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HIV/Microbicides Network Node

Profile

The objective of this project is to develop affordable microbicides through the expression in plant cell culture of two leading antiviral lectins Griffithsin and Scytovirin. The two lectins are under development as candidate microbicides. Data on the safety profile of the molecules in a rabbit model is quite promising and a macaque study was successfully completed, an important aspect since the recent failures of some microbicidal candidates in advanced testing phases. HIV continues to be a major global health problem, and HIV infection is rising more rapidly among women than men in many parts of the world. In 2007, 33 million people around the world were HIV infected (UNAIDS, 2008). Sub-Saharan Africa is home to 67% of all people living with HIV, and more than 60% of these are women. Microbicides are products that prevent the sexual transmission of HIV and other sexually transmitted infections (STIs) when applied topically in the vaginally. In the absence of an effective vaccine against HIV, microbicides are a powerful, viable tool in the fight against HIV transmission. Scytovirin and Griffithsin are under development as topical microbicides to prevent sexual transmission of human immunodeficiency virus (HIV), and an economically feasible means for very large-scale production of these proteins is an urgent priority. These molecules are currently produced in E. coli, where production capacity is limited. Cost, stability and ease of manufacturing are critical for the effectiveness and access of a microbicide by the poor. We believe that a plant based expression system will provide these molecules at a scale and cost that is relevant to the regional demand for an HIV microbicide. Thus we will clone the genes encoding these two molecules, introduce them into plant cell cultures as well as express them transiently in tomato fruit for rapid production. The output will be a prototype for Scytovirin and Griffithsin production under contained conditions that are far more cost effective than mammalian cultures, and human and technological capacity development in the process.

Broad developmental objectives

The broader objective of this project is to curb HIV infection in the Southern African subregion. The CSIR is developing a transgenic plant based platform for the cost effective expression of molecules of interest such as anti-HIV microbicidal proteins. Several microbicides are currently under trial in Southern Africa. Should any of these molecules prove to be effective in curbing HIV, their affordability remains to be seen – cost is a major limitation to adoption of some measures to control infectious disease. It is therefore essential to develop technologies that will allow local production of these molecules at a scale that is relevant to regional demand and a cost that is affordable to regional governments. The CSIR will explore plant based expression systems for microbicidal molecules that are low tech, cheaper and locally developed. Several molecules with capacity to block HIV infection have been identified on a global scale, and the potential of these molecules to curb HIV infection within the region will also be explored. (Pro2000 and BufferGel are not proteinaceous in nature and are therefore not amenable to the production system under exploration).

Purpose

The primary aim of this project is to determine if functional Scytovirin and its analogue SD-1 can be expressed transiently and stably in transgenic tobacco and tomato and carrot cells. We aim to produce adequate amounts of these molecules to allow for generation of data on these molecules against subtype C HIV strains. The effectiveness of the plant derived proteins as microbicides will be tested in cell cultures and, in a second phase of the project, in an in vitro vaginal model system for HIV transmission. In the two MPhil students will be registered, and their work will be upgraded to DPhil research subject to progress made and further funding being secured.

Description

Project The National Cancer Institute has identified the compounds Scytovirin (SVN) and Griffithsin (GRFT) with potent anti-HIV activity (Bokesch, H.R. et al (2003) Biochemistry 42:2578-84 and Mori, T. et al (2004). J of Biol. Chem. 280: 9345-9353 respectively). These molecules bind to sugars on the surface of the envelope glycoprotein preventing viral entry and infection. The two lectins are under development as candidate microbicides. Data on the safety profile of the molecules in a rabbit model is quite promising and a macaque study was successfully completed, an important aspect since the recent failures of some microbicidal candidates in advanced testing phases. To date, these molecules have been shown to have potent anti-HIV predominantly against subtype B and laboratory HIV strains. I this aspect of the project, we will examine the effectiveness of these molecules against subtype C viruses, the predominant strain in Southern Africa. We will test the efficacy of the recombinant Griffithsin and Scytovirin expressed in bacteria against the sub-type C strains. This will be done using a pseudovirion neutralization assay that is routinely performed at the National Institute for Communicable Diseases (NICD) in Johannesburg. If the lectins prove efficacious, the genes encoding the two anti-HIV microbicides will be cloned into expression vectors expressed transiently in tobacco and tomato the plant tissue expressed proteins biochemically characterized, purified and also tested for effectiveness against subtype C HIV viruses. Vectors encoding the effective microbicidal molecules as reflected by transient assays will be also be stably expressed in transgenic tobacco cells, and we will develop purification protocols for plant made microbicidal molecules so that they can proceed to efficacy testing. Outputs envisaged for this project include efficacious microbicidal candidates for further development, technology packages on how this can be routinely accomplished and possibly expanded into production, and human capital development in the form of students trained. Two MSc and 1 PhD students will be enrolled, and two of these students will be seconded for three months at a time to spend time at NCI in the USA learning cutting edge technology in antiviral lectin purification and characterization. The students will be mentored by Prof Lynn Morris and Dr Rachel Chikwamba at CSIR. The Bioprospecting department at the CSIR has identified some medicinal plants with anti-HIV activity based on indigenous knowledge from traditional healers. The feasibility of using some of the plant extracts as microbicides will be explored in bench and in vitro studies, and this work will be led by Dr Vinesh Maharaj.

