ConfidentialMaterials omitted and filed separately with the Securities and Exchange Commission. Asterisks denote omissions.

Confidential Materials omitted and filed separately with the

Securities and Exchange Commission. Asterisks denote omissions.

The safety and immunogenicity of a single dose oral typhoid vaccine in Vietnamese healthy adults and children and identification and preparation of a field site for a Phase III efficacy study

N/A

A breakthrough has been made in the development of an improved oral typhoid vaccine through the application of functional genomics sciences. This has led to the identification of several genes in Salmonella species that are essential for virulence providing new gene targets for attenuation, resulting in the development of a novel live attenuated typhoid vaccine that can be administered orally and is safe and immunogenic at a single dose.

The live attenuated Salmonella typhi strain S. typhi (Ty2 aroC¯ssaV¯) ZH9 contains defined (independently attenuating) non-reverting deletions in two genes, aroC and ssaV. The ssaV gene is encoded on SPI-2, Salmonella Pathogenicity Island 2. SPI-2 encodes a type III secretion system and ssaV encodes a structural component of the secretory apparatus. SPI-2 is required for growth and survival within macrophages, one of the mechanisms by which S. typhi is spread systemically. Therefore, a mutation in this gene will prevent systemic spread of the vaccine strain, an important safety consideration for live vaccines.

The vaccine has been tested in clinical studies involving over 100 US and UK subjects and has shown to have an excellent safety profile (no bacteraemias or persistent shedding detected) and after a single dose, stimulates strong mucosal and systemic immune responses comparable to those stimulated by four doses of the currently licensed vaccine.

A single dose vaccine that does not require a needle for administration would bring enormous healthcare benefits to developing countries where typhoid is endemic and it is difficult to maintain the cold chain. The vaccine offers a new opportunity for the control and possibly the future eradication of typhoid from the wild.

The objectives of the project are to clinically evaluate the vaccine in healthy Vietnamese adults and children in Phase II studies and to set up a field site in preparation for efficacy testing. The project will be carried out in collaboration with the Oxford University Wellcome Trust Unit in Viet Nam, [**] bringing together considerable expertise in vaccinology and specifically on the development of typhoid vaccines.

The STA is critical to the eventual commercialisation of the product in any territory. The commercial market for typhoid vaccines is not large and it is difficult for Microscience to justify funding the whole programme required to gain approval of the product, either as a traveller’s vaccine, or in developing countries, given that the Company has a number of more commercially attractive vaccines in the portfolio.

However, Microscience recognises the importance of this vaccine in providing substantial healthcare benefits in the developing world and would like to ensure that if the vaccine is successful, those benefits are delivered.

The route to commercialisation of the vaccine whether it is for travellers or the developing world will involve carrying out a large efficacy study in a country where typhoid is endemic. The STA proposal is critical for taking the first steps in this process and addressing one of the key technical barriers relating to transfer of vaccines from the developed to the developing world, that is whether the safety and immunogenicity profiles will be similar, particularly in children who will form the bulk of the cohort in the efficacy study. It will be difficult to obtain further investment into the project until this key question has been answered. It is also essential, in parallel to the clinical studies, to establish a field site than can subsequently be used for the efficacy study. Establishing the incidence rate for typhoid will allow detail planning of the efficacy study, particularly in terms of the numbers of subjects that will need to be involved giving an accurate view of what future funding will be required for the efficacy study which will be pivotal for gaining approval of the product.

If the vaccine proves to be successful in the stepping stone studies and in the establishment of the field site in Viet Nam it should provide leverage for obtaining additional funding, either from commercial or NGO sources.

Typhoid fever remains a major disease of the developing world. The spread of bacteria that are resistant to all affordable antibiotics raises the possibility of untreatable typhoid fever and a return to the pre-antibiotic era when 30% of patients died. There is no currently available affordable vaccine that offers long term protection after a single dose. The application of genomic sciences has resulted in a breakthrough in the development of an improved typhoid vaccine that can be given orally and is effective at a single dose. The vaccine has been tested in studies involving over 100 subjects in the US and UK and has shown to have an excellent safety profile and after a single dose, stimulates the body to make immune responses equivalent to those stimulated by four doses of the currently licensed vaccine. A single dose vaccine that does not require a needle for administration would bring enormous healthcare benefits to developing countries where typhoid is endemic and it is difficult to maintain the cold chain. The vaccine offers a new opportunity for the control and possibly the future eradication of typhoid from the wild. The objectives of the project are to clinically evaluate the vaccine in healthy Vietnamese adults and children and to identify and set up a field site in Viet Nam where a future study can be carried out to assess whether it can protect against typhoid fever.

The STA is critical to the eventual commercialisation of the product in any territory. The commercial market for typhoid vaccines is not large and it is difficult for Microscience to justify funding the whole programme required to gain approval of the product, either as a traveller’s vaccine, or in developing countries, given that the Company has a number of more commercially attractive vaccines in the portfolio.

However, Microscience recognises the importance of this vaccine in providing substantial healthcare benefits in the developing world and would like to ensure that if the vaccine is successful, those benefits are delivered.

The route to commercialisation of the vaccine whether it is for travellers or the developing world will involve carrying out a large efficacy study in a country where typhoid is endemic. The STA proposal is critical for taking the first steps in this process and addressing one of the key technical barriers relating to transfer of vaccines from the developed to the developing world, that is whether the safety and immunogenicity profiles will be similar, particularly in children who will form the bulk of the cohort in the efficacy study. It will be difficult to obtain further investment into the project until this key question has been answered. It is also essential, in parallel to the clinical studies, to establish a field site that can subsequently be used for the efficacy study. Establishing the incidence rate for typhoid will allow detail planning of the efficacy study, particularly in terms of the numbers of subjects that will need to be involved, giving an accurate view of what future funding will be required for the efficacy study which will be pivotal for gaining approval of the product.

