Biotech research in health sector: What has academia done?
[Nature India Special Volume: Biotechnology — An agent for sustainable socio-economic transformation]
doi:10.1038/nindia.2016.28 Published online 29 February 2016
There is always a debate as to what basic bioscience research in academic laboratories has contributed to biotech products in India.
The various schemes under India's Biotechnology Industry Research Assistance Council (BIRAC) namely, Biotechnology Ignition Grant Scheme (BIG), Small Business Innovation Research Initiative (SBIRI), Biotechnology Industry Partnership Programme (BIPP) and Contract Research Scheme (CRS), have supported primary R&D in the biotech industry. The number of projects and industries supported would soon reach 400 and 300, respectively. Close to two dozen products have already become realities, mostly in the biogeneric and devices sectors. Most of the projects have been benefited by formal and informal collaborations with the academia, besides intense project monitoring and mentoring by subject experts drawn from various public sector R&D institutions. Substantive basic research in academic institutions has shown potential for major innovations in biotechnology.
Father of proteomics
G N Ramachandran, the doyen of bioscience research in India, established the triple helical structure of collagen using electronic desk calculators at a time, when computers did not exist in India. Issues that arose from this structure determination made him to look at contact distances between C, N, O and H atoms in amino acids and peptides. The concept of limiting distances was applied to polypeptide conformations, leading to the development of the Ramachandran map1. The map was applied to myoglobin, the first crystal structure for any protein obtained at that time and has since become a routine tool for anyone studying protein conformation. One wonders how studies on crystal structures of proteins and functional properties based on conformation would have ever evolved without this basic understanding of Ramachandran angles. It is a pity that GNR, someone who laid the foundation for modern day proteomics, was not awarded the Nobel Prize.
Weapons of mass protection
In today’s context, India is a global leader in vaccines, supplying cheap and affordable vaccines to 150 countries. Most of these represent conventional vaccines, but interestingly many groups in the country have laid the foundation for innovation in this field. The efforts are no less laudable than sending a satellite up in the sky, but, perhaps, are not glamorous enough to catch attention. The fact of the matter is that these efforts would save millions of life.
The credit should go to G P Talwar as the father of modern vaccine research in the country. He is a pioneer who invented the first birth control vaccine in the world. The female vaccine consisted of a heterospecific dimer of the β-subunit of human chorionic gonadotropin linked non-covalently to α-subunit of ovine luteinising hormone and conjugated to tetanus and diphtheria toxin as carriers2. This vaccine never made it for a variety of sociological reasons. However, Talwar's immunotherapeutic leprosy vaccine has been commercialised and has shown efficacy as an adjunct to chemotherapy in tuberculosis and even in some cancers. It is befitting that the vaccine candidate is named Mycobacterium pranii after Pran Talwar.
Cholera and tuberculosis vaccines
One has to start with Sambhu Nath De who discovered the cholera toxin in 1959 and also developed the rabbit ligated ileal loop assay for toxin detection3. Amit Ghosh, presently at National Institute of Cholera and Enteric Diseases (NICED), Kolkata, and his team developed the first Indian-made live oral vaccine from a non-toxigenic strain of V. cholerae 01 El Tor. The strain is devoid of the cholera toxin, and the gene for the ‘B’ subunit of cholera toxin was engineered into the cryptic haemolysis locus. The resulting non-reactogenic VA 1.4 vaccine was found to elicit almost 66% seroconversion in a single oral dose4. This is superior to the 53% seroconversion seen after two doses with the available killed vaccines. Unfortunately, after years of effort, the path for commercialising this 100% made-in-India vaccine is not clear.
Anil Tyagi and his group at the Delhi University South campus expressed several M. tuberculosis antigens in BCG and a detailed evaluation of the candidate vaccines against aerosol infection of M. tuberculosis in guinea pigs using heterologous prime-boost approach (BCG over-expressing 85C followed by BCG overexpressing α-crystalline followed by boosting with crystalline-DNA vaccine) has given a successful candidate vaccine. Adjunct therapy can drastically reduce duration of chemotherapy5. How to take it forward?
Chetan Chitnis at the International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, has taken up the onerous challenge of developing a vaccine against Plasmodium vivax based on the interaction of the Duffy-binding protein with its receptor, enabling invasion into the human erythrocytes. The receptor-binding domain maps to conserved cysteine-rich region, referred to as PvRII. A vaccine against P. vivax malaria is being developed based on PVRII and is being taken through various steps of clinical trial6.
