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The Search for Vaccines Against Helicobacter pylori

P. Monath, MD, K. Lee, PhD, H. Ermak, PhD, Gwendolyn A.

Myers, DVM,

A. Weltzin, PhD, J. sca, PhD, D. , Jr., PhD, Gopalan

Soman, PhD, Hitesh Bhagat,

PhD, A. Ackerman, MD, Harry K. Kleanthous, PhD

[infect Med 15(8):534-535,539-546, 1998. © 1998 SCP Communications, Inc.]

Abstract

Current antibiotic regimens against Helicobacter pylori are effective, but

complex dosing and development of resistance are

concerns. Animal studies and limited clinical trials of H pylori urease and

other bacterial antigens have been conducted, with

promising findings. [infect Med 15(8):534-535,539-546, 1998]

Introduction

One of the most promising recent developments in medicine is the concept that

chronic afflictions, such as peptic ulcer

disease and cancer can be controlled through immunization like classic

infectious diseases. Research on vaccines against

Helicobacter pylori-the leading cause of chronic gastritis and peptic ulcer

disease and a primary risk factor for gastric

adenocarcinoma-began in 1990. The favored approach has been the oral

administration of purified recombinant subunit

proteins of H pylori and a mucosal adjuvant, the labile toxin (LT) of

Escherichia coli. As a single-component vaccine, the

urease protein has shown remarkable prophylactic and therapeutic activity in

animal models and partial therapeutic activity in

humans. A number of other H pylori antigens have been effective in animal

models, and the recent sequencing of the

complete H pylori genome has led to an intensive effort in antigen discovery.

Other research is directed at the comparison of

adjuvants and vaccine delivery systems and toward the immunologic mechanisms

mediating protection. Here, we present

preclinical data, the results of early-stage clinical trials, and directions for

future research on Helicobacter vaccines.

Helicobacter pylori: Medical Impact

A gram-negative spiral bacterium that specifically infects the stomach, H pylori

(Fig. 1) is one of the most prevalent

infections of humankind: Approximately 50% of adults in the industrialized world

and more than 90% of inhabitants of

developing countries are infected.[1] H pylori is thought to be acquired by

person-to-person spread via the fecal-oral and

oral-oral routes, and in some areas it may be waterborne.[2] After oral

ingestion, the bacteria colonize gastric mucus in close

association with gastric epithelial cells (Figs. 2,3). Infection is chronic and

generally lifelong.

In the US, approximately 2.5 million new H pylori infections occur each year. In

Europe, the prevalence and incidence of H

pylori-associated diseases are similar to or higher than those in the US. In

industrialized countries, the incidence of infection

is decreasing overall, although transmission varies with socioeconomic status,

and subpopulations are thus differentially

affected.

H pylori is the cause of chronic gastritis and the vast majority of cases of

peptic ulcer disease.[3-6] Conclusive evidence also

exists for an etiologic role of H pylori infection in dysplasia and metaplasia

of gastric mucosa, distal gastric adenocarcinoma,

and non-Hodgkin's lymphoma of the stomach,[7-12] leading the World Health

Organization to classify the bacteria as a Class I

(definite) carcinogen.[13] Considered in terms of lifetime morbidity, the

illness to infection ratio in the US and Europe may be

estimated at 1:5 for peptic ulceration and 1:200 for gastric adenocarcinoma.

Peptic Ulcer

In the US, approximately 16,000 deaths are attributed annually to complications

of peptic ulcer disease. There are more

than 2 million physician visits per year for duodenal ulcers, 90% of which are

attributable to H pylori, and more than 3

million physician visits per year for gastric ulcers, 60% of which are

attributable to the bacterium.[1,14] In a prospective study,

the risk of developing duodenal ulcer disease in H pylori-infected patients

followed for 10 years exceeded 10%; in contrast,

it was less than 1% in uninfected patients.[15]

Gastric Adenocarcinoma

The incidence of gastric adenocarcinoma in the US is approximately 24,000 cases

per year, with 13,300 deaths,

approximately 60% of which (14,400 cases; 7980 deaths) may be attributed to H

pylori. The risk of developing gastric

cancer is estimated to be 3- to 6-fold higher in infected than in uninfected

individuals.[7,12,16] Gastric cancer is a leading cause

of death in Latin America and Asia. Acquisition of H pylori infection early in

life appears to be associated with early-onset

gastric corpus atrophy and metaplasia and a higher risk of cancer.[17] Ingestion

of dietary carcinogens and deficiencies in

dietary antioxidants are thought to be important cofactors in the genesis of

Helicobacter-related cancer. Helicobacter strain

differences in virulence factors also appear to determine cancer risk.[18]

Rationale for Vaccine Development

Several lines of evidence provide a rationale for the development of a vaccine

against H pylori.

High Illness to Infection Ratio

Compared with illness to infection ratios of other infectious diseases, that of

H pylori-associated peptic ulcer is high (1:5). In

comparison, such ratios are about 1:25 for hepatitis B-associated chronic liver

disease and about 1:10 for Mycobacterium

tuberculosis. Although the ratio for H pylori-associated gastric adenocarcinoma

is lower (1:200), the morbidity and

mortality associated with this disease are substantial.

Vaccines for Prevention

Vaccines have long been regarded as the most effective and economical approach

to the prevention and control of infectious

diseases. Although effective antimicrobial treatment for H pylori is now

employed widely to prevent recurrence in patients

with active or recent duodenal ulcer disease, the ability to treat does not

obviate the need for preventive strategies. Indeed,

most H pylori infections leading to gastric cancer and 20% to 30% of cases of

upper gastrointestinal hemorrhage occur in

individuals who have sustained long-term infections without antecedent symptoms.

For this reason, these individuals do not

present to the physician in time for antimicrobial intervention.

Vaccines as Therapy or Complement

Although the application of vaccines for therapy of infectious diseases is in

its infancy, it has tremendous implications for the

management of chronic infections, such as H pylori, HIV, human papillomavirus,

viral hepatitis, herpesviruses, chlamydia,

and a wide range of parasitic infections.

From a practical perspective, the effectiveness of conventional antimicrobials

has diminished interest in therapeutic vaccines.

However, vaccines used in combination with antibiotics could improve the rate of

treatment success and decrease the

evolution of antimicrobial resistance and disease recurrence. Infection-induced

immunity to H pylori is clearly insufficient to

prevent reinfection, as shown by experiments in animals[19] and limited studies

of humans.[20] In areas of the world with high

rates of transmission of H pylori, re-infection may occur rapidly after

treatment with antimicrobial agents. However, in

industrialized nations, re-infection rates in adults appear to be low, both

overall (0.5%-2%) and in high-risk individuals

(2.5% in spouses of infected persons).[21] However, even in industrialized

nations, re-infection rates in children may be

substantially higher; in 1 study, Oderda and colleagues[22] reported that 18% of

children became re-infected within 18

months of antibiotic therapy.

Convenient Identification ofH pylori

A wide array of simple office-based serologic screening tests and the

noninvasive[13]C-urea breath test are now available for

identifying infected individuals, and new serologic tests that identify H pylori

strains characterized by a higher virulence

phenotype, especially CagA, are under development.[23] These methods could be

used to identify persons with H pylori

gastritis during the first 2 decades of life, thus identifying a population at

future risk of ulcer disease and cancer. If treatment

of the infection is considered, coadministration of a vaccine to prevent

re-infection will be an important component of such a

strategy.

Cost-effectiveness ofH pylori Vaccines

Although there is convincing evidence for the cost-effectiveness of curing H

pylori in patients with duodenal ulcer disease,[24]

the pharmacoeconomics of prophylactic immunization-whether primary

(pre-exposure) immunization or immunization to

prevent re-infection-have not been well defined. Pre-exposure immunization

requires application during infancy or

childhood, depending on age of acquisition of infection in the population at

risk. Since the indication for H pylori vaccination

is the prevention of chronic diseases that occur in the third to the sixth

decade of life, the cost-benefit ratio is influenced by

heavy discounting of future cost savings from disease prevention. However,

childhood immunization to prevent chronic

disease acquired decades later is not without precedent and underlies the

recommendation for universal immunization against

hepatitis B,[25] a disease that causes considerably less cancer morbidity and

mortality than H pylori.[26] In areas where gastric

cancer is a leading cause of death, such as Latin America and Asia, individuals

and society place a high value on investments

that reduce the incidence of this incurable and fatal disease. The World Health

Organization estimates that 550,000 gastric

cancer deaths due to H pylori occur annually,[13] and these deaths must be

considered potentially preventable through

immunization. By way of comparison, 316,000 cases of hepatocellular carcinoma

caused by hepatitis B occur annually, and

many countries are implementing routine childhood immunization policies.

Initial Immunization Trials

Serious consideration of vaccination as a means to control peptic ulcer disease

began around 1990. Pallen and Clayton[27]

suggested that urease would be a candidate antigen for incorporation in an H

pylori vaccine, based in part on findings in

animals and humans immunized with jack-bean urease to suppress ammonia

production in the intestine by ureolytic bacteria.

Czinn and Nedrud[28] showed that H pylori whole-cell sonicates administered

intragastrically to mice and ferrets elicited

serum and intestinal immunoglobulin (Ig) G and IgA antibodies. Subsequent

studies by Chen and coworkers[29,30] and Czinn

and colleagues[31] demonstrated that mice orally immunized with Helicobacter

sonicates or whole cells and cholera toxin

(CT) adjuvant were protected against challenge with Helicobacter felis, a

species capable of infecting murine gastric

mucosa. In addition, passive protection against challenge was demonstrated by

the oral administration of an IgA monoclonal

antibody, suggesting that the principal mediator of protection after active

immunization may be secretory IgA. The protective

monoclonal antibody later was shown to be specific for Helicobacter urease.[32]

In 1994, Michetti and others[33] demonstrated that mice orally immunized with

recombinant H pylori urease were protected

against challenge with H felis. Protective determinants were present on both

subunits (UreA and UreB) of the recombinant

multimeric urease molecule. The recombinant protein is similar to native urease

in multimeric structure, molecular mass

(550kDa), and nano-particulate morphology.[34] The UreB subunit truncated at the

amino terminus,[35] and multimeric urease

that had been aggregated or heat-denatured retained prophylactic activity

(OraVax, unpublished data, 1997). These studies

clearly demonstrated that urease is remarkable among bacterial proteins in its

stability and immunogenicity.

A large body of data has now been accumulated from several laboratories

confirming that H pylori urease administered

mucosally to a variety of animals confers protection against

challenge.[34,36,37] While initial immunization studies utilized H felis

as the challenge bacterium, the subsequent development of mouse models of H

pylori infection led to the confirmation that

urease protected against the human pathogen.[38-40]

In 1994, Doidge and colleagues[41] reported that mice with subchronic H felis

infection cleared or had reduced infection

after oral immunization with H felis whole-cell sonicates. Urease administered

orally to mice experimentally infected with H

felis[42] or ferrets naturally infected with Helicobacter mustelae[43] was shown

to have significant therapeutic activity. These

studies indicated that the up regulation of immunity to specific H pylori

antigens may result in clearance of chronic infection.

The role of mucosal immunity in protection against H pylori in humans is also

supported by a study of infants in West Africa,

where infection usually occurs within the first year of life. Infants of mothers

with high titers of anti-Helicobacter IgA in

breast milk had a significant delay in acquisition of H pylori infection.[44]

Subsequent studies indicate that the principal antigen

recognized by breast milk IgA is urease (J. , MD, The Royal n

Infirmary, Newcastle Upon Tyne, England,

personal communication, 1996).

Approaches to Vaccine Development

Although the feasibility of prophylactic and therapeutic immunization was

established by these initial studies, procedures for

the large-scale production of a safe and effective product are needed (Table I).

The use of whole bacterial cells or cellular

extracts is problematic, and while recombinant subunit vaccines (especially

urease) are attractive alternatives, the

identification of a full complement of protective antigens to be included in a

recombinant vaccine remains a considerable

challenge. However, the greatest problem for vaccine developers is the selection

of an effective method for presenting

antigens to the host's immune system in such a way that protective or

therapeutic immune responses are elicited in the gastric

mucosa. Since the mechanisms by which H pylori evades immunity and the roles of

T and B cells in effector responses are

poorly understood, purely empirical approaches have been applied to screen

antigens, adjuvants, and delivery systems.

Approaches using live H pylori strains, live vectors, and subunit antigens have

also been explored.

LiveH pylori Vaccines

Effective live, attenuated oral vaccines have been developed to protect against

several enteric bacterial infections, including

typhoid, cholera, and Shigella. However, this approach poses certain serious

difficulties in the case of H pylori:

Immunity resulting from infection with wild strains of H pylori does not

result in clearance or provide protection

against superinfection with other H pylori strains, recrudescence after

antibiotic suppression, or re-infection after

successful cure. A live, attenuated vaccine would probably elicit an even

weaker immune response than the wild-type

bacteria. Thus, it would be technically difficult to modify H pylori to

induce effective immunity rather than the evasion

or down-regulation of immunity associated with natural infection.