If the vaccine proves to be successful in the stepping stone studies and in the establishment of the field site in Viet Nam it should provide leverage for obtaining additional funding, either from commercial or NGO sources.

N/A

To date the only source of funding has been through Microscience Ltd, a privately owned UK company working on the development of novel vaccines and immunotherapeutics for prevention and treatment of infectious disease. The Company was founded on vaccine technology developed at Imperial College and was spun out in 1997. To date the Company has raised £[**] in the form of private equity investment. The major investors are Merlin, Apax Partners, Advent Venture Partners and J.P. Morgan Partners. The funding has enabled Microscience to develop five vaccine products, including the oral typhoid vaccine, all of which are now undergoing clinical testing in the U.K. and U.S.

To date approximately £[**] has been spent by Microscience on the development of the oral typhoid vaccine over the past five years. It is estimated that approximately £[**] is still required to complete all the activities required to commercialise the product.

No

The typhoid vaccine candidate contains mutations in aroC and ssaV.

W096/17951 — Contains claims to use of strains harbouring mutations in Salmonella Pathogenicity Island 2, including the ssaV gene.

W000/68261 — Claims to the combination of aroC and ssaV. Claim for the combination being particularly useful for making attenuated mutants of Salmonellae.

The overall objective of the proposal is to translate the Phase I/II clinical findings from an improved oral typhoid vaccine being developed in the UK and US, to a country where typhoid is endemic, in order to prepare for a large field study in which the vaccine can be evaluated for efficacy. This will be achieved by carrying out a Phase II programme in Viet Nam in healthy adult subjects and school age children in order to confirm the safety and immunogenicity of the vaccine in a Vietnamese population, providing assurance that the vaccine is suitable for use in a large efficacy study in the Mekong Delta of Viet Nam. The proposal will address one of the key technical barriers associated with transfer of vaccines from developed to developing countries; the vaccine has so far been tested in healthy subjects in countries where typhoid is not endemic, but it is known that the safety and immunogenicity profiles of vaccines, particularly live vaccines given orally, can be different in subjects where the disease is endemic and where the socio-economic conditions are very different. The proposed project will therefore highlight any differences between the different populations and will provide new scientific data on this issue that will be useful for the development of other vaccines.

The other major objective will be to identify and prepare a site in the Mekong Delta where a large field study can be performed to establish the efficacy of the vaccine. The site development will be carried out in parallel to the Phase II programme described above.

In order to achieve the overall objective the key objectives of the project are as follows:

Initiation of a Phase II programme in Viet Nam in healthy adult volunteers and children:

	•		Identification of site
	•		Clinical protocol development
	•		Regulatory approval from Vietnamese and US authorities
	•		Assay transfer
	•		Manufacture and release of clinical material
	•		Screening and recruitment of subjects

Demonstration of safety, tolerability and immunogenicity in Vietnamese adults and children

	•		Dosing
	•		Monitoring of study (safety and GCP)
	•		Evaluation of safety
	•		Completion of immunoassays
	•		Management and validation of data

Preparation of field site for Phase III study

	•		Identification of site
	•		Establishment of infrastructure
	•		Perform Census
	•		Establishment and provide training in diagnostic tools
	•		Disease surveillance

The proposal could lead to considerable healthcare benefits for both the developing and developed world. The activities outlined in the proposal are the first steps in a clinical evaluation of the vaccine critical for moving to an efficacy study in an endemic area. Such a study will be required for approval of the vaccine in any country since there are no accepted correlates of immunity. All other typhoid vaccines have been approved on efficacy data generated in endemic areas. Typhoid fever remains a very significant global health problem with an estimated 17 to 33 million cases occurring worldwide annually resulting in 600,000 deaths, virtually all these cases occur in the developing world. In the last few years there has been the worrying development and global spread of bacteria that are resistant to all affordable antibiotics. Over 90% of isolates in Southern Viet Nam are resistant to all first line antibiotics making the need for an effective and affordable vaccine more urgent. There are licensed vaccines available to prevent typhoid fever but these are less than ideal for control of typhoid fever in developing countries. The currently licensed oral typhoid vaccine achieves protection in 60-70% of recipients but requires four doses resulting in compliance issues and difficulty in maintaining the cold chain. The single dose injectable vaccine based on the surface polysaccharide Vi is also not ideal as it’s administration requires the use of needles and being a polysaccharride, it is poorly immunogenic in young children. The incidence of typhoid fever is highest in school age and pre-school age children in developing countries.

A single dose vaccine that is immunogenic in young children and does not require a needle for administration would therefore bring enormous healthcare benefits to developing countries where it is difficult to maintain the cold chain that is required for multi-dose vaccines. Furthermore, because S. typhi is a human restricted pathogen, has no animal reservoirs and does not persist in the environment, the vaccine also offers a new opportunity for the control and possibly the future eradication of typhoid from the wild.

Additional healthcare benefits will be derived from setting up of a field site for testing efficacy. Establishing the infrastructure and expertise at a potential field site will also provide information on the general disease burden of the population making it possible to derive other healthcare strategies to address other disease targets.

Finally, the proposal could also lead to the approval of the vaccine for the prevention of typhoid fever in travelers to endemic areas.