Shantha Biotechnics (now a Sanofi company) put India on the global biotech map in 1997 with the first recombinant hepatitis B vaccine using a commercial expression system in Pichia pastoris. This offered a challenge to the imported vaccine selling at Rs 475 a dose. Eventually, UNICEF wanted to buy the vaccine from India for Rs 20 a dose for global distribution. Less advertised, is the same vaccine made by Rangarajan at the Indian Institute of Science (IISc), Bangalore, using the expression system indigenously developed in a local strain of Pichia pastoris. This was transferred to one company in 1998 and two others in 2014. A DNA vaccine comprising of a mammalian expression plasmid in E. coli, encoding rabies surface glycoprotein as a dog rabies vaccine was also developed by Rangarajan7 with all safety data and regulatory approvals.
This could have paved the way for the same vaccine to be used in humans. But it did not make it, since a cheaper vaccine, perhaps, does not make commercial sense. Bhan and his associates at the All India Institute of Medical Sciences, New Delhi, identified a novel reassortant of a human rotavirus strain with a single bovine VP4 gene segment. This new genotype G9P10, referred to as 116E strain, turned out to be a naturally attenuated candidate rotavirus vaccine. Durga Rao and his associates at Indian Institute of Science (IISc), Bangalore, identified another rotaviral strain, I-321 with a completely different set of human and bovine gene segments. This also protected newborns against reinfection. In a large scale trial with infants, 116E was found to be well tolerated giving 53.6% efficacy8, 9. It is possible that a vaccine combination with 116E and 1-321 strains could give even higher levels of protection. Sudanshu Vrati and his colleagues at Translational Health Science and Technology Institute (THSTI), New Delhi developed a Vero cell-derived JE vaccine, inactivated at 22 °C with formaldehyde using an Indian strain, which is projected for scale up under GMP conditions10.
The challenge to control dengue infection is to develop a vaccine that would neutralise all the four known serotypes. Navin Khanna and his colleagues at ICGEB, New Delhi, have developed a novel tetravalent dengue vaccine by fusing the receptor-binding domain III of the four dengue virus serotypes (DEN-1, DEN-2, DEN-3 and DEN-4). The protein expressed in P. Pastoris elicited antibodies in mice and neutralised the infectivity of all the four serotypes, providing the basis for a safe, efficacious and inexpensive tetravalent dengue vaccine11.
Development of influenza vaccine poses a similar challenge. Raghavan Varadarajan and his associates at IISc, Bangalore have developed a thermotolerant, disulphide-free, and trimeric hemagglutinin stem-fragment immunogen, eliciting broadly neutralising antibodies against highly divergent group 1 (H1 and H5 subtypes) and 2 (H3 subtype). A similar immunogen from the unmatched, highly drifted influenza strains offered protection against a lethal heterologous virus challenge in vivo12. A similar challenge is in the generation of broadly neutralising antibodies seen in HIV infected patients with the envelope protein as the immunogen, retaining the native trimeric structure13. Bimal Chakraborti and his colleagues at THSTI, New Delhi have been working with the efficiently cleaved Env expressed on the cell surface as a genetic vaccine to elicit potent, broadly neutralising antibodies and to assess clade specificity as well.
There are many examples where recombinant antigens and MABs have been developed in academic laboratories and transferred to industry for making diagnostic kits. A few examples are those for malaria, HIV and hepatitis B. An innovative example is the development of designer proteins to make a highly sensitive diagnostic kit to detect HCV, HIV, dengue virus along with hepatitis B by Navin Khanna and his associates at ICGEB. In collaboration with the University of Turku, Finland, novel reporter assays based on Terbium-labelled nanoparticles have been developed. Dual assays and 3-in-1 assays to detect HIV, HBV, HCV and a similar 3-in-1 dengue test have all been developed. Some of the products are available in the market.
Although close to 20 biogeneric molecules have been developed in the industry, some strikingly novel molecules and approaches have been developed by the academic community. A good example is the progressive improvement in the production of thrombolytics, starting from native streptokinase, recombinant SK and then on to the third generation clot buster (CSSK) by Girish Sahni and his colleagues at the Institute of Microbial Technology (IMTECH), Chandigarh. The non-specific plasminogen-activation of SK was countered by fusing its core elements with fibrin-binding domains 4 and 5 of human fibronectin14.
CSSK remains inactive in circulation but gets activated by plasmin in the clot. The earlier products have been a commercial success and the clot buster, when fully validated can be a block buster. Kanury Rao and his associates at ICGEB have taken up a novel approach to target host factors to develop molecules against tuberculosis. Based on SiRNA screens of kinases and phosphatases in infected murine macrophages, they identified host molecules essential for pathogen survival15. This has led to the development of inhibitors with potential to become drugs against TB and transferred to industry.