It is likely that a live vaccine would require high doses (possibly >/=109

organisms) and repeated administrations to be

effective. Therefore, high-yield fermentation of H pylori is difficult and

may not be economically feasible at the scale

required for a live vaccine.

H pylori is well adapted to cause chronic, persistent infection in the

host. Since human host responses are highly

variable and uncontrollable, an attenuated vaccine must not cause

persistent infection associated with an inflammatory

response. Regulatory concerns about chronic infection with a vaccine strain

would require long-term follow-up studies

in large populations. The sensitivity of tests for persistence of a vaccine

strain versus wild-type strains in humans is

highly problematic.

A live vaccine would elicit immune responses against a wide range of

antigens, some of which may be undesirable,

due to cross-reactivity with homologous human antigens or stimulation of

delayed-type hypersensitivity responses.

A live vaccine might be used as prophylaxis, but it is difficult to

conceive of its use for treatment of infection.

Despite these concerns, there may be a role for a live, attenuated H pylori

vaccine in an effective prophylactic immunizing

regimen. Preclinical studies in mice have demonstrated that H pylori-specific T

and B cells are recruited to the gastric

mucosa in large numbers only after Helicobacter challenge.[45] In mice immunized

with urease before challenge, the gastric

immune response is effective in clearing most of the challenge organisms, but

without the stimulus provided by the challenge,

the stomach remains immunologically silent. This observation suggests that an

effective immunization might include priming of

intestinal immunity with a subunit antigen, followed by a live, attenuated H

pylori vaccine that would direct the immune

response to the gastric mucosa but would establish only a transient infection

sufficient to target immunity. The sequence of

artificial immunizations in such a model may result in an immune response that

is qualitatively distinct from natural infection.

This concept is currently being explored in our laboratories.

Live Vectors

Recombinant enteric bacterial vectors have been constructed to deliver foreign

antigens. Examples include attenuated strains

of Shigella flexneri, Salmonella typhi, and E coli.[46] These vectors, as well

as others that replicate in the gastrointestinal

tract or invade the body by this route, provide potential approaches to

immunization against mucosal pathogens such as H

pylori. Examples of such vectors include Vibrio cholerae, Lactobacillus species,

Streptococcus gordonii, poxvirus,

adenovirus, poliovirus, rhinovirus, and alphavirus. The ideal live vector is one

that is not replication-deficient or restricted in

its ability to express its own and foreign antigenic determinants. Restriction

of vector replication by anti-vector immunity is a

concern that can potentially be addressed by a combination of 2 antigenically

distinct vectors or a combination of parenteral

priming followed by a live-vector boost or vice versa. Live vectors may preclude

the need for a mucosal adjuvant by

targeting M cells and inductive lymphoid tissues in the gut. Alternatively, the

vectors may be designed to co-express antigens

with immunomodulatory lymphokines. The use of live vectors could also simplify

vaccine administration schedules, since

fewer doses would be required than of a subunit vaccine. The manufacturing

process is also greatly simplified, since protein

purification is unnecessary.

Preliminary studies have been performed in several laboratories with mixed

results, and it is too early to draw conclusions

about the value of live vectors for construction of an effective Helicobacter

vaccine.

Subunit Antigens

Nonliving vaccines include defined subunits, whole-cell or crude preparations,

and DNA-based vaccines. Whole-cell or

crude preparations appear to be effective in animal models and have the

advantage of multiple antigens presenting to the host

without having to isolate, characterize, and prepare individually active

components. This approach is unlikely to be practical

from a scale-up perspective or desirable from a regulatory view, given the

potential problem of autoimmunity due to

Helicobacter antigens, such as blood group antigens (cross-reactive with

human cells).[47,48] DNA-based approaches

are being investigated, but it is too early to assess the feasibility of

generating an effective mucosal (and especially gastric)

immune response by this method.

A nearer-term approach is the delivery of defined H pylori protein antigens in a

formulation designed to elicit protective

responses in the stomach. H pylori bacteria have a number of virulence factors

that are of known importance in chronic

infection, recruitment of inflammatory cells, and damage to mucosal epithelium

(Fig. 1). Among these, prominent is the

urease enzyme, which is implicated in acid tolerance of the bacteria,

colonization, and mucin depletion. As noted,

recombinant urease has been demonstrated to be highly effective in prophylactic

immunization of mice against challenge with

Helicobacter species.[33-41] Evidence of protection has also been obtained in

models using larger animals, including cats and

nonhuman primates.[49-51]

Native urease is a metalloenzyme, dependent for enzymatic activity on Ni2+,

incorporated during intracellular synthesis.[50]

Urease is essential for colonization of the stomach by H pylori; the enzyme

splits urea present in gastric juice to form

ammonia, a strong base that presumably protects the bacterium from inactivation

by gastric acid.[51,52] All strains of H pylori

that infect humans express the urease enzyme. In fact, urease accounts for more

than 6% of the total soluble bacterial protein

of H pylori and is localized, in part, on the surface of the

bacterium.[50,53,54] This makes the urease enzyme an important

target for the immune response elicited by a vaccine. Urease is constitutively

expressed in vivo so that the bacteria would be

exposed to the anti-urease immune response during the entire course of

infection. Moreover, H pylori urease is intrinsically

acid-stable, making it an ideal vaccine for oral application. H pylori urease is

highly conserved at the amino acid sequence

level, and antigenic variation between strains of H pylori urease is not likely

to impair vaccine efficacy. Cross-reactivity

between the ureases of different H pylori clinical isolates and between H pylori

urease and heterologous ureases of H felis

and H mustelae has been demonstrated[52] and is the basis for the heterologous

cross-protection studies.[31] In its native

form, urease is a hexameric structure of large molecular mass (550kDa), composed

of 6 copies of the UreA (30kDa) and

UreB (60kDa) and has a particulate structure of 12nm in diameter,[49,50,55]

favoring uptake by M cells in the gastrointestinal

tract for induction of mucosal immunity.[56]

The vaccine candidate-recombinant urease-is urease antigen produced in

genetically engineered E coli. Antigenically

indistinguishable from native urease, recombinant urease has an identical

particulate structure but is enzymatically

nonfunctional and does not generate toxic ammonia in the presence of urea. This

has been accomplished by cloning and

expressing in E coli only the genes for the structural subunits (ureA and ureB),

omitting all other genes of the operon,[57] and

including those involved in insertion of Ni2+ required for enzymatic function.

After expression in fermentation cultures of E

coli, the recombinant antigen is purified from bacterial lysates and is

subsequently lyophilized in a stabilizer.

Therapeutic Immunization

Treatment of H pylori infection in patients with peptic ulcer disease is now an

accepted health practice in the US[6] and

Europe and is the basis for regulatory labeling of antibiotic-antisecretory drug

combinations. However, antimicrobial therapy

has a number of inherent limitations that might be overcome by use of an

effective vaccine or a combined regimen of

antibiotics and vaccine. On average, primary treatment failures occur in 15% of

patients treated with antibiotics combined

with an antisecretory drug. Poor compliance with complex antibiotic regimens and

antibiotic resistance in H pylori[58-60]

contribute to treatment failures. In contrast to antibiotics, vaccine-induced

immunity is not expected to select for resistant or

more virulent organisms. Since immunologic mechanisms are distinct from those

involved in antimicrobial treatment, vaccines

alone or synergistic activities of vaccines and antimicrobials could achieve the

ultimate goal of 100% cure.

Murine Studies

Using recombinant urease[42,61] and crude cell antigens,[41] therapeutic

activity has been documented in mice, with efficacy

rates (determined by gastric urease activity) between 50% and 94%. When vaccine

and a partially effective antibiotic

regimen were combined, the latter proved to be more effective than either

treatment alone.[62] These studies were conducted

in mice with subchronic H felis infection, the immunization regimen being

applied only a few weeks after infecting the

animals. It is uncertain whether treatment would be as effective in a

chronically infected host. Moreover, the reported cure

rates based on gastric urease or histologic endpoints overestimate the

effectiveness of immunization. In addition, in the

mouse model, H felis is easier to eradicate than H pylori. The results with

vaccine are also supported by the observation

that mice can be cured of H felis with a single antibiotic,[63] whereas multiple

drugs were required to achieve partial cure of

H pylori.[64] When the H pylori mouse model was employed and therapeutic

activity of urease-LT immunization was

measured by quantitative culture, a statistically significant (P = 0.0016)

10-fold reduction in bacterial density (not eradication

of infection) was observed. Interestingly, the LT adjuvant alone appeared to

have some effect in reducing infection, possibly

due to modulation of the immune response to antigens associated with natural

infection.

Ferret Studies

In ferrets, immunization with urease and CT adjuvant resulted in presumptive

cure of chronic H mustelae infection.[43] When

tested 6 weeks after immunization, 30% of the ferrets were cured of infection. A

significant reduction in gastric inflammation

was demonstrated by histopathology in up to 60% of the animals. Interestingly,

gastric inflammation was significantly

reduced in the cured and persistently infected vaccinated animals compared with

infected controls, a finding similar to that

described in the rhesus monkeys.[72] The possibility that vaccines can diminish

the pathologic consequences of Helicobacter

infections deserves further study.

Adjuvants

All preclinical studies reported to date have demonstrated efficacy of

vaccination against Helicobacter infection, using

antigens given mucosally together with CT or LT as a mucosal adjuvant. No

protection was achieved when antigens were

administered without a mucosal adjuvant, even at exceedingly high levels.[34] CT

is not acceptable as a human adjuvant

because it induces diarrhea in humans at microgram levels.[65] LT is less

reactogenic and has been tested clinically.[66] A

possible means to circumvent the reactogenicity of native toxins as adjuvants is

the use of atoxic cholera toxin B subunit

(CTB) spiked with a low dose of native toxin. This combination was shown to be

an effective adjuvant for an H felis

sonicate vaccine, providing protection against H felis challenge.[34] An even

more attractive approach is the use of genetically

detoxified LT molecules, which are enzymatically inactive but still retain

adjuvanticity.[67,68]

Many novel adjuvants have shown promise in preclinical studies with a variety of

other vaccines, including oil emulsions,

saponins, immunostimulating complexes, polyphosphazine, muramyl dipeptide

derivatives, block polymers, vitamin D3,

liposomes, copolymer microspheres, and cytokines. Some data are now available

from clinical trials; more is known about

many of these adjuvants for parenteral than for mucosal routes of

administration. In studies of H pylori urease antigen, a

muramyl dipeptide derivative

(N-acetylglucosaminyl-N-acetyl-muramyl-L-alanyl-D-isoglutamine, GMDP) delivered

orally

did not elicit protection in mice against challenge with H felis.[34] Alum given

parenterally with urease was partially effective

when given prophylactically (OraVax, unpublished data, 1997). An exploration of

various adjuvants for parenteral

immunization with urease and for combined mucosal-parenteral immunization

regimens is currently underway in our

laboratory and that of our partner, Pasteur Merieux Connaught. Preliminary data

indicate that partial protection is achieved

by parenteral injection of antigen with alum and other select adjuvants. Since

adjuvants orient the immune response in a

selective fashion with respect to T-helper subsets, the results of comparative

studies will shed light on the role of Th1 and

Th2 responses in protection. Immunization studies of interleukin-4 knock-out and

gamma-interferon receptor deficient mice

indicate that both Th1 and Th2 responses are required for protective

immunity.[69] This finding is also supported by our

observations of adjuvants having selective immunomodulatory properties.

Clinical Trials

Clinical testing of recombinant urease was initiated by our group in 1994, and

trials of whole-cell and other recombinant

antigens are in the planning stages by others. Our clinical studies were begun

in healthy infected volunteers (rather than

uninfected subjects) because of concern that immunization of naive individuals

may potentiate inflammation upon subsequent

infection. This phenomenon was at that time observed in mice[33,42,70] but

subsequently not observed in cats or monkeys. In

addition, because the immune correlates of protection remain problematic, it was

believed that the direct measurement of a

therapeutic effect in infected subjects would have the greatest clinical

significance.

A limited study was first performed to demonstrate the safety and tolerability

of oral administration of urease without a

mucosal adjuvant.[71] In a randomized, double-blind, placebo-controlled trial

conducted by Kreiss and colleagues,[71] 6

infected asymptomatic adults were administered 4 doses of vaccine-each

consisting of 60mg of recombinant H pylori

urease-by the oral route once a week. Six infected subjects received placebo. As

expected in the absence of an adjuvant,

none of the vaccinated individuals mounted an immune response, and in gastric

biopsies obtained before and 1 month after

vaccination, no change in bacterial density (measured by quantitative culture),

inflammation, or mucosal damage was

observed. No adverse events was attributable to administration of urease.