Modern molecular biology techniques and increasing knowledge of Salmonella pathogenesis have led to the identification of several genes that are essential for in vivo growth and survival of these organisms. (1) This has provided new gene targets for attenuation, leading to the concept that future live vaccine strains can be rationally attenuated by introducing defined mutations into selected genes that are known to be involved in virulence. This will facilitate the development of improved vaccines, particularly in terms of the immunogenicity and therefore the number of doses that have to be given.

Microscience has constructed a Salmonella typhi vaccine strain S. typhi (Ty2 aroC¯ssaV¯) ZH9 that contains defined (independently attenuating) deletions in two genes, aroC and ssaV. (2) The aroC gene encodes chorismate synthase, an enzyme involved in the biosynthesis of aromatic compounds. Aro mutations are well described as being attenuating for Salmonella in humans, (2) and the basis is presumed to be that two aromatic compounds, para-aminobenzoic acid and 2,3-dihydroxyaminobenzoic acid are limiting for growth in mammalian tissue. The ssaV gene is encoded on SPI-2, Salmonella Pathogenicity Island 2, SPI-2 encodes a type III secretion system and ssaV is believed to encode a structural component of the secretory apparatus. SPI-2 mutations have previously been shown to be attenuating in mice, (3) and there is evidence that this is because SPI-2 is required for growth within macrophages, the mechanism by which S. typhi is thought to be spread systemically. Therefore, a mutation in this gene will prevent systemic spread of the vaccine strain.

S. typhi (Ty2 aroC¯ ssaV¯) ZH9 is derived from the virulent S. typhi strain Ty2. This strain has been used as a background strain for constructing candidate typhoid vaccines previously evaluated in volunteers. The currently licensed live Salmonella vaccine is derived from this strain.

S. typhi (Ty2 aroC¯ ssaV¯) ZH9 is being developed as a single dose, orally delivered vaccine that will protect against typhoid fever. (4)

The attenuation of S. typhi (Ty2 aroC¯ ssaV¯) ZH9 has been demonstrated directly by comparing the replication of S. typhi (Ty2 aroC¯ ssaV¯) ZH9 with S. typhi (Ty2 aroC¯) DTY8, which harbours a single aro mutation, in human macrophage-like cells in the presence and absence of aromatic compound supplements. S. typhi (Ty2 aroC¯ ssaV¯) ZH9 was highly attenuated for growth in macrophages since it failed to replicate and bacterial killing was observed, even in the presence of aromatic compounds. (5) This is an important finding, demonstrating the utility of mutations in SPI-2 as an attenuation strategy for Salmonella. This adds an additional level of safety to the strain.

The attenuation of vaccine strain S. typhi (Ty2 aroC¯ ssaV¯) ZH9 has also been demonstrated indirectly by comparing the attenuation of Salmonella typhimurium (S. typhimurium) strains harboring similar mutations in mice. S. typhi does not infect mice. However, genetically-susceptible mice infected with S. typhimurium develop a systemic “typhoid-like” disease and this mouse typhoid model has been used to predict the usefulness of attenuating mutations in S. typhi. This model has been used to demonstrate a safety advantage of a mutation in SPI-2 (such as ssaV) in combination with an aro mutation, over that of an aro mutation alone. (5)

Both IFN-y and IL-12 are required for control of Salmonella infection in mouse and man, therefore, mice deficient in either IL-12 or IFN-y are useful models of immunocompromised humans. The attenuation of a SPI-2 (ssaJ) transposon insertion mutant and an aro (aroA) transposon insertion mutant of S. typhimurium were compared in both IFN-y KO mice and in mice treated wit h IL-12 specific antibodies. No clinical symptoms of disease were observed with the SPI-2 mutant in either class of immunocompromised mouse and clearance from the tissues was observed. In contrast, the aro mutant replicated to high levels in the tissues and caused death of both the IFN- KO and anti-IL 12-treated mice. Further studies sought to assess the attenuation of S. typhimurium harboring defined deletions in aroC and ssaV identical to those present in S. typhi (Ty2 aroC¯ ssaV¯) ZH9. The aroC ssaV double mutant strain S. typhimurium (TML aroC¯ ssaV¯) WTO5 was completely attenuated in IFN — KO mice (100% survival) whereas the single aroC mutant caused up to 100% mortality. (5) Further studies in conventional mice showed that the aroC ssaV double mutant colonized the mouse tissues at lower levels than a single aroC mutant and was cleared more rapidly from the tissues. These data generated in the mouse typhoid model further demonstrate the utility of mutations in SPI-2 in combination with an aro mutation as an attenuation strategy for Salmonella and provide confidence that S. typhi (Ty2 aroC¯ ssaV¯) ZH9 will be highly attenuated in humans with an increased attenuation over single aro mutants.

Give relevant technical background information (continued)

To date S. typhi (Ty2 aroC¯ ssaV¯) ZH9 has been administered to over 80 healthy adult volunteers in three studies. Full details and results of these studies are provided in Appendix A in the form of a published paper and two clinical trial study reports. A summary of the design of the studies and results is provided below:

In the first study (Study MS01.01) 9 subjects (3 per cohort) received doses of either 10⁷, 10⁸ or 10⁹ CFU of a frozen formulation of the vaccine. (4) In the second study (Study MS01.03) 48 subjects (16 per cohort) received doses of 5 x 10⁷, 5 x 10⁸ or 5 x 10⁹ CFU of a freeze-dried formulation of the S. typhi (Ty2 aroC¯ ssaV¯) ZH9 vaccine. In the third study (Study MS01.04) 32 subjects received 5 x10⁹ CFU of freeze-dried S. typhi (Ty2 aroC¯ ssaV¯) ZH9 in one of two different Presentation solutions (16 per group). These studies indicate that the vaccine is highly immunogenic at a single dose with a good safety profile. The immunogenicity achieved with one dose is comparable to that obtained after 4 doses of the currently licensed oral typhoid vaccine.