Some exciting results have been obtained in my laboratory with arteether-curcumin combination to treat parasite recrudescence and cerebral malaria in experimental animals. Apart from the exciting possibility of using curcumin as an adjunct drug in malaria, the study has laid the foundation to use curcumin as an adjunct drug in many infectious diseases more as an immunomodulator than as a direct killing agent, with potential to have long-term protection capability, acting as a vaccine substitute16, 17. We can be proud that curcumin from turmeric, used in ancient Indian medicine, has found validation using modern scientific principles.
By any yardstick, the examples quoted would stand out in terms of innovation with a potential as significant inputs to produce original biotech products in the health sector, beyond biogenerics. This belies the impression that basic research in India is not connected to applications. Each discovery has a story to tell regarding the stumbling blocks that need to be tackled to translate outstanding research into a product.
Will the BIRAC route provide a new opportunity for translating innovative science? While, India needs to have many more such examples the quality of science behind the examples quoted in terms of innovation, novelty, patents and publications definitely meets high global standards. India's Department of Biotechnology (DBT) supported most of these projects and deserves encomiums for the sustained support. But, at this stage a strategy is needed to make these innovative leads into products.
*The author is the former director of Indian Institute of Science (IISc) and currently an honourary professor at the Department of Biochemistry, IISc, Bangalore, India.
2.Talwar, G. P. et al. A vaccine that prevents pregnancy in women. Proc. Natl. Acad. Sci. 91, 8532–8536 (1994)
3. De, S. N. & Chatterjee, D. N. An experimental study of the mechanism of action of Vibrio cholera on the intestinal mucous membrane. J. Pathol. Bacteriol. 66, 559–562 (1953)
4. Kanungo, S. et al. Safety and immunogenicity of a live oral recombinant cholera vaccine VA 1.4: A randomized, placebo controlled trial in healthy adults in a cholera endemic area in Kolkata, India. PLoS ONE 9: e99381 (2014)
5. Chauhan, P et al. Adjunctive immunotherapy with L- crystalline based DNA vaccination reduces tuberculosis chemotherapy period in chronically infected mice. Sci. Rep. 3, 1821 (2013)
6. Chitnis, C & Sharma, A. Targeting the Plasmodium vivax Duffy-binding protein. Trends Parasitol. 24, 29–34 (2008)
7. Biswas, S. et al. Preexposue efficacy of a novel combination DNA and inactivated rabies virus vaccine. Hum. Gene Ther. 12, 1917–1922 (2001)
8. Bhandari, N. et al. Efficacy of a monovalent human-bovine (116E) rotavirus vaccine in Indian infants: a randomized, double-blind, placebo-controlled trial. Lancet 383, 2136–2143 (2014)
9. Glass, R. I. et al. Development of candidate rotavirus vaccines derived from neonatal strains in India. J. Infect. Dis. 192, S30–S35 (2005)
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11. Etemad, B. et al. An envelope Domain III-based chimeric antigen produced in Pichia pastoris elicits neutralizing antibodies against all four dengue virus serotypes. Am. J. Trop. Med. Hyg. 79, 3353–3363 (2008)
12. Mallajosyula, V. V. et al. Influenza hemagglutinin stem-fragment elicits broadly neutralizing antibodies and confers heterologous protection. Proc. Natl. Acad. Sci. USA 111, E2514–2523 (2014)
13. Chakrabarti, B. K. et al. Robust neutralizing antibodies elicited by HIV-1 JRFL envelope glycoprotein trimers in nonhuman primates. J. Virol. 87, 13239–13251 (2013)
14. Aneja, R. et al. Identification of a new exosite involved in catalytic turnover by the streptokinase-plasmin activator complex during human plasminogen activation. J. Biol. Chem. 284, 32642–32650 (2009)
15. Jayaswal, S. et al. Identification of host-dependent survival factors for intracellular Mycobacterium tuberculosis through a siRNA screen. PLoS Pathog. 6: e 1000839 (2010)
16. Dende, C. et al. Simultaneously targeting inflammatory response and parasite sequestration in brain to treat Experimental Cerebral Malaria. Sci. Rep. 5: 12671 (2015)
17. Padmanaban, G. & Rangarajan, P. N. Forum: Curcumin as an adjunct drug for infectious diseases. Trends Pharmacol. Sci. 1282 (2015)