Model for product development of vaccines against neglected tropical diseases: a vaccine against human hookworm

1481www.expert-reviews.com ISSN 1476-0584© 2008 Expert Reviews Ltd10.1586/14760584.7.10.1481

Perspective

Approach to the development of new controls for hookworm diseaseHuman hookworm infection (‘hookworm’) is one of the most important neglected diseases in developing countries [1]. In response to an urgent need for new hookworm control tools [2], the Human Hookworm Vaccine Initiative (HHVI) [201] is developing a human hookworm vaccine to prevent disease caused by heavy infec-tions due to Necator americanus. Candidate anti-gens, manufactured as recombinant proteins and formulated as monovalent vaccines, will be tested individually for safety and their ability to induce an immune response, before being combined and tested for efficacy. Promising candidates include the Na-ASP-2 Hookworm Vaccine [3], designed to protect against the larval

stage of hookworm infection, and the Na-APR-1 Hookworm Vaccine, designed to protect against the adult blood-feeding stage of hookworm [4]. To date, the processes for the production of the recombinant Na-ASP-2 protein have been technology transferred and manufactured successfully under current good manufactur-ing practices (cGMP) at the 60-l scale at both the Walter Reed Army Institute of Research (WRAIR) Pilot Bioproduction Facility (Silver Spring, MD, USA) and at Instituto Butantan (São Paulo, Brazil). Furthermore, Na-ASP-2 Hookworm Vaccine formulated by WRAIR has undergone Phase I testing in the USA, and is currently undergoing Phase I testing in Brazil. Other adult and larval antigens are currently under development [5,6].

Maria Elena Bottazzi† and Ami Shah Brown†Author for correspondence Department of Microbiology, Immunology and Tropical Medicine, The George Washington University 2300 Eye Street NW, Ross Hall, Room 732, Washington, DC 20037, USA Tel.: +1 202 994 2634 Fax: +1 202 994 2913 [email protected]

This article provides an overview of the advances in product development and technology transfer of the vaccine against human hookworm, with particular emphasis on the lessons learned and the challenges of developing a vaccine in the nonprofit sector. The comprehensive approach to vaccine development established by the Human Hookworm Vaccine Initiative (HHVI) identifies key operational and technical aspects that are essential for a successful partnership with a developing country vaccine manufacturer. This article also highlights the importance of a global access roadmap to guide the vaccine development program. The advancement of new products for the control of neglected tropical diseases portends great challenges for global access, including aspects related to vaccine design, product development and manufacture, vaccine introduction and distribution, financing, knowledge dissemination and intellectual property management. With only three vaccines for neglected tropical diseases in clinical trials – hookworm, leishmaniasis and schistosomiasis – we are at the nascent stages of developing vaccines for neglected populations. Product development public–private partnerships, such as the HHVI, continue to show great promise on this front and will eventually provide significant control tools for achieving millennium development goals related to poverty reduction, as well as child and maternal health.

Keywords: hookworm • Necator americanus • neglected tropical diseases • product development • public–private partnerships • recombinant vaccines • technology transfer • vaccine manufacture

Model for product development of vaccines against neglected tropical diseases: a vaccine against human hookwormExpert Rev. Vaccines 7(10), 1481–1492 (2008)

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Perspective Bottazzi & Brown

Latin America and the Caribbean (LAC) are known to have high burdens of neglected tropical diseases (NTDs) [7]. The most disproportionate concentrations of these diseases in LAC can be found to affect the 40 million Brazilians living in extreme pov-erty [8]. Hookworm infection is one of the most common NTDs among this population, occurring in approximately 32.3 million Brazilians. Today, an estimated 65% of LAC’s hookworm burden occurs in Brazil. Other major helminth infections are also coen-demic to hookworm, and include ascariasis (41.7 million cases), trichuriasis (18.9 million cases) and schistosomiasis (1.5 million cases) (Table 1) [7].

In contrast to its high NTD burden and the poverty level of its population, Brazil is ranked number 12 among the top 25 so-called ‘innovative developing countries’ (IDCs) in the world [9]. IDCs rep-resent middle income countries with modest economic capacity but with high innovation capacity [9–11]. With respect to biotechnology, innovation refers to the capacity to develop, manufacture, ensure the safety of and market new health products, and to develop, test and introduce new health policies or strategies that support the products [9]. IDCs are distinguished by their rapidly growing strengths in biotechnology inno-vation, as illustrated by international pat-ents and papers published in international peer-reviewed journals. Furthermore, these countries have growing numbers of biotechnology companies and advanced research institutes with high levels of com-mitment in public and private investments and expenditure in R&D. Finally, IDCs operate in the presence of national regu-latory bodies capable of determining the safety and ethics of testing new investiga-tional products [9]. The other major LAC IDCs include Argentina (ranked number 21), Mexico (ranked number 24) and Cuba (Table 2) [10].

The ability of Brazil to maintain a high IDC ranking is mostly due to the strong commitment by the Brazilian government to expand and enhance the research and biotechnology capacity of the country. A survey on the funding of health research in Brazil between 2000 and 2002 showed that the mean annual expenditure on

R&D reached US$573 million (note that only 3.5% of these funds were from sources outside Brazil). These funds were distributed primarily to academic institutions, the Ministry of Health and its research affiliates (i.e., Oswaldo Cruz Foundation – FIOCRUZ [202]) and to the Science and Technology (DECIT) Division in different Brazilian states. A small percentage was also distributed to private institutions [12]. Furthermore, this year, and as a com-ponent of the Brazil health strategy for 2008–2011 (Programa Mais Saúde: Direito de Todos), the Brazilian federal government announced a new program for national economic development and poverty reduction, known as Programa de Aceleração do Crescimento [203]. The health component of this program will be funded by the Ministry of Health, and DECIT has been charged with developing models for the investment in the development of new vaccines for neglected diseases through capacity building and the strengthening of areas such as technology transfer and scale-up manufacturing, and the establishment of centers for toxicological studies of biologics.