Study MS01.03 was a placebo controlled, double blind, out-patient, single dose, dose escalating study conducted under an Investigational New Drug (IND). The study was designed to determine the safety, tolerability and immunogenicity of three dose levels of the vaccine which consisted of a freeze-dried formulation of the live attenuated vaccine strain S. typhi (Ty2 aroC¯ ssaV¯) ZH9 and excipients. A total of 60 healthy adult volunteers were recruited in 3 cohorts of 20. In each cohort, 16 subjects were randomized to receive vaccine and 4 to receive placebo. The cohorts were dosed sequentially such that the first cohort received a single dose of 5x10⁷ CFU of S. typhi (Ty2 aroC¯ ssaV¯) ZH9 or placebo, the second cohort received 5x10⁸ CFU of S. typhi (Ty2 aroC¯ ssaV¯) ZH9 or placebo and the third cohort received 5x10⁹ CFU of S. typhi (Ty2 aroC¯ ssaV¯) ZH9 or placebo. The vaccine or placebo was administered in 50ml 2% bicarbonate following the prior ingestion of a volume of 2% bicarbonate to neutralise stomach acid.

The important consideration for safety of a live, attenuated strain of S. typhi is that following ingestion the strain is not able to disseminate throughout the body and give rise to bacteraemia or shed persistently from the gut. The safety monitoring was designed to show that this did not occur with MICRO-TY.

The S. typhi (Ty2 aroC¯ ssaV¯) ZH9 vaccine exhibited an excellent safety profile and was found to be well tolerated by all of the subjects dosed. None of the blood or urine cultures taken during the study were positive for S. typhi (Ty2 aroC¯ ssaV¯) ZH9. Shedding of S. typhi (Ty2 aroC¯ ssaV¯) ZH9 in the stools of the subjects did not continue beyond 6 days for any subject, at any of the three dose levels. The number of subjects shedding increased with increasing dose level.

There were no serious adverse events (SAEs) related to study medication and there was no statistically significant difference in the incidence of adverse events (AEs) between subjects receiving the vaccine and those who received placebo.

The immune responses measured were the ability to elicit serum IgG antibody against LPS (humoral immune response), and the presence of gut derived anti-LPS lgA antibody secreting cells in the blood, which be indicative of the priming of the gut mucosa (mucosal immune response). The correlates of protection for typoid are not clearly understood, hence the need to carry out an efficacy study in order to demonstrate protection. However, for the currently licensed oral typhoid vaccine (Vivotif, Bema vaccines) which requires four doses, results of the two immunological assays used in assessing this vaccine have been found to putatively correlate with the protection conferred by different formulations and immunisation schedules of Vivotif in field trials (6,7). The identification of these measurements as putative immunological correlates of protection provides an invaluable tool for use in early clinical trials evaluating new live oral typhoid vaccines. (2,4).

In study MS01.03 all three dose levels of S. typhi (Ty2 aroC¯ ssaV¯) ZH9 were shown to be immunogenic, with the highest dose level being immunogenic in all subjects. In both the 5 x10⁷ and 5 x 10⁸ CFU dose groups 9 (56%) subjects in each group mounted an immune response detected by either an anti-LPS ELISPOT response on Day 7 or an anti-LPS serum IgG response on Day 28. This number increased to 16 (100%) in the 5 x 10⁹ CFU dose group. None of the subjects that received placebo mounted an immunological response against S. typhi (Ty2 aroC¯ ssaV¯) ZH9.

The vaccine presentation used in this study requires preparation in non-chlorinated water and involve pre-dosing with a volume of buffer prior to vaccine administration; this is not a convenient method of preparation and administration. The purpose of study MS01.04 was to assess safety and immunogenicity following administration using a presentation that will be more convenient to prepare and administer; it can be prepared in any water and pre-dosing with a volume of buffer will not be required. This presentation will be suitable for commercialisation. Study MS01.04 was performed in the US under the IND. It was an open label, single oral dose (5 x 10⁹ CFU), out patient study involving 32 healthy adult volunteers. 16 subjects were vaccinated using the presentation used for study MS01.03 (Presentation 1) and 16 were vaccinated using the new presentation (Presentation 2).

The database for this study was locked in February 2004 and the data are currently being analysed. Preliminary results are summarised below:

S. typhi (Ty2 aroC¯ ssaV¯) ZH9 demonstrated an excellent safety profile. There were no serious or severe adverse events reported during the study. No subjects reported a fever considered attributable to the vaccine, there were no bacteraemia, no persistent shedding in stools (no subject shed beyond day 6), and no clinically significant changes in haematological and biochemical profiles.

An immune response was elicited in the majority of subjects following administration of the vaccine and there was no evidence of a difference between the presentation groups in the proportion of subjects showing an immunogenic response detected in either the ELISA or ELISPOT assay; fifteen subjects (94%) who received the vaccine using Presentation 1 showed a response; and fourteen subjects (93%) who received the vaccine using Presentation 2 showed a response.

Microscience is also planning to carry out a clinical trial in South Korean healthy adult volunteers during Q4 of 2004. This will be similar in design to the first study that will be carried out in Vietnam. It is designed to evaluate the safety and immunogenicity of a single dose of the intended commercial dose of the vaccine in 28 healthy Asian volunteers in South Korea. It will be blinded and placebo controlled (16 will receive active and 12 placebo). This study is at an advanced stage of planning and Microscience is now committed to it. It was originally considered that it would be necessary to carry out this study in order to provide comfort to the Vietnamese authorities that safety and immunogenicity had been demonstrated in an Asian population prior to being able to move the programme to Vietnam. However, further discussion with the Wellcome Trust Unit in Vietnam indicates that the initiation of the first study in Vietnam will not be dependent on results from this study. Nevertheless, it is considered that initiating a study in the region ahead of the first Vietnam study will be very helpful in securing regulatory approval in Vietnam and, together with the studies in Vietnam it will also provide valuable information on the uptake of the vaccine in individuals of different genetic backgrounds.

