered either nasally or

by injection to patients in order to produce

the correct immune response.

• Genetic modification of plants to form

transgenic plants that can be used as oral

vaccines (including direct ingestion of the

plant material).

• Virus-assisted expression (VAX Technology),

which is covered by the patents licensed

from the Mycogen agreement. This ap-

proach promotes a high level of expression

of immunologically-active proteins in

plants. The proteins can then be extracted

and purified for injection or nasal appli-

cation, or delivered orally in edible plant

material without a need for purification.

To source appropriate plant material, Axis

works with either academic or commercial col-

laborators on particular targets, selecting the

peptide or protein for the proposed study. The

company obtain the protein sequence from the

collaborator, synthesize the appropriate DNA

sequence and splice it into the company’s sys-

tem. The live virus particles will not grow in

animals and are killed before use as a vaccine.

Significance of new dealCubitt underlines the importance of the

Mycogen agreement in the future work of the

company and believes that the deal opens up

considerably the whole area to Axis. ‘This, com-

bined with the intellectual property we already

own, now gives us the freedom to use several

approaches in our development programme. We

are well advanced on the plant viral route but

now add to this the expression of specific im-

munogenic proteins in plants, the ability to de-

liver large oral doses and the commercial reality

of marketing plant vaccines moves far closer.'

These developments at Axis come at a time

when the pharmaceutical industry is looking for

new therapeutic solutions, preferably with low

production costs. As Cubitt explains, ‘Plants are

fairly alien to the pharmaceutical industry; they’ve

been used for extracting secondary metabolites

but not proteins. The plant is a much more sophis-

ticated chemist than man. What we are doing is

update news PSTT Vol. 1, No. 2 May 1998

48

The cowpea plant (left). When a plant has beensuccessfully inoculated the leaves take on a mottledeffect (right).

Dr Iain Cubitt, Chief Executive Officer, Axis Genetics.

DNA is introduced into the plant by directapplication. Material from the affected plant canthen be extracted to act as stock for reinoculationof plants and further production.

The second IBC conference on strategies and

technologies for drug delivery systems took place

in January in London (UK). Keith Horspool

(Zeneca Pharmaceuticals, Alderley Edge, UK)

opened a session on strategy and licensing by

emphasizing how the nature of pharmaceutical

R&D is rapidly changing in the light of advancing

technologies, and he briefly reviewed examples

such as genome mapping, pharmacogenomics,

combinatorial chemistry, high-throughput

screening and cassette dosing. Horspool affirmed

that, in this highly competitive market, the

timescale for the development of a drug delivery

technology must be dramatically cut and, al-

though the cost of increased productivity will be

high, the associated benefits will provide suitable

compensation. Horspool also suggested that the

major reasons why some new products are fail-

ing to reach the market include a lack of efficacy

and pharmacokinetics problems.

Mike Dey (Rhône-Poulenc Rorer, UK) ex-

plained how to formulate a drug delivery strat-

egy that will ensure technical feasibility and

marketability. He emphasized that the perfor-

mance-to-cost ratio and early market entry are

major goals in the development of a drug deliv-

ery system and that many companies do not

possess the additional technical skills required

to achieve these goals ‘in house'. The alternative

is to employ external experts through arrange-

ments such as partnerships, mergers and con-

sultancy agreements.

Keith Sugden (Reckitt & Coleman, Hull, UK)

provided a comprehensive guide on successfully

negotiating a licensing agreement. It is vital to

have a sound knowledge of the product to be

sold and to know what the corporate aims are

and what is needed as a minimum requirement.

Marketable qualities, such as cost, innovation, ex-

clusivity, geography and speed-to-market, must

be clearly defined. Expectations of the licensing

deal need to be managed realistically with regard

to profit share and the timescale of the deal.

Delivery of proteinsJill Ogden (Andaris, Nottingham, UK) presented

an informative review of the modes of delivery

for peptides and proteins, and the opportunities

that exist for using alternative technologies.

She predicted that, in the next few years, the

growth rate for the number of peptide and pro-

tein drugs on the market will show a significant

increase, citing Epoetin a (Epogen), for

anaemia-associated diseases, and interferon a

(Intron A) as examples of drugs that have ex-

perienced dramatic increases (>20%) in global

sales in recent years. Almost all current peptide

and protein drugs are injectable, and there is

great commercial potential for delivery systems

that utilize alternative routes. Ogden discussed

major issues in pulmonary protein delivery, in-

cluding the potential for systemic therapies,

and she outlined the principle of using spray

dried macromolecules. The industry is also fo-

cused on making extensions to the market life

of a product; improvements in the delivery

regimes can provide this competitive edge.

Michael Flynn (Cortecs, London, UK) discussed

the potential for the oral delivery of peptides

and proteins using lipid systems, and he de-

scribed two proprietary oral calcitonin prepara-

tions, Macritonin™ and Macrulin™, in detail.

Flynn explained that successful oral delivery of

calcitonin and insulin has been demonstrated in

animals, healthy human volunteers and patients

using these systems. Multiple-dose studies of

oral Macritonin have demonstrated its thera-

peutic efficacy, and regulatory filing has now

commenced. (Macrulin will enter multiple-dose

Phase II studies during 1998.)

Coos Verhoef (Leiden/Amsterdam Center for

Drug Research, The Netherlands) discussed use

of absorption enhancers for peptide-drug deliv-

ery. He concentrated on the nasal route as a vi-

able alternative for drugs that are poorly ab-

sorbed after oral administration and explained

that nasally administered peptides are trans-

ported across the epithelium mainly by the

paracellular route. He also provided evidence

that such administration does not hamper the

protective ciliary activity of the mucous epi-

thelium. Strategies for improving the nasal

delivery of peptides and proteins include:

• Synthesis of more stable lipophilic analogues

• Use of peptidase and protease inhibitors

• Application of absorption enhancers

• Use of different formulation approaches,

such as sprays, drops and viscous agents

Verhoef proceeded to describe clinical studies

for the nasal delivery of estradiol using

PSTT Vol. 1, No. 2 May 1998 update conferences

Copyright ©1998 Elsevier Science Ltd. All rights reserved. 1461-5347/98/$19.00. PII: S1461-5347(98)00020-0 49

alien to a lot of the traditional chemists, but we

are just using the plant to work for us.'

Pipeline statusCurrently, three companies have plant proteins

in clinical trials. One of these is a transgenic

plant material, which is in Phase I clinical trials,

and is a traveller’s diarrhoea vaccine using E.coli proteins. It entered trials in the USA on the

basis that it was a plant product (potato), which

is in the diet, and the active protein from E. coliis well characterized. As such, the FDA was able

to recognize this as a low risk product.

At present there is more production of phar-

maceutical products from plants in the USA

than in Europe, where it remains relatively low

profile. Cubitt intends to ensure that the efforts

of Axis change this situation.

Drug delivery: strategies and technologiesKaty Fletcher, Department of Pharmacy, University of Brighton, Moulsecoomb, Brighton, UK BN2 4GJtel: +44 1273 642089, fax +44 1273 679333, e-mail: k.fletcher@brighton.ac.uk