This article provides an overview of the model used by the HHVI for the development of a vaccine against human hook-worm, focusing on the recent progress and challenges in product development (PD) and technology transfer to a public sector vac-cine manufacturer, Instituto Butantan in the IDC of Brazil. In addition, we present information on the relationship between PD activities and the first steps of global access for sustainability. It is envisioned that the lessons learned from the HHVI model for vaccine development in developing countries will provide essential information for successful development of other antipoverty vac-cines, such as schistosomiasis, lymphatic filariasis and trachoma, among others [13].

Table 1. Estimated burden of the major helminthiases in Brazil.

Disease Number of cases Ref.

Trichuriasis 18.9 million [32]

Ascariasis 41.7 million [32]

Hookworm 32.3 million [32]

Schistosomiasis 1.5 million [33]

Modified from [7].

Table 2. Innovative developing countries in Latin America and the Caribbean based on their human development index, gross domestic product per capita, R&D expenditure and number of researchers in R&D.

Region or country

Country classification*

HDI rank‡

GDP per capita (US$)2005§

R&D expenditure (% of GDP)2000–2005¶

Researchers in R&D (per million people)1990–2005¶

OECD 29,860 2.42 3096

LAC 4480 0.56 256

USA G8, OECD 12 41,890 2.68 4605

Argentina Advanced IDC 38 4728 0.41 720

Cuba Upper middle income#

51 0.65

Mexico OECD 52 7454 0.40 268

Brazil Advanced IDC 70 4271 0.98 344*From [10].‡Human Development Index; Human Development Report 2007/2008 [212].§GDP per capita (2005) from the World Development Indicators 2007 [213].¶World Development Indicators 2007; aggregates calculated for the Human Development Report 2007/2008 by the World Bank [212].#Country classification obtained from [212,213].GDP: Gross domestic product; HDI: Human development index; IDC: Innovative developing country; LAC: Latin America and the Carribean; OECD: Organization for Economic Cooperation and Development.

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PerspectiveProduct development for vaccines against neglected diseases

Access to vaccine technology in developing countries: public–private partnerships & developing countries’ vaccine manufacturersDuring the last decade, metrics to determine the advancement of global health indicators have included how much innovation a country achieves with activities related to R&D, technology transfer, biotechnology innovation and intellectual property (IP). This has been especially true for the development of new biologics (drugs and vaccines), which have substantially increased, princi-pally focusing on antigen discovery and preclinical testing, and using primarily animal models in the laboratory.

Historically, biotechnology advances for the development of new biologics in developing countries have been led by market demands [14]. In the early 1980s, considerable biotechnology advances in the LAC IDCs were established primarily through the introduction of national programs and biotechnology initiatives, with the goal of stimulating the application of modern technologies for both the veterinary and health fields [14]. It is important to note that in LAC, unlike most developed countries, biotechnology investment is generally dominated by government agencies, and less from the private sector. These biotechnology initiatives were the first steps to establish coordinated efforts to enhance R&D, stimulate linkages with industries and secure funding from international coopera-tion. For instance, the WHO Tropical Disease Research aimed to target the strengthening and capacity building of infrastruc-ture to conduct research using appropriate technologies in LAC countries, focusing specifically on leading tropical diseases, such as malaria, Chagas disease and leishmaniasis. However, at the end of the 1980s, it was clear that private industries were not interested in investing in the advancement of high-risk products for these diseases (i.e., new drugs or vaccines) [14]. Fortunately, agencies such as the NIH and the CDC in the USA, the United Nations Development Programme, the World Bank, the WHO and its regional offices, as well as private foundations, such as the Bill & Melinda Gates Foundation, continue to have a strong commitment to strengthening capacity in developing coun-tries, aiming to identify technologies and to pursue the development of new products. These initiatives focus on the education and training of in-country scientists, provision of technical assistance and, in limited cases, infrastructure development [15].

The new promise for successful and sustainable biotechnology development in developing countries is now led by a cadre of public–private partnerships (PPPs) espe-cially those devoted to PD, which includes the HHVI. Created with the basic principle to use and adapt procedures from the private sector, the PD–PPPs function with detailed business plans and management structures, they incorporate market research data into

their proposed plans and manage a portfolio of discoveries by secur-ing access to IP [16]. PD–PPPs have led the way in filling the gaps in product development and through creative mechanisms have established models and systems that complement the efforts of traditional research initiatives [17,18].

In addition to PD–PPPs, the alliance between developing coun-tries’ vaccine manufacturers (DCVM), referred to as the mem-bers of the DCVM network [204], has shown great promise for the advancement of products in developing countries. Their goal is to enable a sustainable, consistent, accessible and affordable supply of quality vaccines [19,20]. In order to qualify, vaccine manufacturers have to be either from the public sector of a developing country or owned in majority by a citizen(s) of the developing country where the company produces the vaccine. We believe, in agreement with Jadhav et al. [19,20], that for successful development of novel vac-cines against NTDs, DCVM in the developing world need to be involved. In LAC, there are five vaccine manufacturers that form part of this alliance (Table 3), and Brazil is currently one of the leaders in the region, with Instituto Butantan [205] and Biomanguinhos [206] already manufacturing and distributing more than 80% of vac-cines for the Brazilian population [19–21]. Both entities have attained GMP status as designated by the WHO, and abide by the rules and regulations of the independent and strict national control authority, which ensures international quality levels (Table 3).