The study will be performed at the Clinical Trial Centre based at Seoul National University (SNU), South Korea. Ethics approval submissions have been made and initial discussions have taken place with the Korean FDA. A submission to Korean FDA is being made on the 30^th June 2004. It is planned to dose the first subject during the first week of October 2004. Initiation of studies in Viet Nam is not dependant on data from this study in Korea.

In summary, Microscience has developed a novel single dose oral vaccine that has shown to be safe and immunogenic in healthy U.K. and North American volunteers. There is clearly an unmet medical need in the market for a vaccine that can overcome the compliance issues associated with multi-dose vaccines, provide good immunological memory after a single dose and potentially have increased efficacy. Such a vaccine could potentially have a big impact on the control of typhoid in endemic areas and also represent a big improvement for prevention of typhoid to traveller’s to those areas.

It is considered that the Microscience product is the lead improved typhoid vaccine in development. It has been developed under a Company sponsored IND and is now in Phase II development in outpatient studies in the US. The clinical studies have used a product manufactured from a process that is capable of being commercialised and it is delivered in a commercial presentation that is acceptable to regulatory authorities.

Objective 1. Initiation of a Phase II programme in Viet Nam in healthy adult volunteers and children

	•		Identification of site. Responsibility of the clinical investigators [**]. It is important that phase II safety and immunogenicity data is generated in subjects representative of the target population for the field study. It is therefore intended that a number of the phase II studies will be performed in the endemic region in the Mekong Delta. However, at least the first study, in adult volunteers, will be undertaken under well-controlled conditions at the Oxford University Clinical Research Unit at the Hospital of Tropical Diseases in Ho Chi Minh City. This will ensure that technology is transferred, staff are trained and the regulatory approval process is undertaken as efficiently as possible, prior to moving the programme into the endemic region. Once vaccine safety has been adequately demonstrated in this study approval will be sought to perform subsequent studies in the endemic region.
	•		Clinical protocol development. Individual protocols will be written for each of the studies. The protocols will be owned by Microscience but developed jointly by Microscience and the clinical investigators ([**]).
	•		Regulatory approval. Microscience will be responsible for obtaining regulatory approval for the clinical studies and for field site development. Approval will be sought from both the Vietnamese and US regulatory agencies, as it is intended that the clinical studies will be performed under the US IND that is held for this product by Microscience.
	•		Local ethics approval. The clinical investigators ([**]) will be responsible for obtaining ethics approval from the local Independent Review Board (IRB). Microscience and the clinical investigators ([**]) will be responsible for preparing the documentation required for submission.
	•		Assay transfer. Assay transfer from Microscience to the Hospital for Tropical Diseases is the responsibility of both parties and is expected to take 1 month. The immunoassays being used for these studies have been standardised at Microscience. Assay transfer will ensure that assay performance is comparable at the Hospital for Tropical Diseases, at Microscience and in previous clinical studies. The process involves performing the assays multiple times using reference samples. Transfer will be considered successful once an analyst has performed a pre-defined number of assays that have each met the validity criteria.
	•		Manufacture and release of clinical material. Responsibility of Microscience.

The clinical material will be vaccine, placebo and [**], manufactured to cGMP. [**]

Adult study. It is anticipated that existing batches of vaccine, placebo and [**] will be used for the adult study; only packaging and labelling will be required for this study. Following review of packaging and labelling batch records and receipt of regulatory and ethics approval, the material will be released by Microscience for use in the clinic.

Studies in children. New batches of vaccine and placebo will be manufactured in Q1 2005 for the studies in children. Following packaging and labelling, review of manufacturing batch records and receipt of regulatory and ethics approval, the material will be released by Microscience for use in the clinic.

Screening and recruitment of subjects. Responsibility of the clinical investigators ([**]). To begin once regulatory and ethics approval have been obtained. For each subject screening has to occur within 28 days of dosing. All efforts will be put in place to obtain written consent that is informed and given voluntarily as described in Appendix C.

(d)

Workplan (continued)

Objective 2. Preparation of field site for Phase III study

•

Identification of site. [**] will have joint responsibility.

The decision as to which area will be the site for the efficacy study and therefore for surveillance will be based on known levels of incidence as defined by current government statistics, predicted levels of incidence and the ease with which the required infrastructure can be put in place. The site will be in the Mekong Delta region of Viet Nam. This area has been chosen because population based surveillance studies for typhoid fever have previously been carried out in this region in 1995/1996. (8) It was found that the incidence level was high (overall it was 198 per 10⁹ of the general population). The highest attack rate was among the 5-9 year olds and lowest in the >30 year olds. It was concluded from these studies that typhoid fever is highly endemic in Viet Nam and is a significant disease in both pre-school and school aged children. A region in the Mekong Delta [**] has been selected for this proposal.

A typhoid surveillance study will be conducted in the proposed field-site and data will be collected for at least one year prior to phase III immunization commencing and will continue throughout the duration of the efficacy study. The end-point of the pre-study surveillance will be a rate of incidence of typhoid fever in the study population. These data will be used to determine the number of subjects to be entered into the phase III efficacy study.

	•		Establishment of infrastucture. [**] will have joint responsibility.
	•		Perform Census. [**]will have joint responsibility.