A second trial was conducted to determine the tolerability of coadministration

of urease with a mucosal adjuvant (LT) in

healthy adults with H pylori infection and to obtain preliminary data on

therapeutic activity. Preliminary results of this

trial-which was conducted at the Centre Hospitalier Universitaire, Lausanne, and

at the Center for Vaccine Development,

University of land in Baltimore-were reported by Michetti and colleagues at

the Helicobacter congress in

Copenhagen in 1996. Native LT purified from E coli was supplied by the Naval

Medical Research Institute in Bethesda,

land, which previously reported adjuvant activity in a study involving

cholera vaccine.[66]

The controlled trial involved administration of 4 weekly, graded doses of urease

(20, 60, or 180mg) with LT; placebo

vaccine with LT; or placebo vaccine and placebo adjuvant to groups of 4 or 5

volunteers. The ELISPOT assay for

antibody-secreting cells (ASCs) in peripheral blood was the most sensitive

determinant of immunologic response to the

vaccine. Six of 14 (43%) subjects who received urease, but none of the 10

subjects who received placebo vaccine, had an

increase in IgA or IgG ASCs at 1 or more time points, measured 7 days after each

successive dose of vaccine. Gastric

biopsies were obtained before and 1 month after completion of the immunization

regimen. Differences were determined

between pre- and postimmunization H pylori densities in gastric mucosa. Pairwise

treatment group comparisons were

performed at baseline and on the change from baseline to postimmunization. In

addition, the significance of the mean change

from baseline to postimmunization was assessed within each treatment group.

While the urease-treated groups were not

significantly different from control groups with respect to the change from

baseline to postimmunization, the subjects

receiving active urease experienced, on average, a larger decrease in bacterial

densities from baseline to postimmunization

(P = 0.032) than did subjects receiving placebo (P = 0.425). While the study had

small sample sizes per group and was not

powered to detect significant differences between treatment groups, it provided

the first clinical evidence for a therapeutic

activity of oral urease with LT adjuvant. The duration of the study was not

sufficient to assess whether administration of the

vaccine was associated with a decrease in inflammation, as has been observed in

ferrets[43] and rhesus monkeys.[72]

Conclusions and Future Research

A convincing body of data now exists supporting the potential for successful

immunization against H pylori. However, we

are still at a preliminary stage in clinical development. The best immunogens,

the best mode of presentation, the number of

doses needed, optimal age at immunization, expected benefit, cost-effectiveness,

and other factors involved in vaccine

development require further study.

The complex pathogenesis of this infection,[3,73] including the presence of

antigens on H pylori shared with the host (a

mechanism for immune evasion),[48] demands novel approaches to the development

of a final vaccine formulation. The

selection of defined and well-characterized recombinant subunit antigens appears

to be the most viable approach, and the

urease antigen has so far proved most potent in eliciting protective immunity.

It is reasonable to assume that more than 1

protective component is needed in a vaccine, and a number of such antigens in

addition to urease have now been

discovered. The sequence analysis of the entire H pylori genome by Tomb and

colleagues[74] will enhance antigen discovery

efforts. In addition to antigen composition, a successful vaccine must be

delivered to the host in a manner that elicits

protective (therapeutic) immunity, particularly immunity expressed at the site

of bacterial colonization (gastric mucosa). The

most appropriate means to achieve this end has not yet been fully defined.

Mucosal routes of immunization with a classic

mucosal adjuvant (LT) have yielded the best results, but prophylactic

(therapeutic) activity remains incomplete. Research is

needed on the mechanisms of protective immunity induced by vaccines, on the

protein-specific immune responses to natural

infection, and on the functional role of T cells. Such studies may provide

important data that lead to novel immunization

methods, as well as surrogate tests for protection that are useful in vaccine

trials.

Additional discussion of animal models for the development of Helicobacter

vaccine can be found on Medscape

(www.medscape.com).

Acknowledgments

Original work described in this paper was funded in part by Pasteur Merieux

Connaught (PMC) and by the National

Institutes of Health. The authors are grateful to PMC scientists, particularly

Drs. Pierre Meulien, Marie- Quentin-Millet,

Farukh Rizvi, Bruno Guy, Ling Lissolo, and Veronique Mazarin (PMC, Marcy

l'Etoile, France) for their scientific input

about the work and its interpretation. Drs. Pierre Michetti, Christiana Kreiss,

Irene Couthesy-Theulaz, and Andre Blum

(Centre Hospitalier Universitaire, Lausanne, Switzerland); Kotloff and

Genevieve Losonsky (Center for Vaccine

Development, University of land, Baltimore, Md.); and

(University of land Medical Center,

Baltimore, Md.) conducted the clinical trials reviewed in this paper. Drs.

Czinn and Nedrud (Case-Western

Reserve University, Cleveland, Ohio); Fox (Massachusetts Institute of

Technology, Cambridge, Mass.); Andre

Dubois (Uniformed Services University of the Health Sciences, Bethesda, Md.);

Soike (Tulane University,

Covington, La.); and ph Hill, Christian Stadtlander, Hal Farris, and

Gangemi (Clemson University, Clemson,

S.C.) made significant contributions in many aspects of the testing of

Helicobacter vaccine candidates in animal models. The

authors are especially grateful for the excellent assistance of OraVax

personnel, including ph Simon, Kochi,

Tibbitts, Ingrassia, Gray, Kathleen Georgokopoulos,

Amal Al-Gawari, , Rue Ding,

and Bruce Ekstein.

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[Guest guest]

What really makes me sick is that they are testing efficicy and safety of

the new vaccines on mice, ferrets, monkeys...and even innocent people in

third world countries (West Africa in this case):::not considering that

these people or animals have rights just as we. What makes us superior to

justify risking the lives and health of people in other countries and

animals in labratories? T

Since the begining of time, there has always been some kind of disease to

help stabalize population: the plague, yellow fever, malaria, diptheria.

These diseases eventually subsided on their own (without vaccination) and

another was created in its place. I think there is much truth in the

theory of 'survival of the fittest' ...by trying to control the natural

order of things, you will be confronted with more devestating effects.

Polio, tetanus, malaria--many of these diseases seem to be a thing of the

past; but now we have more serious diseases such as AIDS, luekemia and

cancer at an all time HIGH....

I think Mendelsohn has made a significant point when stating that vaccines

are a medical time bomb.

Thanks for the article ....although I cannot understand much of the

medical jargon.

Lana

Mama of Cody Ukiah

At 09:37 AM 9/6/98 -0600, you wrote:

>From: Mom2Q <Mom2Q@...>

>

>

>Sorry about the error. You have to be registered with Medscape to read

this article. Anyway here it is:

>

>The Search for Vaccines Against Helicobacter pylori

>

> P. Monath, MD, K. Lee, PhD, H. Ermak, PhD, Gwendolyn

A. Myers, DVM,

>A. Weltzin, PhD, J. sca, PhD, D. , Jr., PhD,

Gopalan Soman, PhD, Hitesh Bhagat,

>PhD, A. Ackerman, MD, Harry K. Kleanthous, PhD

>

>[infect Med 15(8):534-535,539-546, 1998. © 1998 SCP Communications, Inc.]

>

>Abstract

>

>Current antibiotic regimens against Helicobacter pylori are effective, but

complex dosing and development of resistance are

>concerns. Animal studies and limited clinical trials of H pylori urease

and other bacterial antigens have been conducted, with

>promising findings. [infect Med 15(8):534-535,539-546, 1998]

>

>Introduction

>

>One of the most promising recent developments in medicine is the concept

that chronic afflictions, such as peptic ulcer

>disease and cancer can be controlled through immunization like classic

infectious diseases. Research on vaccines against

>Helicobacter pylori-the leading cause of chronic gastritis and peptic

ulcer disease and a primary risk factor for gastric

>adenocarcinoma-began in 1990. The favored approach has been the oral

administration of purified recombinant subunit

>proteins of H pylori and a mucosal adjuvant, the labile toxin (LT) of

Escherichia coli. As a single-component vaccine, the

>urease protein has shown remarkable prophylactic and therapeutic activity

in animal models and partial therapeutic activity in

>humans. A number of other H pylori antigens have been effective in animal

models, and the recent sequencing of the

>complete H pylori genome has led to an intensive effort in antigen

discovery. Other research is directed at the comparison of

>adjuvants and vaccine delivery systems and toward the immunologic

mechanisms mediating protection. Here, we present

>preclinical data, the results of early-stage clinical trials, and

directions for future research on Helicobacter vaccines.

>

>Helicobacter pylori: Medical Impact

>

>A gram-negative spiral bacterium that specifically infects the stomach, H

pylori (Fig. 1) is one of the most prevalent

>infections of humankind: Approximately 50% of adults in the industrialized

world and more than 90% of inhabitants of

>developing countries are infected.[1] H pylori is thought to be acquired

by person-to-person spread via the fecal-oral and

>oral-oral routes, and in some areas it may be waterborne.[2] After oral

ingestion, the bacteria colonize gastric mucus in close

>association with gastric epithelial cells (Figs. 2,3). Infection is

chronic and generally lifelong.

>

>In the US, approximately 2.5 million new H pylori infections occur each

year. In Europe, the prevalence and incidence of H

>pylori-associated diseases are similar to or higher than those in the US.

In industrialized countries, the incidence of infection

>is decreasing overall, although transmission varies with socioeconomic

status, and subpopulations are thus differentially

>affected.

>

>H pylori is the cause of chronic gastritis and the vast majority of cases

of peptic ulcer disease.[3-6] Conclusive evidence also

>exists for an etiologic role of H pylori infection in dysplasia and

metaplasia of gastric mucosa, distal gastric adenocarcinoma,

>and non-Hodgkin's lymphoma of the stomach,[7-12] leading the World Health

Organization to classify the bacteria as a Class I

>(definite) carcinogen.[13] Considered in terms of lifetime morbidity, the

illness to infection ratio in the US and Europe may be

>estimated at 1:5 for peptic ulceration and 1:200 for gastric adenocarcinoma.

>

>Peptic Ulcer

>

>In the US, approximately 16,000 deaths are attributed annually to

complications of peptic ulcer disease. There are more

>than 2 million physician visits per year for duodenal ulcers, 90% of which

are attributable to H pylori, and more than 3

>million physician visits per year for gastric ulcers, 60% of which are

attributable to the bacterium.[1,14] In a prospective study,

>the risk of developing duodenal ulcer disease in H pylori-infected

patients followed for 10 years exceeded 10%; in contrast,

>it was less than 1% in uninfected patients.[15]

>

>Gastric Adenocarcinoma

>

>The incidence of gastric adenocarcinoma in the US is approximately 24,000

cases per year, with 13,300 deaths,

>approximately 60% of which (14,400 cases; 7980 deaths) may be attributed

to H pylori. The risk of developing gastric

>cancer is estimated to be 3- to 6-fold higher in infected than in

uninfected individuals.[7,12,16] Gastric cancer is a leading cause

>of death in Latin America and Asia. Acquisition of H pylori infection

early in life appears to be associated with early-onset

>gastric corpus atrophy and metaplasia and a higher risk of cancer.[17]

Ingestion of dietary carcinogens and deficiencies in

>dietary antioxidants are thought to be important cofactors in the genesis

of Helicobacter-related cancer. Helicobacter strain

>differences in virulence factors also appear to determine cancer risk.[18]

>

>Rationale for Vaccine Development

>

>Several lines of evidence provide a rationale for the development of a

vaccine against H pylori.

>

>High Illness to Infection Ratio

>

>Compared with illness to infection ratios of other infectious diseases,

that of H pylori-associated peptic ulcer is high (1:5). In

>comparison, such ratios are about 1:25 for hepatitis B-associated chronic

liver disease and about 1:10 for Mycobacterium

>tuberculosis. Although the ratio for H pylori-associated gastric

adenocarcinoma is lower (1:200), the morbidity and

>mortality associated with this disease are substantial.

>

>Vaccines for Prevention

>

>Vaccines have long been regarded as the most effective and economical

approach to the prevention and control of infectious

>diseases. Although effective antimicrobial treatment for H pylori is now

employed widely to prevent recurrence in patients

>with active or recent duodenal ulcer disease, the ability to treat does

not obviate the need for preventive strategies. Indeed,

>most H pylori infections leading to gastric cancer and 20% to 30% of cases

of upper gastrointestinal hemorrhage occur in

>individuals who have sustained long-term infections without antecedent

symptoms. For this reason, these individuals do not

>present to the physician in time for antimicrobial intervention.

>

>Vaccines as Therapy or Complement

>

>Although the application of vaccines for therapy of infectious diseases is

in its infancy, it has tremendous implications for the

>management of chronic infections, such as H pylori, HIV, human

papillomavirus, viral hepatitis, herpesviruses, chlamydia,

>and a wide range of parasitic infections.

>

>>From a practical perspective, the effectiveness of conventional

antimicrobials has diminished interest in therapeutic vaccines.

>However, vaccines used in combination with antibiotics could improve the

rate of treatment success and decrease the

>evolution of antimicrobial resistance and disease recurrence.