HHVI model for vaccine developmentThe scale and complexity of today’s biomedical research problems increasingly demands that scientists move beyond the confines of their own discipline and explore new organizational models for

Table 3. Members of the Developing Countries Vaccine Manufacturers’ network in Latin America and the Caribbean.

Name Country Description

Bio-Manguinhos (FIOCRUZ)

Brazil Full memberHolds WHO prequalification for one or more of their productsCountry has functional NRA as defined by the WHO

Finlay Institute Cuba Full memberHolds WHO prequalification for one or more of their productsCountry has functional NRA as defined by the WHO

Centro de Ingeniería Genética y Biotecnología (CIGB)

Cuba Full memberHolds WHO prequalification for one or more of their productsCountry has functional NRA as defined by the WHO

Instituto Butantan Brazil Prospective full memberWorking towards attaining status of WHO prequalificationCountry has functional NRA as defined by the WHO

Laboratorios de Biológicos y Reactivos de México, S.A. de C.V (BIRMEX)

Mexico Prospective full memberWorking towards attaining status of WHO prequalificationUndetermined status of a functional NRA

NRA: National Regulatory Authority. Modified from [19,20,211].



team sciences. The HHVI exemplifies how a nonprofit PD–PPP, can achieve successes during the development of a recombinant vaccine for human hookworm infection [13,18,22,23]. The existing collaborations and performance sites among institutions on five different continents (North and South America, Asia, Europe and Australia) are linked in a virtual interactive framework through strong program management.

What differentiates the HHVI from traditional models used by other PD–PPPs is that, through experience, the HHVI has adopted a strategy to minimize its reliance on outside contractors in favor of building, maintaining and sustaining in-house capacity for antigen discovery, product development, preclinical testing, technology trans-fer and clinical development within its seven-center (Sabin–George Washington University–Queensland Institute of Medical Research–London School of Hygiene and Tropical Medicine–Oswaldo Cruz Foundation–Butantan–Institute of Parasitic Diseases) network of collaborators (Table 4). This network primarily comprises of academic institutions, nonprofit entities or governmental organizations [18]. By contrast, other PD–PPPs, such as the Meningitis Vaccine Project (MVP), have relied primarily on contractual agreements and the for-mation of collaborations with commercial companies. This approach has presented the MVP with some major challenges. For instance, the MVP contracted an independent European research group to develop a new conjugation technology, which ultimately did not allow for its technology to be transferred to the manufacturing facil-ity. Faced with this challenge, the MVP resolved the issue by using an alternative conjugation technology from the Center for Biologics Evaluation and Research. In addition, it utilized the contracted services of Serum Institute of India Limited (SIIL), a private sector DCVM. The agreement included the assurance of SIIL to grant back a nonexclusive, sublicensable license to SIIL-owned technology and to sell the vaccine at a fixed low price for Africa but allowed SIIL to sell the vaccine at higher prices elsewhere [24,25].

Comprehensive approach towards successful development & technology transferOne of the major hurdles in the critical path for the development and testing of novel and translational discoveries is overcoming the so-called ‘valley of death’, or the gap in taking a bench discovery from the stage at which it is a laboratory reagent to the point where it shows a clear path to the clinic [18]. The HHVI model applies basic PD–PPP management principles in combination with established collaborative agreements for product development, technology transfer and manufacturing activities. This approach has proven successful to define and sustain process requirements and measure effectiveness, to plan the development and acquisition of necessary capabilities, to further the most promising research, to remove barri-ers, to plan transitions between stages, to track status and coordinate efforts and ultimately translate promising research into the testing and development of safe and efficacious products that are integrated into systems of dissemination and use (Figure 1).

The HHVI has established an operational management frame-work that is based on effective collaborations, continuous com-munications, knowledgeable technical teams, solid science, quality product development and manufacturing strategies, and requisite preclinical and clinical studies. As a first step, a product develop-ment plan (PDP) is prepared for each vaccine target antigen to enter the development pipeline. This document provides overall guidance, a set of specific and achievable goals and the schedules that must be maintained to reach the ultimate objective. It also identifies alternative approaches and the regulations that must be addressed, such as those related to product quality and safety. More specifically, the PDP includes plans for the three stages of vaccine development applied by the HHVI: basic research, product develop-ment and clinical development (Figure 1). Initially, processes need to be in place for the identification, assessment, evaluation and rank-ing of potential target antigens. Once a target antigen is selected,

Table 4. Major organizations of the Human Hookworm Vaccine Initiative: a partnership for product development, technology transfer, clinical testing and global access.

