The Neonatal Rotavirus Vaccine Project (Strain 116E) Rotavac: A Brief Review for the 25th Anniversary of the Indo-US Vaccine Action Program (VAP)

Article contributed by Roger Glass. M.D., Ph.D, Director, Fogarty International Center, NIH

Fogarty Director Dr. Roger I. Glass contributed this article on the Neonatal Rotavirus Vaccine Project as a part of the 25th anniversary of the Indo-U.S. Vaccine Action Program (VAP).

In 1985, Dr. Maharaj Bhan and I met at a WHO meeting on diarrheal diseases in Calcutta. We were lodged at a convent with strict rules so in the evening, we decided to go out for a drink, something not permitted on the premises. Dr. Bhan, then a young assistant professor of pediatrics at the All India Institute of Medical Sciences (AIIMS), mentioned that he had been following an outbreak of rotavirus in the newborn unit at AIIMS but noted surprisingly that the infected neonates did not develop diarrhea. At the time, I was working in the lab of Dr. AI Kapikian at the National Institutes of Health (NIH) and we were characterizing an unusual group of rotaviruses collected from newborns on 4 continents that also did not cause disease. These unusual but perceptive observations by Dr. Bhan led to our collaboration to characterize these neonatal rotavirus strains and determine whether newborns asymptomatically infected with these strains were protected against subsequent severe disease with rotavirus.

After three years of informal collaboration, the Indo-U.S. Vaccine Action Program (VAP) was launched by the Department of Biotechnology (DBT), and the National Institute of Allergy and Infectious Diseases (NIAID), NIH, and our proposal was approved. In 1989-90, DBT along with NIH and USAID provided the first funding, seed money that was meant to grow programs and products. Dr. Bimal Das from AIIMS visited CDC to characterize the AIIMS newborn strain. Dr. Jon Gentsch at CDC had just developed RT­ PCR typing methods to determine the G- and P- genotypes of common human rotavirus strains. Surprisingly, the Indian neonatal strains could not be typed. After intense investigation, the strain turned out to be a novel reassortant of a human rotavirus strain with a single VP4 gene segment replacement of bovine origin. The AIIMS strain, now called 116E, was characterized as genotype G9P10, which had not been seen previously. Follow up of infected neonates by Dr. Jayshree Ayer at AIIMS indicated that these neonatal infants mounted a robust serum and mucosal immune response to rotavirus. Moreover, study of a cohort of infected and non-infected infants by Drs. Bhan and Judy Lew (from CDC) indicated that those newborns infected were protected against subsequent severe rotavirus disease. This observation immediately raised the question of whether strain 116E might be a naturally attenuated candidate rotavirus vaccine.

Further studies of the epidemiology of rotavirus in India changed the way we think about the epidemiology of rotavirus infections in low vs. high income settings. In India, Drs. Madhu Ramachandran and Vivek Jain demonstrated that the diversity of rotavirus strains circulating in India at any time was greater than that seen in the rest of the world, and Indian children were more likely to be infected with several strains rather than a single strain. Rotavirus infections are highly seasonal in the United States and Europe but occur year round in India. Eighty percent of children in India were infected in the first year of life, and severe infections were fatal for about one in 200 children. Dr. Jain estimated that India witnessed about 100,000 deaths per year from rotavirus in contrast to the United States where fewer than 100 children die each year!

As we began our work on rotavirus in New Delhi, another group led by Drs. Durga Rao in Bangalore and Harry Greenberg at Stanford University began a similar effort, also funded through the VAP. Durga had returned to India after completing his post-doc at NIH and was exploring new research avenues to pursue. He saw an advertisement from DBT announcing the Indo-U.S. VAP and the importance of rotavirus. Fortuitously, he had prior experience working with Bluetongue virus, a close relative of rotavirus. Durga contacted Dr. Greenberg and visited Stanford University to establish a collaboration on rotavirus. Harry had played an important role in the initial discovery and development of the first generation of rotavirus vaccines (Rotashield) and the VAP support enabled the two to begin a highly productive collaboration. Durga had also identified a distinct asymptomatic neonatal rotavirus strain from Bangalore, identified as 1-321, which had a completely different constellation of human and bovine gene segments than the 116E strain. Neonatal infection with 1-321 also seemed to protect newborns against subsequent severe diarrhea on reinfection, so a healthy and productive competition evolved between the two groups as they worked to characterize these two interesting strains, both supported by the VAP.