A census will be performed in the study area. [**] Each of these households and each individual residing in the household is given a unique identification number (ID#), which is used for all interactions that occur as part of the study. ID numbers will be allocated based on the serial number of the census form, and the sequential order within each household. The aims of the census will be as follows: To assign a unique study number to each household

	•		To assign a unique study identification number of each individual resident in the household
	•		To obtain base-line data on socio-economic status, health seeking behaviour, prior typhoid vaccine usage and potential typhoid risk factors
	•		To provide the household members with information on the project

	•		Establish and provide training in diagnostic tools. [**] will have joint responsibility.
	•		Disease surveillance, [**] will have joint responsibility.

The surveillance system will rely on patients attending existing healthcare facilities in the endemic region (government health care facilities and participating private healthcare physicians). This healthcare facility-based passive surveillance system will be aimed at detecting the majority of reported cases of typhoid fever among study participants seeking medical care. Medical personnel will interview, examine, and obtain a venous blood specimen for laboratory investigation from all patients living in the study area with [**]

All relevant clinical information will be recorded using standard procedures and will include: date and time of examination; name; study ID number; age and gender; full address; name of head of household; number of days since disease began; symptoms and signs of the disease. The venous blood sample will be taken to identify the presence of S. typhi by blood culture. Serum will also be taken for typhoid fever serological assays.

Typhoid fever proven cases will be given antibiotic treatment as appropriate. It is anticipated that S. typhi resistance patterns will be monitored regularly throughout the study.

A crucial feature of the surveillance programme will be the accurate identification of all patients attending healthcare facilities, who are study participants. To accomplish this, each clinical supervisor will attempt to identify patient ID numbers using a computer search programme (for names, age ranges, dates of birth, sex, names of the head of households).

Additionally, all culture-proven cases and positive serological cases will be visited at home to confirm the identification of the patient; to assess clinical progress; to assess any typhoid fever-related disability; to determine typhoid carrier status and to apply a verbal autopsy when needed. Culture-proven typhoid fever cases will be visited [**] after onset of illness. Follow-up questionnaires will be completed at each visit. Stool samples will also be collected at the end of the post-immunization [**] year follow-up on all typhoid fever cases during a home visit, with the aim of identifying typhoid carriers.

Objective 3. Demonstration of safety, tolerability and immunogenicity in Vietnamese adults and children

The clinical programme will involve Vietnamese subjects of a broad age range (approximately 30-5 years) and will be run as a series of age-descending studies as it will be important to demonstrate that the vaccine is safe when administered to adults and adolescents prior to administering it to children. Volunteers will be stratified into three different age brackets: adults (18-30 years); older children (10-18 years); young children (5-10 years).

A. Study to demonstrate safety, tolerability and immunogenicity in healthy Vietnamese adults

The purpose of the study is to evaluate the immunogenicity and safety of [**] S. typhi (Ty2 aroC¯ ssaV¯ ) ZH9 oral typhoid vaccine in approximately [**] healthy adult volunteers (age 18 – 30 years inclusive) from Viet Nam. The study will be a single centre, single-blind, placebo-controlled, randomised study. The study schedule is show in Table 12.1. The study design will be such that there will be two groups of subjects, Group 1 will receive vaccine and Group 2 will receive placebo.

Table 12.1 Study schedule for study in healthy adult volunteers

Visit

Screen

1

2

3

4

5

6

Day

-28 to – 2

0

1

7

10

14

28

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Objectives/key tasks

•

Dosing. Responsibility of the clinical investigators ([**]).

Following completion of the screening assessments subjects who satisfy the study entry criteria will be randomised to one of the two treatment groups and will receive medication on Day 0. Subjects will be split into at least [**] cohorts for dosing. The time between dosing each cohort may be at least [**] weeks; it may therefore take [**] weeks to complete dosing of all subjects. The size of the cohorts will be governed by the number of [**] samples that can be processed on one occasion. In each cohort some subjects will receive vaccine and some will receive placebo. The vaccine will be nominal dose of [**] typhoid vaccine administered in [**] of presentation solution. Placebo will also be administered in [**] of presentation solution. Vaccine will be prepared in the pharmacy and administered to the subjects within [**] minutes of preparation.

Subjects will return to the investigative site on study Days [**] for assessment of safety and immunogenicity and to provide blood, urine and stool samples as required. Subjects will record their temperatures on a Diary Card for the first [**] days following dosing and will return to the clinic for additional visits should they develop fever.

•

Evaluation of safety. Responsibility for the clinical investigators ([**]).

The primary safety endpoints for the study are the proportion of subjects:

•

reporting adverse events during the study, particularly a fever of more than [**]° C, attributable to the study medication.

The secondary safety endpoints are the proportion of subjects:

	•		withdrawn from the study due to adverse events, including bacteraemia attributed to study medication
	•		demonstrating bacteraemia, attributable to the study medication
	•		demonstrating persistent faecal shedding ([**]) of S. typhi (Ty2 aroC¯ ssaV¯ ) ZH9, in stools
	•		with changes in laboratory parameters from Day 0 to post treatment which are considered clinically significant

•

Completion of immunoassays. Responsibility of the clinical investigators ([**]).

As in previous studies in the UK and US, immunogenicity will be assessed by measuring immune response against S. typhi lipopolysaccharide (LPS); using the [**] to measure numbers of circulating antibody secreting cells producing anti-LPS IgA, and an ELISA assay to measure serum IgG response against LPS. Subjects will be considered to have an immune response if they achieve the following: a [**].