Institution Location Major activities

Sabin Vaccine Institute Washington, DC, USA Program management, grants and contract management, regulatory affairs, quality assurance, clinical development and oversight

The George Washington University School of Medicine and Health Sciences, Department of Microbiology, Immunology and Tropical Medicine

Washington, DC, USA Antigen discovery, preclinical testing, process development, assay development, lot release, stability testing, quality control and technology transfer

Queensland Institute of Medical Research, Division of Infectious Diseases and Immunology

Brisbane, Australia Antigen discovery and assay development

London School of Hygiene and Tropical Medicine, Division of Infectious Diseases and Epidemiology

London, UK Epidemiology and mathematical modeling

Institute of Parasitic Diseases, Chinese Centers for Disease Control and Prevention

Shanghai, China Preclinical testing

Rene Rachou Research Centre, Oswaldo Cruz Foundation (FIOCRUZ-CPqRR)

Belo Horizonte, Brazil Field epidemiology, clinical trials and clinical immunology assay development and testing

Instituto Butantan São Paulo, Brazil Technology transfer and current good manufacturing practices manufacturing


Perspective

Vaccine development strategy

Stage 1: Basic research

Data assessment and evaluation• Disease target• Host–pathogen relationships• Portfolio of target candidate antigens

Recombinant expressionsystem selection• Cloning, selection, characterization and generation of cell banks• Evaluation of yield and purity

Preparation and filing of regulatorydocumentations• cGMP manufacture of drug substance and drug products• Lot release testing and stability programs• Development of GLP toxicology protocols• Development, preparation and filing of applications to regulatory authorities (USA and Brazil)• Development, preparation and filing of ethical applications• Preparation and design of clinical protocols

Clinical testing

Quality management plan

Process development• Development, optimization and scale-up of fermentation and purification processes

Technology transfer• Transfer of cell bank production processes• Transfer of fermentation and purification processes• Transfer of assays or characterization and release• Transfer of formulation processes

Quality control andproduct evaluation• Development and qualification of assays• Stability evaluation (temperature and pH incursions)• Formulation studies• Immunological potency studies• Proof-of-concept in laboratory animals

Rationale and feasibility• Assessment for target selection

Selection, ranking andscoring process• Molecular biology• Structure–function• Immunoepidemiology• Proof-of-concept animal challenge studies

Stage 2: Product development Stage 3: Clinical development

Product development for vaccines against neglected diseases

the PDP details which expression system will be selected; which processes will be developed and optimized for its production; a technology transfer plan for the scale-up and cGMP manufac-ture; which assays will be developed, qualified and used during in-process, lot release and stability testing; details of the formula-tion studies and the preclinical evaluations; plans for toxicology testing under good laboratory practices; and finally provides the strategy for the preparation and filing of the regulatory application leading into a clinical development plan. A quality management plan is included at each stage, specifically reviewing and approving standard operating procedures, analytical instrument qualifica-tion and method qualification, documentation of change control, record review, approval or rejection of materials, discrepancies/out of specification/investigations and corrective actions.

Technology transfer to an IDC manufacturerThe HHVI faces formidable manufacturing challenges for produc-ing a product intended for the world’s poorest people. Foremost among these challenges is hookworm’s neglected disease status and, therefore, the limited commercial market that exists for such a

vaccine, making development and production by a for-profit com-pany, such as a major vaccine manufacturer, extremely unlikely. This situation has required the HHVI to consider innovative mechanisms for mid- to large-scale manufacture.

In the absence of significant commercial markets, the HHVI’s global access strategy is to partner with vaccine manufacturers in IDCs that already have some sophistication in manufacturing and distributing vaccines and other biologics. From the IDC nations, Brazil, China, India and Indonesia account for almost a half of the world’s hookworm burden, and also have varying degrees of vac-cine manufacturing capacity [9,26]. The selection of Brazil as the HHVI’s first IDC partner was based on the presence of endemic hookworm, as well as regions of high hookworm transmission in addition to its technological capability to manufacture and distribute vaccines.

Vaccine development partnerships in BrazilAs noted previously, the HHVI has chosen Brazil as its first IDC partner for the mid- to large-scale production and clini-cal testing of the hookworm vaccine. The partnership, which

Figure 1. The Human Hookworm Vaccine Initiative model for vaccine development. cGMP: Current good manufacturing processes; GLP: Good laboratory practice.



includes two public sector Brazilian organizations (FIOCRUZ and Instituto Butantan) aspires to provide sustainable develop-ment through Brazilian ownership of the hookworm vaccine technology and a commitment by the Brazilians to its long-term product and clinical development. In addition, to ensure that the hookworm vaccine will be accessible to all countries and popu-lations in need, the HHVI–Brazil partnership encourages and allows competitiveness in the production market and facilitates collaborative strategies (i.e., South–South collaborations with Portuguese-speaking countries).

In 1989, Instituto Butantan, which is located in São Paulo and is one of Brazil’s two major vaccine manufacturers, created the Butantan Foundation, charged with securing the financial sus-tainability of the Instituto Butantan. The Foundation facilitated the construction of new vaccine production facilities, such as new plants for producing the diphtheria, pertussis and tetanus (DPT) and recombinant hepatitis B vaccines (HBVs) [27]. As a result, a relatively sophisticated vaccine manufacturing infrastructure is in place. As mentioned previously, Instituto Butantan produces approximately 500 million doses of vaccines that are utilized in Brazil, including its own yeast-derived recombinant HBV. It is important to note that Instituto Butantan does not simply formulate pre-existing bulk drug substance produced by North American and European manufacturers. For example, Instituto Butantan’s HBV was re-engineered in a novel Hensenula yeast strain (the Merck HBV is expressed in Saccharomyces cervisiae). Other vaccines in early product development include those for dengue, under a nonexclusive license from the NIH; human papillomavirus, under a loose affiliation with the University of Colorado (CO, USA); rotavirus, using a clone licensed from the NIH; and pneumococcal infection in collaboration with the Children’s Hospital at Harvard Medical School (MA, USA). A new combination rabies–leishmaniasis canine vaccine is also undergoing pilot manufacturing and testing. Finally, they are also completing a technology transfer agreement with Sanofi Pasteur for influenza vaccine development. Instituto Butantan and the Butantan Foundation are both nonprofit entities whose major focus is the application of biotechnology to public health [21]. In summary, HHVI selected to partner with Instituto Butantan based on the following criteria:

History of high-level commitment by Brazilian federal and state •government to R&D, access to new technology and sustainable vaccine development and production;

Participation in the DCVM network and application of •standards and requirements set by the WHO;

History of other successful collaborations;•

Established infrastructure and experienced personnel;•

Prior experience in recombinant vaccine technology using yeast •expression systems.