Beneficiaries

The greater population of sub-Saharan are the beneficiaries of this project. Primary beneficiaries are women and girls of sexually active age groups. In particular, this project will empower women and girls to protect themselves against HIV infection. In the predominantly patriarchal societies of the subregion, women and girls do not have the social or economic power to influence their partners’ sexual behaviour. Microbicides are a powerful women controlled tool against infection that they can use without their partners consent.

Results

The expected outcome of this work can be summarised as the establishment of develop technical know-how on the development candidate microbicides for cost effectively in plant expression systems via the following steps

• Effectiveness of the two purified microbicidal candidate molecules on HIV-1 subtype C viruses determined.
• Genes encoding the three anti-HIV microbicides expressed transiently in tobacco and tomato.
• Transiently expressed proteins biochemically characterized, purified and tested for effectiveness against subtype C HIV viruses.
• Genes encoding the effective microbicidal molecules expressed in transgenic tobacco and tomato.
• Development of purification protocols for plant made microbicidal molecules.

In the implementation of the technical work described, at least three post graduate students will be trained in all facets of research design and implementation and they will acquire higher degrees (two MSc and 1 PhD).

Sustainability

Compatibility with strategic goals for the Finnish Development Cooperation

The Finnish government’s development policy objectives are reduction of poverty, prevention and mitigation of environmental problems, and promotion of equality, democracy and human rights constitute the basic elements in the promotion of global peace and security. The aim of this work is to develop candidate microbicidal proteins to curb the sexual transmission of HIV, one of the largest menaces to global health. The production of a cost effective microbicide will make a major contribution to the global fight against HIV/AIDS. A plane cell based expression system will allow production of large amounts of Griffithsin and SVN, requiring minimal purification at a relatively low cost. Development of such a system in South Africa will facilitate availability of the microbicidal compound in a region of the world where it is urgently required. This is in line with the Finnish government’s policy goals, impacting directly on poverty reduction. HIV/AIDS affects the productive age groups of populations of countries in the sub-region, burdening the rudimentary health delivery systems and impacting negatively on economic growth. A healthy population is also conducive to economic prosperity, peace and security.

Participation and ownership

Because of the highly technical nature of this phase of the project, the participation of members of the public was limited. However, consultations with the South African government agencies (such as Department of Science and Technology (DST) Health Technologies, LIFElab and others have indicated that this is a high priority area for research and development. Globally, there are large initiatives to develop and test microbicidal candidates (e.g. International Partnerships on Microbicides). The next 36 months for which we are asking support is a preliminary explorative phase that did not require a consultative process. If the molecules prove effective and the production system proves viable, consultation with regional governments and civic groups will be intensified to design the way forward to facilitate progressing these molecules into human good manufacturing practices and clinical testing. The CSIR views this project as a “public good” outcomes project on which it does not aim to profit.

The CSIR will own this technology and will make it available to the developing countries under mutually agreed terms.

Sustainability plan

At this stage the CSIR is requesting support for 3 years for preliminary/developmental phase to determine if the efficacy of these molecules against subtype C HIV viruses and the feasibility/cost-effectiveness of making these molecules. Additional funding beyond this activity will be sought to develop the technology further. Efforts will be made to seek funding from the South African National Biotechnology Regional Innovation Centres (BRICs) to put the plant expressed into clinical testing. Discussions will also be initiated with the DST Health Technologies group. Should the technology prove to be viable, it will be licensed out under terms and conditions that will allow sustainable production of the molecule at a cost that leaves them available to regional governments, a different phase of funding will be required for these aspects.