Infection-induced immunity to H pylori is clearly insufficient to

>prevent reinfection, as shown by experiments in animals[19] and limited

studies of humans.[20] In areas of the world with high

>rates of transmission of H pylori, re-infection may occur rapidly after

treatment with antimicrobial agents. However, in

>industrialized nations, re-infection rates in adults appear to be low,

both overall (0.5%-2%) and in high-risk individuals

>(2.5% in spouses of infected persons).[21] However, even in industrialized

nations, re-infection rates in children may be

>substantially higher; in 1 study, Oderda and colleagues[22] reported that

18% of children became re-infected within 18

>months of antibiotic therapy.

>

>Convenient Identification ofH pylori

>

>A wide array of simple office-based serologic screening tests and the

noninvasive[13]C-urea breath test are now available for

>identifying infected individuals, and new serologic tests that identify H

pylori strains characterized by a higher virulence

>phenotype, especially CagA, are under development.[23] These methods could

be used to identify persons with H pylori

>gastritis during the first 2 decades of life, thus identifying a

population at future risk of ulcer disease and cancer. If treatment

>of the infection is considered, coadministration of a vaccine to prevent

re-infection will be an important component of such a

>strategy.

>

>Cost-effectiveness ofH pylori Vaccines

>

>Although there is convincing evidence for the cost-effectiveness of curing

H pylori in patients with duodenal ulcer disease,[24]

>the pharmacoeconomics of prophylactic immunization-whether primary

(pre-exposure) immunization or immunization to

>prevent re-infection-have not been well defined. Pre-exposure immunization

requires application during infancy or

>childhood, depending on age of acquisition of infection in the population

at risk. Since the indication for H pylori vaccination

>is the prevention of chronic diseases that occur in the third to the sixth

decade of life, the cost-benefit ratio is influenced by

>heavy discounting of future cost savings from disease prevention. However,

childhood immunization to prevent chronic

>disease acquired decades later is not without precedent and underlies the

recommendation for universal immunization against

>hepatitis B,[25] a disease that causes considerably less cancer morbidity

and mortality than H pylori.[26] In areas where gastric

>cancer is a leading cause of death, such as Latin America and Asia,

individuals and society place a high value on investments

>that reduce the incidence of this incurable and fatal disease. The World

Health Organization estimates that 550,000 gastric

>cancer deaths due to H pylori occur annually,[13] and these deaths must be

considered potentially preventable through

>immunization. By way of comparison, 316,000 cases of hepatocellular

carcinoma caused by hepatitis B occur annually, and

>many countries are implementing routine childhood immunization policies.

>

>Initial Immunization Trials

>

>Serious consideration of vaccination as a means to control peptic ulcer

disease began around 1990. Pallen and Clayton[27]

>suggested that urease would be a candidate antigen for incorporation in an

H pylori vaccine, based in part on findings in

>animals and humans immunized with jack-bean urease to suppress ammonia

production in the intestine by ureolytic bacteria.

>Czinn and Nedrud[28] showed that H pylori whole-cell sonicates

administered intragastrically to mice and ferrets elicited

>serum and intestinal immunoglobulin (Ig) G and IgA antibodies. Subsequent

studies by Chen and coworkers[29,30] and Czinn

>and colleagues[31] demonstrated that mice orally immunized with

Helicobacter sonicates or whole cells and cholera toxin

>(CT) adjuvant were protected against challenge with Helicobacter felis, a

species capable of infecting murine gastric

>mucosa. In addition, passive protection against challenge was demonstrated

by the oral administration of an IgA monoclonal

>antibody, suggesting that the principal mediator of protection after

active immunization may be secretory IgA. The protective

>monoclonal antibody later was shown to be specific for Helicobacter

urease.[32]

>

>In 1994, Michetti and others[33] demonstrated that mice orally immunized

with recombinant H pylori urease were protected

>against challenge with H felis. Protective determinants were present on

both subunits (UreA and UreB) of the recombinant

>multimeric urease molecule. The recombinant protein is similar to native

urease in multimeric structure, molecular mass

>(550kDa), and nano-particulate morphology.[34] The UreB subunit truncated

at the amino terminus,[35] and multimeric urease

>that had been aggregated or heat-denatured retained prophylactic activity

(OraVax, unpublished data, 1997). These studies

>clearly demonstrated that urease is remarkable among bacterial proteins in

its stability and immunogenicity.

>

>A large body of data has now been accumulated from several laboratories

confirming that H pylori urease administered

>mucosally to a variety of animals confers protection against

challenge.[34,36,37] While initial immunization studies utilized H felis

>as the challenge bacterium, the subsequent development of mouse models of

H pylori infection led to the confirmation that

>urease protected against the human pathogen.[38-40]

>

>In 1994, Doidge and colleagues[41] reported that mice with subchronic H

felis infection cleared or had reduced infection

>after oral immunization with H felis whole-cell sonicates. Urease

administered orally to mice experimentally infected with H

>felis[42] or ferrets naturally infected with Helicobacter mustelae[43] was

shown to have significant therapeutic activity. These

>studies indicated that the up regulation of immunity to specific H pylori

antigens may result in clearance of chronic infection.

>The role of mucosal immunity in protection against H pylori in humans is

also supported by a study of infants in West Africa,

>where infection usually occurs within the first year of life. Infants of

mothers with high titers of anti-Helicobacter IgA in

>breast milk had a significant delay in acquisition of H pylori

infection.[44] Subsequent studies indicate that the principal antigen

>recognized by breast milk IgA is urease (J. , MD, The Royal

n Infirmary, Newcastle Upon Tyne, England,

>personal communication, 1996).

>

>Approaches to Vaccine Development

>

>Although the feasibility of prophylactic and therapeutic immunization was

established by these initial studies, procedures for

>the large-scale production of a safe and effective product are needed

(Table I). The use of whole bacterial cells or cellular

>extracts is problematic, and while recombinant subunit vaccines

(especially urease) are attractive alternatives, the

>identification of a full complement of protective antigens to be included

in a recombinant vaccine remains a considerable

>challenge. However, the greatest problem for vaccine developers is the

selection of an effective method for presenting

>antigens to the host's immune system in such a way that protective or

therapeutic immune responses are elicited in the gastric

>mucosa. Since the mechanisms by which H pylori evades immunity and the

roles of T and B cells in effector responses are

>poorly understood, purely empirical approaches have been applied to screen

antigens, adjuvants, and delivery systems.

>Approaches using live H pylori strains, live vectors, and subunit antigens

have also been explored.

>

>LiveH pylori Vaccines

>

>Effective live, attenuated oral vaccines have been developed to protect

against several enteric bacterial infections, including

>typhoid, cholera, and Shigella. However, this approach poses certain

serious difficulties in the case of H pylori:

>

> Immunity resulting from infection with wild strains of H pylori does

not result in clearance or provide protection

> against superinfection with other H pylori strains, recrudescence

after antibiotic suppression, or re-infection after

> successful cure. A live, attenuated vaccine would probably elicit an

even weaker immune response than the wild-type

> bacteria. Thus, it would be technically difficult to modify H pylori

to induce effective immunity rather than the evasion

> or down-regulation of immunity associated with natural infection.

>

> It is likely that a live vaccine would require high doses (possibly

>/=109 organisms) and repeated administrations to be

> effective. Therefore, high-yield fermentation of H pylori is

difficult and may not be economically feasible at the scale

> required for a live vaccine.

>

> H pylori is well adapted to cause chronic, persistent infection in

the host. Since human host responses are highly

> variable and uncontrollable, an attenuated vaccine must not cause

persistent infection associated with an inflammatory

> response. Regulatory concerns about chronic infection with a vaccine

strain would require long-term follow-up studies

> in large populations. The sensitivity of tests for persistence of a

vaccine strain versus wild-type strains in humans is

> highly problematic.

>

> A live vaccine would elicit immune responses against a wide range of

antigens, some of which may be undesirable,

> due to cross-reactivity with homologous human antigens or stimulation

of delayed-type hypersensitivity responses.

>

> A live vaccine might be used as prophylaxis, but it is difficult to

conceive of its use for treatment of infection.

>

>Despite these concerns, there may be a role for a live, attenuated H

pylori vaccine in an effective prophylactic immunizing

>regimen. Preclinical studies in mice have demonstrated that H

pylori-specific T and B cells are recruited to the gastric

>mucosa in large numbers only after Helicobacter challenge.[45] In mice

immunized with urease before challenge, the gastric

>immune response is effective in clearing most of the challenge organisms,

but without the stimulus provided by the challenge,

>the stomach remains immunologically silent. This observation suggests that

an effective immunization might include priming of

>intestinal immunity with a subunit antigen, followed by a live, attenuated

H pylori vaccine that would direct the immune

>response to the gastric mucosa but would establish only a transient

infection sufficient to target immunity. The sequence of

>artificial immunizations in such a model may result in an immune response

that is qualitatively distinct from natural infection.

>This concept is currently being explored in our laboratories.

>

>Live Vectors

>

>Recombinant enteric bacterial vectors have been constructed to deliver

foreign antigens. Examples include attenuated strains

>of Shigella flexneri, Salmonella typhi, and E coli.[46] These vectors, as

well as others that replicate in the gastrointestinal

>tract or invade the body by this route, provide potential approaches to

immunization against mucosal pathogens such as H

>pylori. Examples of such vectors include Vibrio cholerae, Lactobacillus

species, Streptococcus gordonii, poxvirus,

>adenovirus, poliovirus, rhinovirus, and alphavirus. The ideal live vector

is one that is not replication-deficient or restricted in

>its ability to express its own and foreign antigenic determinants.

Restriction of vector replication by anti-vector immunity is a

>concern that can potentially be addressed by a combination of 2

antigenically distinct vectors or a combination of parenteral

>priming followed by a live-vector boost or vice versa. Live vectors may

preclude the need for a mucosal adjuvant by

>targeting M cells and inductive lymphoid tissues in the gut.

Alternatively, the vectors may be designed to co-express antigens

>with immunomodulatory lymphokines. The use of live vectors could also

simplify vaccine administration schedules, since

>fewer doses would be required than of a subunit vaccine. The manufacturing

process is also greatly simplified, since protein

>purification is unnecessary.

>

>Preliminary studies have been performed in several laboratories with mixed

results, and it is too early to draw conclusions

>about the value of live vectors for construction of an effective

Helicobacter vaccine.

>

>Subunit Antigens

>

>Nonliving vaccines include defined subunits, whole-cell or crude

preparations, and DNA-based vaccines. Whole-cell or

>crude preparations appear to be effective in animal models and have the

advantage of multiple antigens presenting to the host

>without having to isolate, characterize, and prepare individually active

components. This approach is unlikely to be practical

>from a scale-up perspective or desirable from a regulatory view, given the

potential problem of autoimmunity due to

>Helicobacter antigens, such as blood group antigens (cross-reactive

with human cells).[47,48] DNA-based approaches

>are being investigated, but it is too early to assess the feasibility of

generating an effective mucosal (and especially gastric)

>immune response by this method.

>

>A nearer-term approach is the delivery of defined H pylori protein

antigens in a formulation designed to elicit protective

>responses in the stomach. H pylori bacteria have a number of virulence

factors that are of known importance in chronic

>infection, recruitment of inflammatory cells, and damage to mucosal

epithelium (Fig. 1). Among these, prominent is the

>urease enzyme, which is implicated in acid tolerance of the bacteria,

colonization, and mucin depletion. As noted,

>recombinant urease has been demonstrated to be highly effective in

prophylactic immunization of mice against challenge with

>Helicobacter species.[33-41] Evidence of protection has also been obtained

in models using larger animals, including cats and

>nonhuman primates.[49-51]

>

>Native urease is a metalloenzyme, dependent for enzymatic activity on

Ni2+, incorporated during intracellular synthesis.[50]

>Urease is essential for colonization of the stomach by H pylori; the

enzyme splits urea present in gastric juice to form

>ammonia, a strong base that presumably protects the bacterium from

inactivation by gastric acid.[51,52] All strains of H pylori

>that infect humans express the urease enzyme. In fact, urease accounts for

more than 6% of the total soluble bacterial protein

>of H pylori and is localized, in part, on the surface of the

bacterium.[50,53,54] This makes the urease enzyme an important

>target for the immune response elicited by a vaccine. Urease is

constitutively expressed in vivo so that the bacteria would be

>exposed to the anti-urease immune response during the entire course of

infection. Moreover, H pylori urease is intrinsically

>acid-stable, making it an ideal vaccine for oral application. H pylori

urease is highly conserved at the amino acid sequence

>level, and antigenic variation between strains of H pylori urease is not

likely to impair vaccine efficacy. Cross-reactivity

>between the ureases of different H pylori clinical isolates and between H

pylori urease and heterologous ureases of H felis

>and H mustelae has been demonstrated[52] and is the basis for the

heterologous cross-protection studies.[31] In its native

>form, urease is a hexameric structure of large molecular mass (550kDa),

composed of 6 copies of the UreA (30kDa) and

>UreB (60kDa) and has a particulate structure of 12nm in

diameter,[49,50,55] favoring uptake by M cells in the gastrointestinal

>tract for induction of mucosal immunity.[56]

>

>The vaccine candidate-recombinant urease-is urease antigen produced in

genetically engineered E coli. Antigenically

>indistinguishable from native urease, recombinant urease has an identical

particulate structure but is enzymatically

>nonfunctional and does not generate toxic ammonia in the presence of urea.