In order to launch the Brazilian collaboration, a codevelopment agreement was signed between the HHVI and Instituto Butantan in 2006. The essential components of this document are similar to the critical components of such an agreement as identified

by the Center for the Management of Intellectual Property in Health Research and Development and described by Oehler in 2004 [28], and include:

Definition of the nature of the final product•

Role of each party in the development process•

Resources (financial, personnel and institutional) each party •will invest

Process by which the project will be managed•

Interim goals (milestones) and timetable•

Provisions for sharing in success or failure•

The main objectives set forth in the agreement included coinvestment in the construction of a production plant for pilot scale manufacturing up to 60-l scale and the development of processes for the 60-l scale production of the Na-ASP-2 hook-worm vaccine. Ultimately, this agreement envisions that Instituto Butantan will produce enough vaccine for Phase I, II and III clinical trials in Brazil.

Furthermore, parallel to the partnership with Instituto Butantan, an agreement was also established with FIOCRUZ, headquar-tered in Rio de Janeiro and with a branch institute located in Belo Horizonte (Centro de Pesquisa Rene Rachou [207]) in the state of Minas Gerais. This agreement includes the codevelopment of the epidemiological assessments and clinical testing of the hookworm vaccines. This approach will enhance the ease of testing vaccines at local field sites within the IDC nation, hence promoting in-country collaborations between the IDC vaccine manufacturer (Instituto Butantan) and IDC clinical researchers (at FIOCRUZ) who are typically based at universities or government research institutes.

Planning for global accessAmong the major challenges that require consideration for attaining successful global access of a hookworm vaccine are aspects related to vaccine design, vaccine development and dis-tribution, vaccine introduction, financing, knowledge dissemi-nation and IP management. The HHVI has devised a potential strategy for the low-cost development and distribution of a vac-cine with essentially no commercial market. This strategy is based on two principles:

The human hookworm vaccine will be made available at • affordable prices to those most in need in the developing world

Knowledge gained through discovery will be promptly made •available to the broader scientific community

The technology required to produce the human hookworm vaccine is at an equivalent level of sophistication to that required for more expensive recombinant vaccines, but through bypassing the large Northern vaccine manufacturers and instead developing a recombinant vaccine through a nonprofit PPP in collaboration with an IDC vaccine manufacturer, the HHVI plans to produce a recombinant product at a fraction of the usual cost. Therefore, the HHVI focuses on the use of processes that maximize manu-facturing yields and minimize costs, both essential elements for a product intended for the world’s poor. Some examples include the



use of high-yield yeast expression vectors and protein purification protocols that use low-cost column resins. Formulations will ini-tially be prepared as a single dose (primarily for ease of conduct-ing clinical testing in remote rural areas); however, ultimately, multidose vials will be used for wide-scale delivery. Finally, to enhance immunogenicity, the HHVI works towards obtaining restricted licenses for low-cost adjuvant technologies.

In summary, the HHVI is manufacturing a product that spe-cifically targets the world’s poorest people, rather than one that is intended primarily for North American and European markets and that then slowly trickles down to developing country popula-tions over a period of decades. This new paradigm may be applied to a large of number of the antipoverty vaccines for NTDs [13].

IP & licensing strategiesThe approach used by the HHVI to handle IP is based on its abil-ity to patent, with the assumption that doing so would maximize the downstream public health benefits. This approach is similar to the one used by other PD–PPPs, including the MVP [25]. In addition, it is expected that this approach would also support partnering with selected IDC vaccine manufacturers, since pat-ents are a valuable tool with which to encourage investment. A patent for the ‘hookworm vaccine’ has been filed in the USA (7303752 B2-December 4, 2007 [101]) and in the IDC nations of Brazil, China, India, Mexico and South Africa. Since these are IDC nations with tremendous hookworm burden, filing patent applications will ultimately help to secure freedom of action to manufacture the human hookworm vaccine as desired. In addi-tion, it will assist in documenting and publicizing the HHVI project’s activities, and in those important regions, avoid late-stage surprise developments in which third parties purport to have rights or interest in what is proposed for the final vaccine. Furthermore, the HHVI recently submitted a second patent filing for a bivalent human hookworm vaccine. The HHVI IP man-agement plan also includes the codevelopment agreement with Instituto Butantan and identification of specific milestones to grant a sublicense to Instituto Butantan and other potential IDC manufacturers, which will include commitments to public-sector manufacture and distribution.

The transfer of partial ownership of the vaccine to the IDC of Brazil (‘Southern ownership’) will provide Brazil a greater incen-tive for taking over the industrial scale production, advanced clinical testing (e.g., Phase III) and eventual distribution of the vaccine. This provides a built-in mechanism for the sustainable development of the vaccine against hookworm by laying the foundation for initiating in-country demonstration programs, (i.e., vaccine introduction trials and effectiveness evaluations in the host IDC). Ultimately, the goal is that through these partner ships, the HHVI can help to ensure an adequate and cost-competitive hookworm vaccine supply.