[**]. ELISA assays will be performed on frozen serum samples. It is anticipated that data from the pivotal immunoassays, [**] to detect secretory IgA against LPS and ELISA to detect serum IgG against LPS, will be available for review within [**] weeks of dosing the first subject.

	•		Monitoring of study (safety and GCP). Responsibility of Microscience.
	•		Management and validation of data. Responsibility of Microscience.

Once a full dataset is available from safety assessments and from the pivotal immunoassays, the data will be evaluated by Microscience, the clinical investigators, the regulatory agencies and by the local ethics committee to establish whether it provides adequate confidence to progress to evaluation of safety and immunogenicity in children.

B. Study to demonstrate safety, tolerability and immunogenicity in Vietnamese children.

This section of the programme will involve a series of age-descending studies. The purpose is to evaluate safety and immunogenicity of the vaccine in children from 5-18 years. Approximately [**] children will be involved, stratified into 2 age groups: 10-18 years, approximately [**] subjects; 5-10 years, approximately [**] subjects.

The studies will be single centre, single-blind, placebo-controlled, randomised studies. The basic study schedules will be as for the adult study shown in Table 12.1.

Objectives/key tasks for each study

	•		Dosing. Responsibility of the clinical investigators ([**]). Group 10-18 – years it is anticipated that data from the adult study will support the use of the same dose level, [**] dose, and formulation of vaccine as was administered to adults.
			Group 5-10 years — evaluation of safety data from the study in 10-18 year olds will determine whether dose escalation will be required for this study. Dose escalation will require an additional group of subjects who will receive a lower vaccine dose level (likely to be [**]).
	•		Evaluation of safety. Responsibility of the clinical investigators ([**]). As for adult study.
	•		Completion of Immunoassays. Responsibility of the clinical investigators ([**]).
			The assays used will be the same as for the adult study.
	•		Monitoring of study (safety and GCP). Responsibility of Microscience.
	•		Management and validation of data. Responsibility of Microscience.

Once a full dataset is available from safety assessments and from the pivotal immunoassays, the data will be evaluated to establish whether it is adequate to support entry of children into the phase III field study.
 

(a) Intellectual property and freedom to operate

The product is a mutated Salmonella bacterium. The ssaV gene and the aroC genes are deleted. The ssaV gene was identified as part of the Salmonella Pathogenicity Island-2 (SPI-2) using Signature Tagged Mutagenesis (STM). Both the method (STM) and the pathogenicity island, including its genes and attenuated mutants, are claimed in WO96/17951. The particular combination of an ssaV mutation with an aroC mutation is claimed in WO00/68261.

Relevant claims relating to this product and directed to genes of the SPI-2 region and attenuated mutants were initially present in WO 96/17951 and WO 00/68261. The major claims As mentioned above, these claims were subsequently filed in divisional applications which have now proceeded to grant in both the US and the EPO. In Europe, the claims provide coverage for particular DNA sequences from Salmonella virulence genes; antisense nucleic acids; bacteria having mutations in one or more particular virulence genes (one of these being ssaV); virulence genes; promoters of virulence genes; polypeptides; pharmaceutical compositions and methods of identifying compounds which reduce the ability of a microorganism to adapt to a particular environment involving selecting compounds which interfere with the function of a gene covered by the earlier claim. Similar claims have been granted to Microscience and Imperial in the US.

A further European divisional application has also been filed. This divisional application, no. 01205191.8, is also registered in the joint names of Imperial and Microscience. It is at a relatively early stage of prosecution but could, conceivably, provide useful additional peripheral protein in relation to this product. This divisional application is directed to particular DNA sequences isolated from a Salmonella genome and virulence genes containing such DNA; a method of identifying a compound which reduces the ability of a microorganism to adapt to a particular environment, and compounds identified by the method; the use of such a compound in the manufacture of a medicament for treating infection of a host organism with a particular microorganism; molecules which selectively interact with and inhibit a virulence gene; use of such compounds in manufacturing medicaments for treating hosts which have or are susceptible to an infection with the microorganism; mutant bacteria; vaccines and pharmaceutical compositions; methods of making mutant microrganisms; and methods of making a pharmaceutical formulation of a mutant microorganism. Certain claims, eg relating to DNA sequences or to antisense nucleic acids capable of interacting with and inhibiting virulence genes, have had only ‘technolocial background’ references cited against them in the EPO search report, indicating that at least some of the claimed subject matter is likely to be patentable.

The typhoid vaccine product is also covered by international application no. PCT/GB00/01749. This application has entered a very wide range of national phases. This reflects the number of territories in which typhoid is endemic.

In summary, Microscience has already obtained effective protection for its typhoid vaccine product. Further related protection can also be expected in many territories.

The market is driven by the number of travellers to the typhoid risk regions. During 2000, there were a total of approximately 40 million visits from the US, Europe and Japan to destinations with a risk of typhoid in Africa, Asia and Central and South America. However, it must be noted that this number has been negatively impacted by world events since then.

However, the greatest need for the vaccine is in the endemic areas themselves. Typhoid fever remains a very significant global health problem with an estimated 17 to 33 million cases occurring worldwide annually resulting in 600,000 deaths throughout the world, virtually all these cases occur in the developing world. In the last few years there has been the worrying development and global spread of bacteria that are resistant to all affordable antibiotics. Over 90% of isolates in Southern Viet Nam are resistant to all first line antibiotics making the need for an effective and affordable vaccine more urgent. There are licensed vaccines available to prevent typhoid fever but these are less than ideal for control of typhoid fever in developing countries.