This has been accomplished by cloning and

>expressing in E coli only the genes for the structural subunits (ureA and

ureB), omitting all other genes of the operon,[57] and

>including those involved in insertion of Ni2+ required for enzymatic

function. After expression in fermentation cultures of E

>coli, the recombinant antigen is purified from bacterial lysates and is

subsequently lyophilized in a stabilizer.

>

>Therapeutic Immunization

>

>Treatment of H pylori infection in patients with peptic ulcer disease is

now an accepted health practice in the US[6] and

>Europe and is the basis for regulatory labeling of

antibiotic-antisecretory drug combinations. However, antimicrobial therapy

>has a number of inherent limitations that might be overcome by use of an

effective vaccine or a combined regimen of

>antibiotics and vaccine. On average, primary treatment failures occur in

15% of patients treated with antibiotics combined

>with an antisecretory drug. Poor compliance with complex antibiotic

regimens and antibiotic resistance in H pylori[58-60]

>contribute to treatment failures. In contrast to antibiotics,

vaccine-induced immunity is not expected to select for resistant or

>more virulent organisms. Since immunologic mechanisms are distinct from

those involved in antimicrobial treatment, vaccines

>alone or synergistic activities of vaccines and antimicrobials could

achieve the ultimate goal of 100% cure.

>

>Murine Studies

>

>Using recombinant urease[42,61] and crude cell antigens,[41] therapeutic

activity has been documented in mice, with efficacy

>rates (determined by gastric urease activity) between 50% and 94%. When

vaccine and a partially effective antibiotic

>regimen were combined, the latter proved to be more effective than either

treatment alone.[62] These studies were conducted

>in mice with subchronic H felis infection, the immunization regimen being

applied only a few weeks after infecting the

>animals. It is uncertain whether treatment would be as effective in a

chronically infected host. Moreover, the reported cure

>rates based on gastric urease or histologic endpoints overestimate the

effectiveness of immunization. In addition, in the

>mouse model, H felis is easier to eradicate than H pylori. The results

with vaccine are also supported by the observation

>that mice can be cured of H felis with a single antibiotic,[63] whereas

multiple drugs were required to achieve partial cure of

>H pylori.[64] When the H pylori mouse model was employed and therapeutic

activity of urease-LT immunization was

>measured by quantitative culture, a statistically significant (P = 0.0016)

10-fold reduction in bacterial density (not eradication

>of infection) was observed. Interestingly, the LT adjuvant alone appeared

to have some effect in reducing infection, possibly

>due to modulation of the immune response to antigens associated with

natural infection.

>

>Ferret Studies

>

>In ferrets, immunization with urease and CT adjuvant resulted in

presumptive cure of chronic H mustelae infection.[43] When

>tested 6 weeks after immunization, 30% of the ferrets were cured of

infection. A significant reduction in gastric inflammation

>was demonstrated by histopathology in up to 60% of the animals.

Interestingly, gastric inflammation was significantly

>reduced in the cured and persistently infected vaccinated animals compared

with infected controls, a finding similar to that

>described in the rhesus monkeys.[72] The possibility that vaccines can

diminish the pathologic consequences of Helicobacter

>infections deserves further study.

>

>Adjuvants

>

>All preclinical studies reported to date have demonstrated efficacy of

vaccination against Helicobacter infection, using

>antigens given mucosally together with CT or LT as a mucosal adjuvant. No

protection was achieved when antigens were

>administered without a mucosal adjuvant, even at exceedingly high

levels.[34] CT is not acceptable as a human adjuvant

>because it induces diarrhea in humans at microgram levels.[65] LT is less

reactogenic and has been tested clinically.[66] A

>possible means to circumvent the reactogenicity of native toxins as

adjuvants is the use of atoxic cholera toxin B subunit

>(CTB) spiked with a low dose of native toxin. This combination was shown

to be an effective adjuvant for an H felis

>sonicate vaccine, providing protection against H felis challenge.[34] An

even more attractive approach is the use of genetically

>detoxified LT molecules, which are enzymatically inactive but still retain

adjuvanticity.[67,68]

>

>Many novel adjuvants have shown promise in preclinical studies with a

variety of other vaccines, including oil emulsions,

>saponins, immunostimulating complexes, polyphosphazine, muramyl dipeptide

derivatives, block polymers, vitamin D3,

>liposomes, copolymer microspheres, and cytokines. Some data are now

available from clinical trials; more is known about

>many of these adjuvants for parenteral than for mucosal routes of

administration. In studies of H pylori urease antigen, a

>muramyl dipeptide derivative

(N-acetylglucosaminyl-N-acetyl-muramyl-L-alanyl-D-isoglutamine, GMDP)

delivered orally

>did not elicit protection in mice against challenge with H felis.[34] Alum

given parenterally with urease was partially effective

>when given prophylactically (OraVax, unpublished data, 1997). An

exploration of various adjuvants for parenteral

>immunization with urease and for combined mucosal-parenteral immunization

regimens is currently underway in our

>laboratory and that of our partner, Pasteur Merieux Connaught. Preliminary

data indicate that partial protection is achieved

>by parenteral injection of antigen with alum and other select adjuvants.

Since adjuvants orient the immune response in a

>selective fashion with respect to T-helper subsets, the results of

comparative studies will shed light on the role of Th1 and

>Th2 responses in protection. Immunization studies of interleukin-4

knock-out and gamma-interferon receptor deficient mice

>indicate that both Th1 and Th2 responses are required for protective

immunity.[69] This finding is also supported by our

>observations of adjuvants having selective immunomodulatory properties.

>

>Clinical Trials

>

>Clinical testing of recombinant urease was initiated by our group in 1994,

and trials of whole-cell and other recombinant

>antigens are in the planning stages by others. Our clinical studies were

begun in healthy infected volunteers (rather than

>uninfected subjects) because of concern that immunization of naive

individuals may potentiate inflammation upon subsequent

>infection. This phenomenon was at that time observed in mice[33,42,70] but

subsequently not observed in cats or monkeys. In

>addition, because the immune correlates of protection remain problematic,

it was believed that the direct measurement of a

>therapeutic effect in infected subjects would have the greatest clinical

significance.

>

>A limited study was first performed to demonstrate the safety and

tolerability of oral administration of urease without a

>mucosal adjuvant.[71] In a randomized, double-blind, placebo-controlled

trial conducted by Kreiss and colleagues,[71] 6

>infected asymptomatic adults were administered 4 doses of vaccine-each

consisting of 60mg of recombinant H pylori

>urease-by the oral route once a week. Six infected subjects received

placebo. As expected in the absence of an adjuvant,

>none of the vaccinated individuals mounted an immune response, and in

gastric biopsies obtained before and 1 month after

>vaccination, no change in bacterial density (measured by quantitative

culture), inflammation, or mucosal damage was

>observed. No adverse events was attributable to administration of urease.

>

>A second trial was conducted to determine the tolerability of

coadministration of urease with a mucosal adjuvant (LT) in

>healthy adults with H pylori infection and to obtain preliminary data on

therapeutic activity. Preliminary results of this

>trial-which was conducted at the Centre Hospitalier Universitaire,

Lausanne, and at the Center for Vaccine Development,

>University of land in Baltimore-were reported by Michetti and

colleagues at the Helicobacter congress in

>Copenhagen in 1996. Native LT purified from E coli was supplied by the

Naval Medical Research Institute in Bethesda,

>land, which previously reported adjuvant activity in a study involving

cholera vaccine.[66]

>

>The controlled trial involved administration of 4 weekly, graded doses of

urease (20, 60, or 180mg) with LT; placebo

>vaccine with LT; or placebo vaccine and placebo adjuvant to groups of 4 or

5 volunteers. The ELISPOT assay for

>antibody-secreting cells (ASCs) in peripheral blood was the most sensitive

determinant of immunologic response to the

>vaccine. Six of 14 (43%) subjects who received urease, but none of the 10

subjects who received placebo vaccine, had an

>increase in IgA or IgG ASCs at 1 or more time points, measured 7 days

after each successive dose of vaccine. Gastric

>biopsies were obtained before and 1 month after completion of the

immunization regimen. Differences were determined

>between pre- and postimmunization H pylori densities in gastric mucosa.

Pairwise treatment group comparisons were

>performed at baseline and on the change from baseline to postimmunization.

In addition, the significance of the mean change

>from baseline to postimmunization was assessed within each treatment

group. While the urease-treated groups were not

>significantly different from control groups with respect to the change

from baseline to postimmunization, the subjects

>receiving active urease experienced, on average, a larger decrease in

bacterial densities from baseline to postimmunization

>(P = 0.032) than did subjects receiving placebo (P = 0.425). While the

study had small sample sizes per group and was not

>powered to detect significant differences between treatment groups, it

provided the first clinical evidence for a therapeutic

>activity of oral urease with LT adjuvant. The duration of the study was

not sufficient to assess whether administration of the

>vaccine was associated with a decrease in inflammation, as has been

observed in ferrets[43] and rhesus monkeys.[72]

>

>Conclusions and Future Research

>

>A convincing body of data now exists supporting the potential for

successful immunization against H pylori. However, we

>are still at a preliminary stage in clinical development. The best

immunogens, the best mode of presentation, the number of

>doses needed, optimal age at immunization, expected benefit,

cost-effectiveness, and other factors involved in vaccine

>development require further study.

>

>The complex pathogenesis of this infection,[3,73] including the presence

of antigens on H pylori shared with the host (a

>mechanism for immune evasion),[48] demands novel approaches to the

development of a final vaccine formulation. The

>selection of defined and well-characterized recombinant subunit antigens

appears to be the most viable approach, and the

>urease antigen has so far proved most potent in eliciting protective

immunity. It is reasonable to assume that more than 1

>protective component is needed in a vaccine, and a number of such antigens

in addition to urease have now been

>discovered. The sequence analysis of the entire H pylori genome by Tomb

and colleagues[74] will enhance antigen discovery

>efforts. In addition to antigen composition, a successful vaccine must be

delivered to the host in a manner that elicits

>protective (therapeutic) immunity, particularly immunity expressed at the

site of bacterial colonization (gastric mucosa). The

>most appropriate means to achieve this end has not yet been fully defined.

Mucosal routes of immunization with a classic

>mucosal adjuvant (LT) have yielded the best results, but prophylactic

(therapeutic) activity remains incomplete. Research is

>needed on the mechanisms of protective immunity induced by vaccines, on

the protein-specific immune responses to natural

>infection, and on the functional role of T cells. Such studies may provide

important data that lead to novel immunization

>methods, as well as surrogate tests for protection that are useful in

vaccine trials.

>

>Additional discussion of animal models for the development of Helicobacter

vaccine can be found on Medscape

>(www.medscape.com).

>

>Acknowledgments

>

>Original work described in this paper was funded in part by Pasteur

Merieux Connaught (PMC) and by the National

>Institutes of Health. The authors are grateful to PMC scientists,

particularly Drs. Pierre Meulien, Marie- Quentin-Millet,

>Farukh Rizvi, Bruno Guy, Ling Lissolo, and Veronique Mazarin (PMC, Marcy

l'Etoile, France) for their scientific input

>about the work and its interpretation. Drs. Pierre Michetti, Christiana

Kreiss, Irene Couthesy-Theulaz, and Andre Blum

>(Centre Hospitalier Universitaire, Lausanne, Switzerland); Kotloff

and Genevieve Losonsky (Center for Vaccine

>Development, University of land, Baltimore, Md.); and

(University of land Medical Center,

>Baltimore, Md.) conducted the clinical trials reviewed in this paper. Drs.

Czinn and Nedrud (Case-Western

>Reserve University, Cleveland, Ohio); Fox (Massachusetts Institute

of Technology, Cambridge, Mass.); Andre

>Dubois (Uniformed Services University of the Health Sciences, Bethesda,

Md.); Soike (Tulane University,

>Covington, La.); and ph Hill, Christian Stadtlander, Hal Farris, and

Gangemi (Clemson University, Clemson,

>S.C.) made significant contributions in many aspects of the testing of

Helicobacter vaccine candidates in animal models. The

>authors are especially grateful for the excellent assistance of OraVax

personnel, including ph Simon, Kochi,

> Tibbitts, Ingrassia, Gray, Kathleen

Georgokopoulos, Amal Al-Gawari, , Rue Ding,

>and Bruce Ekstein.