Knowledge disseminationThe HHVI has adopted a policy of consistent publication of its sci-entific findings in the peer-reviewed literature and in a timely man-ner. Since its inception in the year 2000, the HHVI has published

approximately 100 papers, with many of them in the highest impact journals, such as the New England Journal of Medicine, Public Library of Science Medicine, Lancet, Federation of American Societies for Experimental Biology Journal, Journal of Biological Chemistry, Journal of Molecular Biology, Journal of Infectious Diseases and Infection and Immunity. In order to promote even further the HHVI’s policy of open access to its scientific program, the HHVI’s Director has partnered with the Public Library of Science to establish PLoS Neglected Tropical Diseases, a journal dedicated to publishing the highest quality articles (with rigorous peer review) on all scientific, medical and public-health aspects of the NTDs, including relevant public policy.

Recent advances & lessons learned after technology transfer of Na-ASP-2 recombinant protein to Instituto ButantanThe HHVI was successful in transferring the fermentation, purification at the 60-l scale and vaccine formulation processes for the Na-ASP-2 Hookworm Vaccine to the WRAIR Pilot Bioproduction Facility [3]. In 2006, the implementation phase of the HHVI–Brazilian DCVM partnership started with tech-nology transfer for manufacture of the recombinant Na-ASP-2 hookworm protein. Based on Instituto Butantan’s previous success in manufacturing the HBV vaccine, also known as Butang®, at approximately US$0.28 per vaccine dose [29], there is optimism that the Na-ASP-2 hookworm vaccine, whose pro-tein is likewise expressed in yeast but in addition is secreted and can be purified with a high yield, could be developed at a similar cost.

To initiate the technology transfer process in Brazil, and based on the codevelopment agreement, the HHVI charged a technical team with the purpose of following the process development and technology transfer activities. First, the HHVI used the techni-cal staff with expertise in scale-up and technology transfer activi-ties to establish a plan for the scale-up of the production process. Second, technical exchange visits were conducted between scientists based at George Washington University and Instituto Butantan. Third, a comprehensive ‘technology transfer’ package was com-piled, which detailed project goals, historical process data, process changes, applicable regulatory guidelines and requirements, and documentation requirements. Fourth, processes and procedures conducted at Instituto Butantan were verified through parallel testing of quality, integrity and purity of the material at George Washington University.

Operational effectiveness & communicationsAs mentioned above, the HHVI structure includes a strong manage-ment team to cover the functional operations of the different aspects of vaccine development. For example, the HHVI has dedicated teams for global vaccine operations, product development, clini-cal development, quality assurance and regulatory affairs. Overall, these teams collectively manage effective collaborations and ensure consistent communications between, and within, the technical units. In addition, they also provide continuous assessment on whether the quality plan is followed as well as the evaluation of each



activity-driven milestones and ‘go/no go’ criteria or decision points. Management tools are key for the effectiveness of the management group. For instance, Gantt charts are used as project planning and tracking tools for the review of timelines and to evaluate project performance. As the project progresses, these charts are updated accordingly. In addition, the vaccine candidate’s potential benefits are evaluated and accurately weighed against any potential risks. This approach is used during the technology transfer activities.

Initially, the HHVI–Instituto Butantan collaboration started with performing an in-country assessment and evaluating the capabilities at the technical and operational levels. Capabilities, such as processing and manufacturing practices, the availability of reliable sources of raw materials and storage capacity and main-tenance of cold chain, were assessed systematically. During an initial site visit to Instituto Butantan, the HHVI evaluated the following criteria:

Basic scientific knowledge of trained personnel•

Physical infrastructure with appropriate equipment to sustain the •level and scale of production and approved cGMP certification

Availability of good-quality raw material supplies and reagents•

Established quality assurance/quality control policies and •procedures

Institutional and national regulatory oversight•

Based on this assessment, the process requirements and the means to measure the effectiveness of the technology transfer activities were defined. This assessment also included a review of the needs for the acquisition of capabilities or special production equipment. Furthermore, the HHVI created a communication strategy that includes the use of email, teleconferences and in-person visits both in Brazil and the USA. To guide these communications, detailed agendas are generated to address the current status of technical activities. To track the follow-up action items, a point of contact is identified who coordinates each action. Ultimately, the goal is to maintain a consistent message of the overall mission, goals and objectives, assess the general knowledge, skills and training of the personnel, keep tight timelines, streamline resource utilization, set priorities and address any cross-cultural issues.

Building & strengthening technical capacityThe technology transfer process was initiated through a series of scientific exchanges between HHVI scientists and Instituto Butantan scientists. The primary milestone was to transfer proc-ess development at the 10-l scale, with joint efforts leading to the cGMP manufacture of the Na-ASP-2 recombinant protein at the 60-l scale.