In 1993 Gray Handley, then U.S. Health Attache in Delhi, encouraged us to patent the 116E strain as a candidate vaccine. The 1-321 strain was not patented in the United States. Dr. George Curlin worked through the VAP to have NIAID prepare two pilot lots of vaccine - for 116E and 1-321 - that were tested for safety in U.S. adults through the NIH Vaccine Testing and Evaluation Units, managed at the time by Dr. Regina Rabinovich. After a long delay due to suspected contamination of the cell bank, the candidate vaccines were both found to be safe in adults and ready for further study in children and infants. At this point, 1998, Dr. Kate Aultman, the NIAID Project Officer, together with DBT's Dr. T.S. Rao hosted a VAP meeting in Bangalore to bring investigators with candidate vaccines together with a group of Indian vaccine manufacturers. At the meeting, presided over by U.S. Secretary of Health Donna Shalala, we met Dr. Krishna Ella, CEO of Bharat Biotech. At the time he had not made a single vaccine although he was working on a hepatitis B surface antigen candidate vaccine. His vision, enthusiasm and "can-do" attitude was infectious, and while he had only one small vaccine production facility in Hyderabad, he promised to have a factory in place within nine months of our first visit to his plant.

In 1999, the Gates Foundation funded PATH for $25 million to work on vaccine introduction in Andhra Pradesh. A $6 million set-aside in the grant allowed us to begin the project at Bharat with support from Dr. Jim Maynard of PATH. The VAP had initially funded the two related rotavirus programs but only one could eventually go forward to full development. The two groups joined forces to move both neonatal strain candidates forward into early safety and immunogenicity trials in humans with the understanding that somewhere along the way, one of the strains would outperform the other and be selected for final development. Our teams combined and gained great strength by our willingness to share resources, intellectual capacity and expertise in a true partnership. It has been an amazing ride!

Bharat had no previous experience in making a viral vaccine in cell culture so initial progress was painfully slow. Their key laboratory staff members were trained at CDC by Drs. Jon Gentsch and Baoming Jiang in methods and tests required to prepare the virus as a candidate vaccine. Dr. Harshavardhan assembled a team for the project while the facilities in Hyderabad were being constructed. The Greenberg lab at Stanford helped make several critical antibody reagents to allow accurate quantification of viral yield in cell culture and Dr. Greenberg, who had recently spent two years in the private sector bringing the live attenuated influenza vaccine to licensure, provided helpful "real-world" expertise in areas of development, project management and regulatory affairs. In 2003, Mr. Sai Prasad joined as project manager to speed up the development process. Then, tragedy struck. Mycoplasma contamination was found in the new facility and all strain, cells, and a pilot lot had to be discarded. The lab was thoroughly cleaned, and all efforts began anew. Problems arose immediately when the Bharat team could not readapt the vaccine strain to grow in Vero cells. We dispatched Dr. Jiang to Hyderabad and in two weeks, he was able to recover the vaccine virus, adapt it to grow to reasonably high titer, and the game continued. This experience turned back the clock, but got everyone together, with new experience and confidence. Dr. Gillian Challoner Larson, a consultant, assisted in these difficult times and helped to shape the team through her stubbornly strict adherence to international standards. These were not easy lessons to teach.

As part of the program, we established sentinel hospital surveillance for rotavirus diarrhea in five cities in India to begin to get a better assessment of the burden of disease. Dr. Umesh Parashar, a graduate of AIIMS, now at CDC, discussed this proposal which was severely underfunded with Dr. Nirmal Ganguly, then DG of the Indian Council of Medical Research Nirmal, responded that this surveillance should be funded by the ICMR since it was of national interest and he more than doubled the funding to begin a national surveillance activity Dr. Pratima Ray and Dr. Gagandeep Kang joined this effort and developed strong involvement in the laboratory issues.

Between 2003 and 2008, the program experienced some major changes in leadership and organization. The PATH team was reorganized twice before Dr. John Boslego joined. John brought real vaccine development experience from his time at Merck and brought in Dr. Georges Thiry who helped with project management and Dr. Duncan Steele who had just left WHO and was an expert on rotavirus clinical investigation and epidemiology. The program was back on track Bharat reorganized and Dr. Krishna Mohan assumed leadership of the project joining Mr. Sai Prasad and Dr. Harshavardhan. NIH assisted with advisors on GCP and data management (Ms. Beth Horigan and Dr. Bob Kohberger). Dr. Nita Bhandari, the most wonderfully capable and compulsive epidemiologist, moved her team from AIIMS to the Society for Applied Studies (SAS) to facilitate conducting the trials with freedom and independence from the rules of the academic center. Dr. Bhan became Secretary of the Department of Biotechnology, one of the first groups in the VAP that supported the activity; CDC remained involved in rotavirus surveillance and in assisting with clinical design and laboratory issues related to the trials. Dr. Greenberg remained a valued partner from Stanford providing basic virology expertise as well as real world vaccine development knowledge and Dr. Sudanshu Vrati opened a rotavirus vaccine lab at India's National Institute of Immunology to support clinical studies by establishing validated studies.