>

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> treatment against Helicobacter infection in mice. Gastroenterology

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>

> 43.Cuenca R, Blanchard TG, Czinn SJ, et al: Therapeutic immunization

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> 44. JE, Austin S, Dale A, et al: Protection by human milk IgA

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> 45.Ermak TH, Kleanthous HK, Myers G, et al: Oral immunization of mice

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>

> 46.Noriega FR, Losonsky G, Wang JY, et al: Further characterization of

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> CVD1203 as a mucosal Shigella vaccine and a live-vector vaccine for

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> 47.Negrini R, Savio A, Poiesi C, et al: Antigenic mimicry between

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> pathogenesis of body atrophic gastritis. Gastroenterology

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> 48.Appelmelk BJ, Simoons-Smit I, Negrini R, et al: Potential role of

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> lipopolysaccharide and host blood group antigens in

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> 49.Mobley HLT, Foxall PA: H pylori urease, in Hunt R, Tytgat G (eds):

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> 50.Mobley HLT, Island MD, Hausinger RP: Molecular biology of microbial

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> 51.Ferrero RL, Lee A: The importance of urease in acid protection for

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> 52.Hawtin PR, Stacey AR, Newell DG: Investigation of the structure and

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> 53.Phadnis SH, Parlow MH, Levy M, et al: Surface localization of

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> 54.Gootz TD, - G, Clancy J, et al: Immunological and molecular

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> 55.Dunn BE, CP, - G, et al: Purification and

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> 56.Neutra MR, Kraehenbuhl J-P: Transepithelial transport of proteins by

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> 57.Labigne A, Cussac V, Courcoux P: Shuttle cloning and nucleotide

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> 58.Noach LA, Langenberg WL, Bertola MA, et al: Impact of metronidazole

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> 60.Parasakthi N, Goh KL: Primary and acquired resistance to

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> 61.Kleanthous H, Ermak T, Pappo J, et al: Oral immunization with

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> 62.Kleanthous H, Tibbitts T, Bakios J, et al: Oral immunization with

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> 63.Hook-Nikanne J, Aho P, Karkkainen P, et al: The Helicobacter felis

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> 64.Dubois A, Fiala N, Heman-Ackah LM, et al: Natural gastric infection

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> 65.Levine MM, Black RE, Clements ML, et al: Volunteer studies in

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> 66. DA, Baqar S, Oplinger M, et al: Safety and adjuvant activity of

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> 67.Dickinson BL, Clements JD: Dissociation of Escherichia coli

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> 70.Ermak TH, Ding R, Ekstein B, et al: Oral immunization of mice with

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> gastritis after challenge with H felis is due to the presence of

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> 1997.

>

> 71.Kreiss C, Buclin T, Cosma M, et al: Safety of oral immunization with

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> Helicobacter pylori infection. Lancet 347:1630-1631, 1996.

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> 72.Dubois A, Lee CK, Fiala N, et al: Immunization against natural

Helicobacter pylori infection in rhesus monkeys.

> Gastroenterology, 1998. In press.

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> 73.Labigne A, de Reuse H: Determinants of Helicobacter pylori

pathogenicity. Infect Agents Dis 5:191-202, 1996.

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> 74.Tomb JF, White O, Kerlavage AR, et al: The complete genome sequence

of the gastric pathogen Helicobacter

> pylori. Nature 388(6642):539-547, 1997.

>

>

>

>

>------------------------------------------------------------------------

>

[Guest guest]

In the same vein, check this book out: " The Fourth Horseman: A Short

History of Plagues and Diseases " by Nikiforuk. I may not have the

title exactly right. It's a fascinating and horrifying read, and talks

about the devastating consequences of messing with the natural order of

things.

Kate

At 01:42 AM 9/7/98 -0400, Mark & Lana Clifton wrote:

>From: Mark & Lana Clifton <mclifton@...>

>

>What really makes me sick is that they are testing efficicy and safety of

>the new vaccines on mice, ferrets, monkeys...and even innocent people in

>third world countries (West Africa in this case):::not considering that

>these people or animals have rights just as we. What makes us superior to

>justify risking the lives and health of people in other countries and

>animals in labratories? T

>

>Since the begining of time, there has always been some kind of disease to

>help stabalize population: the plague, yellow fever, malaria, diptheria.

>These diseases eventually subsided on their own (without vaccination) and

>another was created in its place. I think there is much truth in the

>theory of 'survival of the fittest' ...by trying to control the natural

>order of things, you will be confronted with more devestating effects.

>Polio, tetanus, malaria--many of these diseases seem to be a thing of the

>past; but now we have more serious diseases such as AIDS, luekemia and

>cancer at an all time HIGH....

>

>I think Mendelsohn has made a significant point when stating that vaccines

>are a medical time bomb.

>

>Thanks for the article ....although I cannot understand much of the

>medical jargon.

>

>Lana

>Mama of Cody Ukiah

>

>

>

>

>

>At 09:37 AM 9/6/98 -0600, you wrote:

>>From: Mom2Q <Mom2Q@...>

>>

>>

>>Sorry about the error. You have to be registered with Medscape to read

>this article. Anyway here it is:

>>

>>The Search for Vaccines Against Helicobacter pylori

>>

>> P. Monath, MD, K. Lee, PhD, H. Ermak, PhD, Gwendolyn

>A. Myers, DVM,

>>A. Weltzin, PhD, J. sca, PhD, D. , Jr., PhD,

>Gopalan Soman, PhD, Hitesh Bhagat,

>>PhD, A. Ackerman, MD, Harry K. Kleanthous, PhD

>>

>>[infect Med 15(8):534-535,539-546, 1998. © 1998 SCP Communications, Inc.]

>>

>>Abstract

>>

>>Current antibiotic regimens against Helicobacter pylori are effective, but

>complex dosing and development of resistance are

>>concerns. Animal studies and limited clinical trials of H pylori urease

>and other bacterial antigens have been conducted, with

>>promising findings. [infect Med 15(8):534-535,539-546, 1998]

>>

>>Introduction

>>

>>One of the most promising recent developments in medicine is the concept

>that chronic afflictions, such as peptic ulcer

>>disease and cancer can be controlled through immunization like classic

>infectious diseases. Research on vaccines against

>>Helicobacter pylori-the leading cause of chronic gastritis and peptic

>ulcer disease and a primary risk factor for gastric

>>adenocarcinoma-began in 1990. The favored approach has been the oral

>administration of purified recombinant subunit

>>proteins of H pylori and a mucosal adjuvant, the labile toxin (LT) of

>Escherichia coli. As a single-component vaccine, the

>>urease protein has shown remarkable prophylactic and therapeutic activity

>in animal models and partial therapeutic activity in

>>humans. A number of other H pylori antigens have been effective in animal

>models, and the recent sequencing of the

>>complete H pylori genome has led to an intensive effort in antigen

>discovery. Other research is directed at the comparison of

>>adjuvants and vaccine delivery systems and toward the immunologic

>mechanisms mediating protection. Here, we present

>>preclinical data, the results of early-stage clinical trials, and

>directions for future research on Helicobacter vaccines.

>>

>>Helicobacter pylori: Medical Impact

>>

>>A gram-negative spiral bacterium that specifically infects the stomach, H

>pylori (Fig. 1) is one of the most prevalent

>>infections of humankind: Approximately 50% of adults in the industrialized

>world and more than 90% of inhabitants of

>>developing countries are infected.[1] H pylori is thought to be acquired

>by person-to-person spread via the fecal-oral and

>>oral-oral routes, and in some areas it may be waterborne.[2] After oral

>ingestion, the bacteria colonize gastric mucus in close

>>association with gastric epithelial cells (Figs. 2,3). Infection is

>chronic and generally lifelong.

>>

>>In the US, approximately 2.5 million new H pylori infections occur each

>year. In Europe, the prevalence and incidence of H

>>pylori-associated diseases are similar to or higher than those in the US.

>In industrialized countries, the incidence of infection

>>is decreasing overall, although transmission varies with socioeconomic

>status, and subpopulations are thus differentially

>>affected.

>>

>>H pylori is the cause of chronic gastritis and the vast majority of cases

>of peptic ulcer disease.[3-6] Conclusive evidence also

>>exists for an etiologic role of H pylori infection in dysplasia and

>metaplasia of gastric mucosa, distal gastric adenocarcinoma,

>>and non-Hodgkin's lymphoma of the stomach,[7-12] leading the World Health

>Organization to classify the bacteria as a Class I

>>(definite) carcinogen.[13] Considered in terms of lifetime morbidity, the

>illness to infection ratio in the US and Europe may be

>>estimated at 1:5 for peptic ulceration and 1:200 for gastric adenocarcinoma.

>>

>>Peptic Ulcer

>>

>>In the US, approximately 16,000 deaths are attributed annually to

>complications of peptic ulcer disease. There are more

>>than 2 million physician visits per year for duodenal ulcers, 90% of which

>are attributable to H pylori, and more than 3

>>million physician visits per year for gastric ulcers, 60% of which are

>attributable to the bacterium.[1,14] In a prospective study,

>>the risk of developing duodenal ulcer disease in H pylori-infected

>patients followed for 10 years exceeded 10%; in contrast,

>>it was less than 1% in uninfected patients.[15]

>>

>>Gastric Adenocarcinoma

>>

>>The incidence of gastric adenocarcinoma in the US is approximately 24,000

>cases per year, with 13,300 deaths,

>>approximately 60% of which (14,400 cases; 7980 deaths) may be attributed

>to H pylori. The risk of developing gastric

>>cancer is estimated to be 3- to 6-fold higher in infected than in

>uninfected individuals.[7,12,16] Gastric cancer is a leading cause

>>of death in Latin America and Asia. Acquisition of H pylori infection

>early in life appears to be associated with early-onset

>>gastric corpus atrophy and metaplasia and a higher risk of cancer.[17]

>Ingestion of dietary carcinogens and deficiencies in

>>dietary antioxidants are thought to be important cofactors in the genesis

>of Helicobacter-related cancer. Helicobacter strain

>>differences in virulence factors also appear to determine cancer risk.[18]

>>

>>Rationale for Vaccine Development

>>

>>Several lines of evidence provide a rationale for the development of a

>vaccine against H pylori.

>>

>>High Illness to Infection Ratio

>>

>>Compared with illness to infection ratios of other infectious diseases,

>that of H pylori-associated peptic ulcer is high (1:5). In

>>comparison, such ratios are about 1:25 for hepatitis B-associated chronic

>liver disease and about 1:10 for Mycobacterium

>>tuberculosis. Although the ratio for H pylori-associated gastric

>adenocarcinoma is lower (1:200), the morbidity and

>>mortality associated with this disease are substantial.

>>

>>Vaccines for Prevention

>>

>>Vaccines have long been regarded as the most effective and economical

>approach to the prevention and control of infectious

>>diseases. Although effective antimicrobial treatment for H pylori is now

>employed widely to prevent recurrence in patients

>>with active or recent duodenal ulcer disease, the ability to treat does

>not obviate the need for preventive strategies. Indeed,

>>most H pylori infections leading to gastric cancer and 20% to 30% of cases

>of upper gastrointestinal hemorrhage occur in

>>individuals who have sustained long-term infections without antecedent

>symptoms. For this reason, these individuals do not

>>present to the physician in time for antimicrobial intervention.

>>

>>Vaccines as Therapy or Complement

>>

>>Although the application of vaccines for therapy of infectious diseases is

>in its infancy, it has tremendous implications for the

>>management of chronic infections, such as H pylori, HIV, human

>papillomavirus, viral hepatitis, herpesviruses, chlamydia,

>>and a wide range of parasitic infections.

>>

>>>From a practical perspective, the effectiveness of conventional

>antimicrobials has diminished interest in therapeutic vaccines.

>>However, vaccines used in combination with antibiotics could improve the

>rate of treatment success and decrease the

>>evolution of antimicrobial resistance and disease recurrence.

>Infection-induced immunity to H pylori is clearly insufficient to

>>prevent reinfection, as shown by experiments in animals[19] and limited

>studies of humans.[20] In areas of the world with high

>>rates of transmission of H pylori, re-infection may occur rapidly after

>treatment with antimicrobial agents. However, in

>>industrialized nations, re-infection rates in adults appear to be low,

>both overall (0.5%-2%) and in high-risk individuals

>>(2.5% in spouses of infected persons).[21] However, even in industrialized

>nations, re-infection rates in children may be

>>substantially higher; in 1 study, Oderda and colleagues[22] reported that

>18% of children became re-infected within 18

>>months of antibiotic therapy.

>>

>>Convenient Identification ofH pylori

>>

>>A wide array of simple office-based serologic screening tests and the

>noninvasive[13]C-urea breath test are now available for

>>identifying infected individuals, and new serologic tests that identify H

>pylori strains characterized by a higher virulence

>>phenotype, especially CagA, are under development.[23] These methods could

>be used to identify persons with H pylori

>>gastritis during the first 2 decades of life, thus identifying a

>population at future risk of ulcer disease and cancer. If treatment

>>of the infection is considered, coadministration of a vaccine to prevent

>re-infection will be an important component of such a

>>strategy.