Critical to the success of these technology transfer activities was an understanding of the importance of adapting scale-up process develop ment and manufacturing technologies in order to ensure compatibility in terms of equipment, methods and philoso-phies between the two organizations. Therefore, it was essential to exchange critical documentation, including batch production records, standard operating procedures, process flow diagrams,

sample plans and tables of specifications. This effort began with the export of a research cell bank (yeast host system) for the pro-duction of recombinant Na-ASP-2 at the 10-l scale. Fermentation and purification runs were done in parallel in the USA and in Brazil, and served as the proof of principle that processes were reproducible at both sites. For instance, the process development team first traveled to São Paolo, where they worked jointly with the Instituto Butantan team dedicated to the hookworm vaccine project and performed an initial 10-l run. The results obtained were used to jointly plan for the optimization of fermentation and purification processes in order to maximize yield and reduce costs. In addition, a quality control team assisted by transferring the techniques for in-process testing and final release of the produced protein to assess identity and purity.

After the teams developed a reproducible process for the fermen-tation and purification (10-l scale) of the Na-ASP-2 recombinant protein, the next step included the export of the cGMP Master Cell Bank previously manufactured at WRAIR. This transfer was done after regulatory approvals were obtained from both the Comissão Técnica Nacional de Biossegurança (CTNBio) [208] and from the Agência Nacional de Vigilância Sanitária (ANVISA; see later) [209]. In addition, and based on the results obtained from the yield and purity derived from the 10-l scales, the HHVI created a plan to design the scale-up processes to the 60-l level. Significant differences in the equipment and produc-tion procedures, including differences in buffer preparation and the use of continuous flow centrifugation for cell removal versus microfiltration and ultra filtration, were identified and additional changes to the purification process were developed jointly. Briefly, after fermentation, cells were separated and washed to recover the expressed protein in the supernatant. Different strategies for cell separation were developed using tangential microfiltration with hollow fibers, continuous centrifugation or expanded bed chromatography. The purification process used alternate steps of ultrafiltration, for concentration and buffer exchange, and anion and cation exchange chromatography for purification, followed by a gel filtration polishing step.

A successful cGMP 60-l run was performed by Instituto Butantan and yielded approximately 16 g of purified bulk Na-ASP-2 recombinant protein. The lot has been successfully released and is currently under long-term stability testing. Plans are in progress for the formulation and fill of a Na-ASP-2 Hookworm Vaccine lot to be tested in a downstream Phase I trial performed by FIOCRUZ in the Minas Gerais state of Brazil.

Quality managementWith the importance of producing quality products and processes and the increase in regulatory scrutiny of all aspects of the product development and manufacturing cycle, the HHVI developed a plan detailing the approaches to managing quality. This plan includes the basic quality control responsibilities divided by each technical unit, with quality control procedures built into the processes as part of the ongoing, daily work activities. In addition, the plan includes quality assurance responsibilities, which provide the higher level, external, objective assessment of the systems (structures, responsibilities,



procedures and standards) in place during the operations. Therefore, the HHVI generated a quality agreement with Instituto Butantan and has performed several quality assurance assessment visits.

Interaction with regulatory agenciesOne of the challenges during vaccine development in IDCs relates to the regulatory timelines. In Brazil, the regulatory proc-ess is conducted according to the International Conference on Harmonization, good clinical practice and regional regulations [30]. Regulatory approvals generally pass through two ethical evaluations, one institutional and one national, also known as the Comissão Nacional de Ética em Pesquisa [210]. In addition, there is one national methodological evaluation performed by ANVISA. For manufacturing processes approvals are obtained from CTNBio and ANVISA. The HHVI has developed a full regulatory plan for the development of a vaccine against hookworm in Brazil. This plan includes a review of the Brazilian laws and resolutions.

Expert commentaryGiven the HHVI experience with technology transfer thus far, several valuable lessons have been learned as detailed below.

Programmatic strategiesA team with strong program and operational management is neces-sary to prevent disaggregated development processes. This team is essential for the frequent and consistent communication and for the evaluation of timelines. In addition, it ensures the smooth transition and linkage between activities related to basic research, early proc-ess development, GMP production, quality control and regulatory assurances, assessment of decision points and, most importantly, the allocation of appropriate resources to each activity or task.

Capacity building & strengthening of technical expertiseThe HHVI model for technology transfer with a DCVM is based primarily on combining the expertise of industry-experi-enced staff, with knowledge of biotechnology and pharmaceuti-cal procedures and practices, and traditional scientists from the academic and nonprofit sector. The transfer of the Na-ASP-2 Hookworm Vaccine technology required persistence and creativ-ity by the HHVI to explain and educate Instituto Butantan on the difference between requirements for licensure and pilot-scale manufacture for clinical trials. In addition, the communica-tion between teams of competent and experienced scientists, with expertise in fermentation and protein purification, assay development, quality control and quality assurance, program management and operations, has been critical to ensure that all components of technology transfer process were incorporated and that any problems encountered were addressed efficiently and effectively.

The goal during these communications was not only to success-fully transfer a low-cost, robust process for production and quality control testing, but at the same time share technical information to strengthen the Brazilian capacity for transitioning potential new vaccine targets through downstream development.

Quality managementA detailed plan to manage the quality of processes and activities performed during technology transfer and manufacturing is essen-tial to ensure the success of a vaccine development program. Several factors need to be taken into consideration for successful quality management:

The ability to implement recommendations to improve and •maintain quality

Table 5. Summary of technology transfer strategies and coordination.