The current market for typhoid vaccines is about $100 million in US and Europe and around $120 million worldwide. Provided its efficacy is superior to existing vaccines, it is well tolerated and it is an oral single dose, Microscience’s vaccine should be competitively positioned to gain share in the travellers’ market from the existing typhoid vaccines.

c) Managing, monitoring and reporting the project

The overall management of the project will be the responsibility of Microscience. Microscience has a full time experienced, project manager who leads the existing project team and has been responsible for the development of the oral typhoid vaccine to date. The project team members include experienced development staff from CMC (chemistry manufacturing and control) pre-clinical, regulatory and clinical. The team is further supported by external specialists such as clinical research organisations that handle monitoring, GCP (good clinical practice) compliance and clinical data bases. It is intended that the scientific co-applicants will be integrated into the existing project team and the tasks coordinated through this structure which already has a proven track record in developing this programme. Regular project meetings will be held which will monitor the progress of the project against the project plan and deal with issues as they arise. Project reports will be issues on a monthly basis to the Wellcome Trust in a format to be agreed.

d) Planned commercial exit

The commercial market for typhoid vaccines is not large and it is difficult for Microscience to justify funding the whole programme required to gain approval of the product, either as a travellers vaccine, or in developing countries, given that the Company has a number of more commercially attractive vaccines in the portfolio. Acambis, who had a similar product in development, recently announced that they are not going to invest further resources in the project because they do not believe that they will generate the required return on investment.

However, Microscience recognises the importance of this vaccine in providing substantial healthcare benefits in the developing world and would like to ensure that if the vaccine is successful, those benefits are delivered.

The route to commercialisation of the vaccine whether it is for travellers or the developing world will involve carrying out a large efficacy study in a country where typhoid is endemic. The STA proposal is critical for taking the first steps in this process and addressing one of the key issues relating to transfer of vaccines from the developed to the developing world, that is whether the safety and immunogenicity profiles will be similar. It will be difficult to obtain further investment into the project until this key question has been answered.

If the vaccine proves to be successful in the stepping stone studies in Viet Nam it should provide leverage for obtaining additional funding, either from commercial or NGO sources.

It is therefore intended to use these data to facilitate interactions with other NGO funding groups in order to provide funding for the Phase III efficacy study that it is intended to initiate in 2006. All studies in Viet Nam will be carried out under a US IND as well as under the appropriate authority in Viet Nam in order that it is possible to eventually submit a Biologics License Application to the FDA for approval of the product in [**]. In parallel, with appropriate funding and technology transfer activities it should also be possible to get the product manufactured and approved in developing countries such as Vietnam where the vaccine has the potential to deliver considerable health care benefits.

      

	1.		Chatfield, S.N., Roberts, M., Li, J-L, Starns, A. and Dougan, G. 1994. The use of live attenuated Salmonella for oral vaccination. In: Recombinant Vaccines in Vaccine Development: Dev. Biol. Stand. Basel, Karger 82, 35-42.
	2.		Tacket, C.O, Sztein, M.B., Losonsky, G.A., Wasserman, S.S., Nataro, J.P., Edelman R., Pickard, D., Dougan, G., Chatfield, S.N. and Levine, M.M. 1997. Safety of live oral Salmonella typhi vaccine strains with deletions in htrA and aroC aroD and immune response in humans. Infect. Immun. 65, (2) p.452-456.
	3.		Shea, J.E, Buezon, C.R, Gleeson C, Mundy R, Holden, D.W. Influence of the Salmonella typhimurium Pathogencity isaland 2 type III secretion system on bacterial growth in the mouse. Infect. Immun. 1999, 67(1): 213-219.
	4.		Hindle Z, Chatfield SN, Phillimore J, Bentley M, Johnson J, Cosgrove CA, Ghaem-Maghami M, Sexton A, Khan M, Brennan FR, Everest P, Wu T, Pickard D, Holden DW, Dougan G, Griffin GE, House D, Santangelo JD, Khan SA, Shea JE, Feldman RG, Lewis DJ Characterization of Salmonella enterica derivatives harboring defined aroC and Salmonella pathogenicity island 2 type III secretion system (ssaV) mutations by immunization of healthy volunteers. Infect. Immun. 2002 Jul:70(7):3457-67..
	5.		Shahid A Khan, Richard Stratford, Tao Wu, Nicola McKelvie, Trevor Bellaby, Zoe Hindle, Katharine A Sinha, Shayne Eltze, Piero Mastronel, Derek Pickard, Gordon Dougan, Steven N Chatfield, Frank R Brennan Salmonella typhi and S. typhimurium derivatives harbouring deletions in aromatic biosynthesis and Salmonella pathogenicity island-2 (SPI-2) genes as vaccines and vectors Vaccine 21 (2003) 538-548
	6.		Kantele, A. Antibody secreting cells in the evaluation of the immunogencicty of an oral vaccine. Vaccine. 1990 (8) 321-326
	7.		Ferreccio. C, Levine. M.M, Rodriguez, H, Contreras. R, Chilean Typhoid Committee.
			J. Infect. Dis 1989 (159) 4 766-769
	8.		Feng-Ying C. Lin, Vo Anh Ho, Phan Van Bay, Hguyen Thi Thanh Thuy, Dolores Brlyla, Tran Cong Thanh, Ha Ba Khiem, Dang Duc Trach and John B. Robbins. The epidemiology of typhoid fever in the Dong Tap Province, Mekong Delta Region of Vietnam. J. Trop. Med. Hyg. 2000 62 (5), 2000 644-648.

     Copies of references provided in Appendix D.

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A consultancy agreement already exists with the International Vaccine Institute (dated 02-12-03) which relates to the project. This was set up in order for Microscience to receive advice on the development of clinical strategy and to establish the field site in Vietnam. This is attached as Appendix E.