>>

>>Cost-effectiveness ofH pylori Vaccines

>>

>>Although there is convincing evidence for the cost-effectiveness of curing

>H pylori in patients with duodenal ulcer disease,[24]

>>the pharmacoeconomics of prophylactic immunization-whether primary

>(pre-exposure) immunization or immunization to

>>prevent re-infection-have not been well defined. Pre-exposure immunization

>requires application during infancy or

>>childhood, depending on age of acquisition of infection in the population

>at risk. Since the indication for H pylori vaccination

>>is the prevention of chronic diseases that occur in the third to the sixth

>decade of life, the cost-benefit ratio is influenced by

>>heavy discounting of future cost savings from disease prevention. However,

>childhood immunization to prevent chronic

>>disease acquired decades later is not without precedent and underlies the

>recommendation for universal immunization against

>>hepatitis B,[25] a disease that causes considerably less cancer morbidity

>and mortality than H pylori.[26] In areas where gastric

>>cancer is a leading cause of death, such as Latin America and Asia,

>individuals and society place a high value on investments

>>that reduce the incidence of this incurable and fatal disease. The World

>Health Organization estimates that 550,000 gastric

>>cancer deaths due to H pylori occur annually,[13] and these deaths must be

>considered potentially preventable through

>>immunization. By way of comparison, 316,000 cases of hepatocellular

>carcinoma caused by hepatitis B occur annually, and

>>many countries are implementing routine childhood immunization policies.

>>

>>Initial Immunization Trials

>>

>>Serious consideration of vaccination as a means to control peptic ulcer

>disease began around 1990. Pallen and Clayton[27]

>>suggested that urease would be a candidate antigen for incorporation in an

>H pylori vaccine, based in part on findings in

>>animals and humans immunized with jack-bean urease to suppress ammonia

>production in the intestine by ureolytic bacteria.

>>Czinn and Nedrud[28] showed that H pylori whole-cell sonicates

>administered intragastrically to mice and ferrets elicited

>>serum and intestinal immunoglobulin (Ig) G and IgA antibodies. Subsequent

>studies by Chen and coworkers[29,30] and Czinn

>>and colleagues[31] demonstrated that mice orally immunized with

>Helicobacter sonicates or whole cells and cholera toxin

>>(CT) adjuvant were protected against challenge with Helicobacter felis, a

>species capable of infecting murine gastric

>>mucosa. In addition, passive protection against challenge was demonstrated

>by the oral administration of an IgA monoclonal

>>antibody, suggesting that the principal mediator of protection after

>active immunization may be secretory IgA. The protective

>>monoclonal antibody later was shown to be specific for Helicobacter

>urease.[32]

>>

>>In 1994, Michetti and others[33] demonstrated that mice orally immunized

>with recombinant H pylori urease were protected

>>against challenge with H felis. Protective determinants were present on

>both subunits (UreA and UreB) of the recombinant

>>multimeric urease molecule. The recombinant protein is similar to native

>urease in multimeric structure, molecular mass

>>(550kDa), and nano-particulate morphology.[34] The UreB subunit truncated

>at the amino terminus,[35] and multimeric urease

>>that had been aggregated or heat-denatured retained prophylactic activity

>(OraVax, unpublished data, 1997). These studies

>>clearly demonstrated that urease is remarkable among bacterial proteins in

>its stability and immunogenicity.

>>

>>A large body of data has now been accumulated from several laboratories

>confirming that H pylori urease administered

>>mucosally to a variety of animals confers protection against

>challenge.[34,36,37] While initial immunization studies utilized H felis

>>as the challenge bacterium, the subsequent development of mouse models of

>H pylori infection led to the confirmation that

>>urease protected against the human pathogen.[38-40]

>>

>>In 1994, Doidge and colleagues[41] reported that mice with subchronic H

>felis infection cleared or had reduced infection

>>after oral immunization with H felis whole-cell sonicates. Urease

>administered orally to mice experimentally infected with H

>>felis[42] or ferrets naturally infected with Helicobacter mustelae[43] was

>shown to have significant therapeutic activity. These

>>studies indicated that the up regulation of immunity to specific H pylori

>antigens may result in clearance of chronic infection.

>>The role of mucosal immunity in protection against H pylori in humans is

>also supported by a study of infants in West Africa,

>>where infection usually occurs within the first year of life. Infants of

>mothers with high titers of anti-Helicobacter IgA in

>>breast milk had a significant delay in acquisition of H pylori

>infection.[44] Subsequent studies indicate that the principal antigen

>>recognized by breast milk IgA is urease (J. , MD, The Royal

>n Infirmary, Newcastle Upon Tyne, England,

>>personal communication, 1996).

>>

>>Approaches to Vaccine Development

>>

>>Although the feasibility of prophylactic and therapeutic immunization was

>established by these initial studies, procedures for

>>the large-scale production of a safe and effective product are needed

>(Table I). The use of whole bacterial cells or cellular

>>extracts is problematic, and while recombinant subunit vaccines

>(especially urease) are attractive alternatives, the

>>identification of a full complement of protective antigens to be included

>in a recombinant vaccine remains a considerable

>>challenge. However, the greatest problem for vaccine developers is the

>selection of an effective method for presenting

>>antigens to the host's immune system in such a way that protective or

>therapeutic immune responses are elicited in the gastric

>>mucosa. Since the mechanisms by which H pylori evades immunity and the

>roles of T and B cells in effector responses are

>>poorly understood, purely empirical approaches have been applied to screen

>antigens, adjuvants, and delivery systems.

>>Approaches using live H pylori strains, live vectors, and subunit antigens

>have also been explored.

>>

>>LiveH pylori Vaccines

>>

>>Effective live, attenuated oral vaccines have been developed to protect

>against several enteric bacterial infections, including

>>typhoid, cholera, and Shigella. However, this approach poses certain

>serious difficulties in the case of H pylori:

>>

>> Immunity resulting from infection with wild strains of H pylori does

>not result in clearance or provide protection

>> against superinfection with other H pylori strains, recrudescence

>after antibiotic suppression, or re-infection after

>> successful cure. A live, attenuated vaccine would probably elicit an

>even weaker immune response than the wild-type

>> bacteria. Thus, it would be technically difficult to modify H pylori

>to induce effective immunity rather than the evasion

>> or down-regulation of immunity associated with natural infection.

>>

>> It is likely that a live vaccine would require high doses (possibly

>>/=109 organisms) and repeated administrations to be

>> effective. Therefore, high-yield fermentation of H pylori is

>difficult and may not be economically feasible at the scale

>> required for a live vaccine.

>>

>> H pylori is well adapted to cause chronic, persistent infection in

>the host. Since human host responses are highly

>> variable and uncontrollable, an attenuated vaccine must not cause

>persistent infection associated with an inflammatory

>> response. Regulatory concerns about chronic infection with a vaccine

>strain would require long-term follow-up studies

>> in large populations. The sensitivity of tests for persistence of a

>vaccine strain versus wild-type strains in humans is

>> highly problematic.

>>

>> A live vaccine would elicit immune responses against a wide range of

>antigens, some of which may be undesirable,

>> due to cross-reactivity with homologous human antigens or stimulation

>of delayed-type hypersensitivity responses.

>>

>> A live vaccine might be used as prophylaxis, but it is difficult to

>conceive of its use for treatment of infection.

>>

>>Despite these concerns, there may be a role for a live, attenuated H

>pylori vaccine in an effective prophylactic immunizing

>>regimen. Preclinical studies in mice have demonstrated that H

>pylori-specific T and B cells are recruited to the gastric

>>mucosa in large numbers only after Helicobacter challenge.[45] In mice

>immunized with urease before challenge, the gastric

>>immune response is effective in clearing most of the challenge organisms,

>but without the stimulus provided by the challenge,

>>the stomach remains immunologically silent. This observation suggests that

>an effective immunization might include priming of

>>intestinal immunity with a subunit antigen, followed by a live, attenuated

>H pylori vaccine that would direct the immune

>>response to the gastric mucosa but would establish only a transient

>infection sufficient to target immunity. The sequence of

>>artificial immunizations in such a model may result in an immune response

>that is qualitatively distinct from natural infection.

>>This concept is currently being explored in our laboratories.

>>

>>Live Vectors

>>

>>Recombinant enteric bacterial vectors have been constructed to deliver

>foreign antigens. Examples include attenuated strains

>>of Shigella flexneri, Salmonella typhi, and E coli.[46] These vectors, as

>well as others that replicate in the gastrointestinal

>>tract or invade the body by this route, provide potential approaches to

>immunization against mucosal pathogens such as H

>>pylori. Examples of such vectors include Vibrio cholerae, Lactobacillus

>species, Streptococcus gordonii, poxvirus,

>>adenovirus, poliovirus, rhinovirus, and alphavirus. The ideal live vector

>is one that is not replication-deficient or restricted in

>>its ability to express its own and foreign antigenic determinants.

>Restriction of vector replication by anti-vector immunity is a

>>concern that can potentially be addressed by a combination of 2

>antigenically distinct vectors or a combination of parenteral

>>priming followed by a live-vector boost or vice versa. Live vectors may

>preclude the need for a mucosal adjuvant by

>>targeting M cells and inductive lymphoid tissues in the gut.

>Alternatively, the vectors may be designed to co-express antigens

>>with immunomodulatory lymphokines. The use of live vectors could also

>simplify vaccine administration schedules, since

>>fewer doses would be required than of a subunit vaccine. The manufacturing

>process is also greatly simplified, since protein

>>purification is unnecessary.

>>

>>Preliminary studies have been performed in several laboratories with mixed

>results, and it is too early to draw conclusions

>>about the value of live vectors for construction of an effective

>Helicobacter vaccine.

>>

>>Subunit Antigens

>>

>>Nonliving vaccines include defined subunits, whole-cell or crude

>preparations, and DNA-based vaccines. Whole-cell or

>>crude preparations appear to be effective in animal models and have the

>advantage of multiple antigens presenting to the host

>>without having to isolate, characterize, and prepare individually active

>components. This approach is unlikely to be practical

>>from a scale-up perspective or desirable from a regulatory view, given the

>potential problem of autoimmunity due to

>>Helicobacter antigens, such as blood group antigens (cross-reactive

>with human cells).[47,48] DNA-based approaches

>>are being investigated, but it is too early to assess the feasibility of

>generating an effective mucosal (and especially gastric)

>>immune response by this method.

>>

>>A nearer-term approach is the delivery of defined H pylori protein

>antigens in a formulation designed to elicit protective

>>responses in the stomach. H pylori bacteria have a number of virulence

>factors that are of known importance in chronic

>>infection, recruitment of inflammatory cells, and damage to mucosal

>epithelium (Fig. 1). Among these, prominent is the

>>urease enzyme, which is implicated in acid tolerance of the bacteria,

>colonization, and mucin depletion. As noted,

>>recombinant urease has been demonstrated to be highly effective in

>prophylactic immunization of mice against challenge with

>>Helicobacter species.[33-41] Evidence of protection has also been obtained

>in models using larger animals, including cats and

>>nonhuman primates.[49-51]

>>

>>Native urease is a metalloenzyme, dependent for enzymatic activity on

>Ni2+, incorporated during intracellular synthesis.[50]

>>Urease is essential for colonization of the stomach by H pylori; the

>enzyme splits urea present in gastric juice to form

>>ammonia, a strong base that presumably protects the bacterium from

>inactivation by gastric acid.[51,52] All strains of H pylori

>>that infect humans express the urease enzyme. In fact, urease accounts for

>more than 6% of the total soluble bacterial protein

>>of H pylori and is localized, in part, on the surface of the

>bacterium.[50,53,54] This makes the urease enzyme an important

>>target for the immune response elicited by a vaccine. Urease is

>constitutively expressed in vivo so that the bacteria would be

>>exposed to the anti-urease immune response during the entire course of

>infection. Moreover, H pylori urease is intrinsically

>>acid-stable, making it an ideal vaccine for oral application. H pylori

>urease is highly conserved at the amino acid sequence

>>level, and antigenic variation between strains of H pylori urease is not

>likely to impair vaccine efficacy. Cross-reactivity

>>between the ureases of different H pylori clinical isolates and between H

>pylori urease and heterologous ureases of H felis

>>and H mustelae has been demonstrated[52] and is the basis for the

>heterologous cross-protection studies.[31] In its native

>>form, urease is a hexameric structure of large molecular mass (550kDa),

>composed of 6 copies of the UreA (30kDa) and

>>UreB (60kDa) and has a particulate structure of 12nm in

>diameter,[49,50,55] favoring uptake by M cells in the gastrointestinal

>>tract for induction of mucosal immunity.[56]

>>

>>The vaccine candidate-recombinant urease-is urease antigen produced in

>genetically engineered E coli. Antigenically

>>indistinguishable from native urease, recombinant urease has an identical

>particulate structure but is enzymatically

>>nonfunctional and does not generate toxic ammonia in the presence of urea.