Strategy Description

Programmatic and technical communications

Regular email and telephone contact between lead scientists for updates on progress of the pilot plant development, including equipment, biologics and reagents purchase, equipment assembly and functioning, and regulatory approvals. Problems encountered and queries are addressed by the associated unit team at HHVIYearly in-person meetings for general overview of timelines and next steps

Assessment visits Initial familiarization visits with the staff, and facilities and assessment visits of pilot plant, equipment, animal facility and regulatory compliance:– Equipment checks– Fermentation and purification engineering runs– Procedure modifications– Assay support

Facilities and equipment

Standardization of different equipment to ensure similar performance and the purchase of similar equipment to HHVI or updated models

Personnel Minimum of yearly current good manufacturing practices and good laboratory practices training of personnel

Documentation Exchange of documents including batch production records and standard operating procedures process flow diagrams, sample plans and tables of specifications

Timelines Regular review and concurrent adjustment of timelines by operational management and technical staff at HHVI and Instituto Butantan

Quality management

Quality control, assurance and compliance procedures are being coordinated with the quality assurance department at Instituto Butantan

HHVI: Human Hookworm Vaccine Initiative.



The ability to maintain objectivity and unbiased oversight•

Use personnel with established technical expertise for the •routine compliance review

The ability to understand and keep abreast of the Brazilian •ethical and regulatory requirements

The ability to shift resources and priorities quickly•

The ability to ensure consistency in standards, practices and cri-•teria across the organization and the implementation of quality

The ability to carry the goals and objectives efficiently and •economically

We believe that challenges of technology transfer of a vaccine production process to a DCVM can be overcome through strong partnerships and collaborations, through an increase in shared IP and ownership of discoveries and, most importantly, using a framework for operational management and careful technical planning. The technology transfer strategies applied by HHVI and Instituto Butantan are detailed in Table 5.

Five-year viewThe PD–PPPs continue to be the driving forces for research and development of new drugs and vaccines against NTDs. They have been instrumental in bridging the gaps between scientific discover-ies and the transition into development and testing by primarily creating ‘push’ mechanisms [31]. In the next 5 years we predict that these push forces, composed of scientific and technological advances, management and coordination support and the creation of stronger partnerships with DCVM, will provide additional incentives for commitments to product development funding. However, in order for product pipelines to become sustainable and yield new vaccines, governments and the general public must commit and recognize that there is a real health need for these vaccines. This, in our view, will continue to be a challenge.

The model used by the HHVI specifically addresses this chal-lenge by initiating the process of advocacy for the need of a hookworm vaccine very early in the development process. The

HHVI’s Global Access Roadmap details the strategy that will permit a rapid deployment of the human hookworm vaccine to the developing world once efficacy has been shown. It emphasizes two core principles:

The global health solution, which in this case is the human •hookworm vaccine, must be made available at affordable prices to those most in need in the developing world

The knowledge gained through discovery must be made •available to the broader scientific community promptly

Specifically, the HHVI outlines a strategy for the low-cost development and distribution of a vaccine with essentially no commercial market. It is anticipated that from day 1, the human hookworm vaccine will be made available for less than US$1 per dose, and possibly much less. This will be accomplished by developing a recombinant vaccine in collaboration with an IDC vaccine manufacturer. In addition, the HHVI’s decision to patent IP is based on the assumption that doing so would support its global access strategy, since patents are a valuable tool to encour-age investment and help to secure freedom of action, in addition to documenting and publicizing the HHVI project’s activities and avoid late-stage surprise developments in which third par-ties purport to have rights or interest in what is proposed for the final vaccine.

AcknowledgementsThe authors wish to thank Dr Peter J Hotez for his critical review of this manuscript.

Financial & competing interests disclosureThese studies were supported by the Sabin Vaccine Institute’s Human Hookworm Vaccine Initiative funded by the Bill & Melinda Gates Foundation. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Key issues

Initiatives to improve and develop new control strategies for neglected tropical diseases (NTDs) have become health priorities due to • the recognized burden of these diseases and how they are the basic factor contributing to poverty and poor public health in the developing world.

Product development public–private partnerships continue to focus their initiatives on the challenges within disease-endemic countries • and implement them using a combination of business-like practices, traditional basic science approaches and applied product development technology exchanges. This approach will continue to provide the ‘push’ for the development of new products for NTDs.

Early partnering between vaccine developers and manufacturing facilities will provide a more solid platform for successful and • sustainable technology transfer, scale-up and manufacturing of recombinant biologics in the nonprofit sector.

To ensure that a vaccine will ultimately emerge on the market, a detailed global access strategy should be developed early in • development, including plans for vaccine design, development and distribution, introduction and financing. Furthermore, this strategy should include detailed plans for knowledge dissemination, sharing of intellectual property and the pursuit of patents in innovative developing countries.

With the advances in genomics, immunomics and proteomics to understand host–pathogen relationships, with new epidemiological • studies to map distribution and burden of diseases, and with the emergence of novel adjuvant platform technologies, critical information is being generated for the successful selection and design of affordable vaccines against infectious diseases.



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AffiliationsMaria Elena Bottazzi, PhD •Associate Professor and Director of Product Development, the Human Hookworm Vaccine Initiative, Department of Microbiology, Immunology and Tropical Medicine, The George Washington University, 2300 Eye Street NW, Ross Hall Room 732, Washington, DC 20037, USA Tel.: +1 202 994 2634 Fax: +1 202 994 2913 [email protected]

Ami Shah Brown, PhD, MPH •Assistant Professor and Director of Vaccine Operations, the Human Hookworm Vaccine Initiative, Albert B Sabin Vaccine Institute, 1889 F Street, NW, Suite 200S, Washington, DC 20006, USA Tel.: +1 202 842 8478 Fax: +1 202 842 7693 [email protected]

Documents

Model for product development of vaccines against neglected tropical diseases: a vaccine against human hookworm