>This has been accomplished by cloning and

>>expressing in E coli only the genes for the structural subunits (ureA and

>ureB), omitting all other genes of the operon,[57] and

>>including those involved in insertion of Ni2+ required for enzymatic

>function. After expression in fermentation cultures of E

>>coli, the recombinant antigen is purified from bacterial lysates and is

>subsequently lyophilized in a stabilizer.

>>

>>Therapeutic Immunization

>>

>>Treatment of H pylori infection in patients with peptic ulcer disease is

>now an accepted health practice in the US[6] and

>>Europe and is the basis for regulatory labeling of

>antibiotic-antisecretory drug combinations. However, antimicrobial therapy

>>has a number of inherent limitations that might be overcome by use of an

>effective vaccine or a combined regimen of

>>antibiotics and vaccine. On average, primary treatment failures occur in

>15% of patients treated with antibiotics combined

>>with an antisecretory drug. Poor compliance with complex antibiotic

>regimens and antibiotic resistance in H pylori[58-60]

>>contribute to treatment failures. In contrast to antibiotics,

>vaccine-induced immunity is not expected to select for resistant or

>>more virulent organisms. Since immunologic mechanisms are distinct from

>those involved in antimicrobial treatment, vaccines

>>alone or synergistic activities of vaccines and antimicrobials could

>achieve the ultimate goal of 100% cure.

>>

>>Murine Studies

>>

>>Using recombinant urease[42,61] and crude cell antigens,[41] therapeutic

>activity has been documented in mice, with efficacy

>>rates (determined by gastric urease activity) between 50% and 94%. When

>vaccine and a partially effective antibiotic

>>regimen were combined, the latter proved to be more effective than either

>treatment alone.[62] These studies were conducted

>>in mice with subchronic H felis infection, the immunization regimen being

>applied only a few weeks after infecting the

>>animals. It is uncertain whether treatment would be as effective in a

>chronically infected host. Moreover, the reported cure

>>rates based on gastric urease or histologic endpoints overestimate the

>effectiveness of immunization. In addition, in the

>>mouse model, H felis is easier to eradicate than H pylori. The results

>with vaccine are also supported by the observation

>>that mice can be cured of H felis with a single antibiotic,[63] whereas

>multiple drugs were required to achieve partial cure of

>>H pylori.[64] When the H pylori mouse model was employed and therapeutic

>activity of urease-LT immunization was

>>measured by quantitative culture, a statistically significant (P = 0.0016)

>10-fold reduction in bacterial density (not eradication

>>of infection) was observed. Interestingly, the LT adjuvant alone appeared

>to have some effect in reducing infection, possibly

>>due to modulation of the immune response to antigens associated with

>natural infection.

>>

>>Ferret Studies

>>

>>In ferrets, immunization with urease and CT adjuvant resulted in

>presumptive cure of chronic H mustelae infection.[43] When

>>tested 6 weeks after immunization, 30% of the ferrets were cured of

>infection. A significant reduction in gastric inflammation

>>was demonstrated by histopathology in up to 60% of the animals.

>Interestingly, gastric inflammation was significantly

>>reduced in the cured and persistently infected vaccinated animals compared

>with infected controls, a finding similar to that

>>described in the rhesus monkeys.[72] The possibility that vaccines can

>diminish the pathologic consequences of Helicobacter

>>infections deserves further study.

>>

>>Adjuvants

>>

>>All preclinical studies reported to date have demonstrated efficacy of

>vaccination against Helicobacter infection, using

>>antigens given mucosally together with CT or LT as a mucosal adjuvant. No

>protection was achieved when antigens were

>>administered without a mucosal adjuvant, even at exceedingly high

>levels.[34] CT is not acceptable as a human adjuvant

>>because it induces diarrhea in humans at microgram levels.[65] LT is less

>reactogenic and has been tested clinically.[66] A

>>possible means to circumvent the reactogenicity of native toxins as

>adjuvants is the use of atoxic cholera toxin B subunit

>>(CTB) spiked with a low dose of native toxin. This combination was shown

>to be an effective adjuvant for an H felis

>>sonicate vaccine, providing protection against H felis challenge.[34] An

>even more attractive approach is the use of genetically

>>detoxified LT molecules, which are enzymatically inactive but still retain

>adjuvanticity.[67,68]

>>

>>Many novel adjuvants have shown promise in preclinical studies with a

>variety of other vaccines, including oil emulsions,

>>saponins, immunostimulating complexes, polyphosphazine, muramyl dipeptide

>derivatives, block polymers, vitamin D3,

>>liposomes, copolymer microspheres, and cytokines. Some data are now

>available from clinical trials; more is known about

>>many of these adjuvants for parenteral than for mucosal routes of

>administration. In studies of H pylori urease antigen, a

>>muramyl dipeptide derivative

>(N-acetylglucosaminyl-N-acetyl-muramyl-L-alanyl-D-isoglutamine, GMDP)

>delivered orally

>>did not elicit protection in mice against challenge with H felis.[34] Alum

>given parenterally with urease was partially effective

>>when given prophylactically (OraVax, unpublished data, 1997). An

>exploration of various adjuvants for parenteral

>>immunization with urease and for combined mucosal-parenteral immunization

>regimens is currently underway in our

>>laboratory and that of our partner, Pasteur Merieux Connaught. Preliminary

>data indicate that partial protection is achieved

>>by parenteral injection of antigen with alum and other select adjuvants.

>Since adjuvants orient the immune response in a

>>selective fashion with respect to T-helper subsets, the results of

>comparative studies will shed light on the role of Th1 and

>>Th2 responses in protection. Immunization studies of interleukin-4

>knock-out and gamma-interferon receptor deficient mice

>>indicate that both Th1 and Th2 responses are required for protective

>immunity.[69] This finding is also supported by our

>>observations of adjuvants having selective immunomodulatory properties.

>>

>>Clinical Trials

>>

>>Clinical testing of recombinant urease was initiated by our group in 1994,

>and trials of whole-cell and other recombinant

>>antigens are in the planning stages by others. Our clinical studies were

>begun in healthy infected volunteers (rather than

>>uninfected subjects) because of concern that immunization of naive

>individuals may potentiate inflammation upon subsequent

>>infection. This phenomenon was at that time observed in mice[33,42,70] but

>subsequently not observed in cats or monkeys. In

>>addition, because the immune correlates of protection remain problematic,

>it was believed that the direct measurement of a

>>therapeutic effect in infected subjects would have the greatest clinical

>significance.

>>

>>A limited study was first performed to demonstrate the safety and

>tolerability of oral administration of urease without a

>>mucosal adjuvant.[71] In a randomized, double-blind, placebo-controlled

>trial conducted by Kreiss and colleagues,[71] 6

>>infected asymptomatic adults were administered 4 doses of vaccine-each

>consisting of 60mg of recombinant H pylori

>>urease-by the oral route once a week. Six infected subjects received

>placebo. As expected in the absence of an adjuvant,

>>none of the vaccinated individuals mounted an immune response, and in

>gastric biopsies obtained before and 1 month after

>>vaccination, no change in bacterial density (measured by quantitative

>culture), inflammation, or mucosal damage was

>>observed. No adverse events was attributable to administration of urease.

>>

>>A second trial was conducted to determine the tolerability of

>coadministration of urease with a mucosal adjuvant (LT) in

>>healthy adults with H pylori infection and to obtain preliminary data on

>therapeutic activity. Preliminary results of this

>>trial-which was conducted at the Centre Hospitalier Universitaire,

>Lausanne, and at the Center for Vaccine Development,

>>University of land in Baltimore-were reported by Michetti and

>colleagues at the Helicobacter congress in

>>Copenhagen in 1996. Native LT purified from E coli was supplied by the

>Naval Medical Research Institute in Bethesda,

>>land, which previously reported adjuvant activity in a study involving

>cholera vaccine.[66]

>>

>>The controlled trial involved administration of 4 weekly, graded doses of

>urease (20, 60, or 180mg) with LT; placebo

>>vaccine with LT; or placebo vaccine and placebo adjuvant to groups of 4 or

>5 volunteers. The ELISPOT assay for

>>antibody-secreting cells (ASCs) in peripheral blood was the most sensitive

>determinant of immunologic response to the

>>vaccine. Six of 14 (43%) subjects who received urease, but none of the 10

>subjects who received placebo vaccine, had an

>>increase in IgA or IgG ASCs at 1 or more time points, measured 7 days

>after each successive dose of vaccine. Gastric

>>biopsies were obtained before and 1 month after completion of the

>immunization regimen. Differences were determined

>>between pre- and postimmunization H pylori densities in gastric mucosa.

>Pairwise treatment group comparisons were

>>performed at baseline and on the change from baseline to postimmunization.

>In addition, the significance of the mean change

>>from baseline to postimmunization was assessed within each treatment

>group. While the urease-treated groups were not

>>significantly different from control groups with respect to the change

>from baseline to postimmunization, the subjects

>>receiving active urease experienced, on average, a larger decrease in

>bacterial densities from baseline to postimmunization

>>(P = 0.032) than did subjects receiving placebo (P = 0.425). While the

>study had small sample sizes per group and was not

>>powered to detect significant differences between treatment groups, it

>provided the first clinical evidence for a therapeutic

>>activity of oral urease with LT adjuvant. The duration of the study was

>not sufficient to assess whether administration of the

>>vaccine was associated with a decrease in inflammation, as has been

>observed in ferrets[43] and rhesus monkeys.[72]

>>

>>Conclusions and Future Research

>>

>>A convincing body of data now exists supporting the potential for

>successful immunization against H pylori. However, we

>>are still at a preliminary stage in clinical development. The best

>immunogens, the best mode of presentation, the number of

>>doses needed, optimal age at immunization, expected benefit,

>cost-effectiveness, and other factors involved in vaccine

>>development require further study.

>>

>>The complex pathogenesis of this infection,[3,73] including the presence

>of antigens on H pylori shared with the host (a

>>mechanism for immune evasion),[48] demands novel approaches to the

>development of a final vaccine formulation. The

>>selection of defined and well-characterized recombinant subunit antigens

>appears to be the most viable approach, and the

>>urease antigen has so far proved most potent in eliciting protective

>immunity. It is reasonable to assume that more than 1

>>protective component is needed in a vaccine, and a number of such antigens

>in addition to urease have now been

>>discovered. The sequence analysis of the entire H pylori genome by Tomb

>and colleagues[74] will enhance antigen discovery

>>efforts. In addition to antigen composition, a successful vaccine must be

>delivered to the host in a manner that elicits

>>protective (therapeutic) immunity, particularly immunity expressed at the

>site of bacterial colonization (gastric mucosa). The

>>most appropriate means to achieve this end has not yet been fully defined.

>Mucosal routes of immunization with a classic

>>mucosal adjuvant (LT) have yielded the best results, but prophylactic

>(therapeutic) activity remains incomplete. Research is

>>needed on the mechanisms of protective immunity induced by vaccines, on

>the protein-specific immune responses to natural

>>infection, and on the functional role of T cells. Such studies may provide

>important data that lead to novel immunization

>>methods, as well as surrogate tests for protection that are useful in

>vaccine trials.

>>

>>Additional discussion of animal models for the development of Helicobacter

>vaccine can be found on Medscape

>>(www.medscape.com).

>>

>>Acknowledgments

>>

>>Original work described in this paper was funded in part by Pasteur

>Merieux Connaught (PMC) and by the National

>>Institutes of Health. The authors are grateful to PMC scientists,

>particularly Drs. Pierre Meulien, Marie- Quentin-Millet,

>>Farukh Rizvi, Bruno Guy, Ling Lissolo, and Veronique Mazarin (PMC, Marcy

>l'Etoile, France) for their scientific input

>>about the work and its interpretation. Drs. Pierre Michetti, Christiana

>Kreiss, Irene Couthesy-Theulaz, and Andre Blum

>>(Centre Hospitalier Universitaire, Lausanne, Switzerland); Kotloff

>and Genevieve Losonsky (Center for Vaccine

>>Development, University of land, Baltimore, Md.); and

>(University of land Medical Center,

>>Baltimore, Md.) conducted the clinical trials reviewed in this paper. Drs.

> Czinn and Nedrud (Case-Western

>>Reserve University, Cleveland, Ohio); Fox (Massachusetts Institute

>of Technology, Cambridge, Mass.); Andre

>>Dubois (Uniformed Services University of the Health Sciences, Bethesda,

>Md.); Soike (Tulane University,

>>Covington, La.); and ph Hill, Christian Stadtlander, Hal Farris, and

> Gangemi (Clemson University, Clemson,

>>S.C.) made significant contributions in many aspects of the testing of

>Helicobacter vaccine candidates in animal models. The

>>authors are especially grateful for the excellent assistance of OraVax

>personnel, including ph Simon, Kochi,

>> Tibbitts, Ingrassia, Gray, Kathleen

>Georgokopoulos, Amal Al-Gawari, , Rue Ding,

>>and Bruce Ekstein.

>>

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