Trials of the NIH and then the Bharat pilot lots provided some promising results. Nita Bhandari and her group at SAS demonstrated that both 116E and 1-321 were safe in adults and infants. She also found that the immune response to 116E in infants (89 percent) was greater than that seen for Rotavirus in India (about 55 percent). At the same time, the other neonatal vaccine candidate, strain 1-321, was much less immunogenic, and so further development efforts for this strain were dropped However, Dr. Greenberg remained as a valued partner advising on further development of the 116E candidate. Despite the encouraging immunogenicity results, it took two years to develop the protocol and clinical development plan for the final pivotal trials and secure the financial support needed to begin the trials of 116E. Funds were raised from the Gates Foundation through PATH, the DBT, and the Government of Norway, with additional inputs from NIAID and CDC through the VAP.

The pivotal phase three trial of the vaccine was launched in March 2011 with recruitment at three outstanding sites - in Delhi (SAS; Dr. Temsunaro Rongsen-Chandola), Pune (KEM; Dr. Ashish Bavdekar) and Vellore (CMC; Dr. G. Kang). The trial is being coordinated by the Clinical Operations Management Unit led by Dr. Nita Bhandari (SAS), Project Director, and supported by Ms. Kalpana Antony (PATH), Project Coordinator and Dr. Sunita Taneja (SAS), Data Management and Biostatistics. By November 2011, about 6,800 infants had been enrolled and randomized to receive either the vaccine or placebo and follow up is now in progress. The trial will be unblinded when 85 cases of severe rotavirus diarrhea are identified through careful and accurate surveillance and testing. If the results demonstrate a high level of safety and efficacy, Bharat will submit the portfolio to the Drug Controller General of India for consideration for licensure.

If licensed, this vaccine will hopefully be used first to prevent rotavirus diarrhea in India and the approximately 100,000 deaths and several hundred thousand hospitalizations and clinic visits estimated to occur from this most common infection of children. The product will also be the first totally new vaccine developed entirely in India for more than a century, made with an Indian strain, an Indian manufacturer and Indian clinical trials, through an Indo-U.S. partnership initiated by the VAP and strongly supported by the Gates Foundation, the Indian DBT and some strong collaborators at SAS, CDC, NIH, Stanford, PATH, Bharat Biotech and the Government of Norway. Results should become available in 2013.

Along the way, this collaboration led to a number of important additional advances in support of vaccine development in India by Bharat. The qualified Vero cells brought in for this program were subsequently used as substrate for a rabies vaccine made with a strain provided through the collaboration from CDC; this product is now licensed and marketed in India. Through the NIH connection, Bharat received methods to develop the Vi typhoid vaccine of Dr. John Robbins, now licensed in India, as well as the typhoid conjugate vaccine that is in final stages of development. The group of Dr. Bhandari, which began at AIIMS, has established itself as an independent and highly successful NGO conducting clinical trials with 116E and other products and protocols. Additionally, a newly formed Translational Research Center has been established in Gurgaon as the home for a vaccine support laboratory headed by Dr. Sudhanshu Vrati. Clearly, this long-term collaboration has had spinoff effects that have been incredibly valuable but never could have been anticipated in advance. The training of several dozen junior researchers and publication of more than 40 papers is striking. Most important is the prospect that this project could well lead to a novel vaccine for the prevention of rotavirus diarrhea in India and beyond. It would also be a vaccine affordable by all at a starting cost of about $1.00 per dose, and with the hope of WHO prequalification that would make it accessible to GAVI and UNICEF. We still have a way to go but prospects for success are most exciting and we could have definitive results in late 2012 or early in 2013. The total cost of the project, to bring a new vaccine from earliest discovery to market, will be less than $50 million, less than a tenth of the cost of bringing a similar live attenuated viral vaccine to market in an industrialized setting.

This project could not have advanced with the help of many people and institutions. We have to acknowledge the steadfast support of Bill and Melinda Gates who recognized the importance of rotavirus early on and committed funds for this project from the beginning of its commercial development through the pivotal trials. DBT and NIH have co-hosted the VAP and provided early and continuing support to build a long-term collaboration between groups. Krishna and Suchitra Ella signed on to the project never appreciating the complexity of developing a product that would have a protracted clinical time line and have built a wonderful team to make this happen. PATH and John Boslego stepped in to help speed the clinical development program. The list of people to thank goes on and will be recorded at a future date but there have been many. Without the skills and support of the entire menagerie, and despite the difficulties of bringing a new manufacturer up to international standards, we are very close to determining whether or not this vaccine really works and can do what we feel will make a huge difference to improve the health of children in India and beyond.

[The rotavirus research group mourns the loss of Dr. Bob Kohberger who assisted in the development of the clinical trial program.]

Program Trainees and Advisors

• India: AIIMS
• Dr. Jayshree Ayer
• Dr. Bimal Das
• Dr. Madhu Ramachandran
• Dr. Pratima Ray
• Dr. Rajiv Bahl
• Dr. Durga Rao
• United States: CDC
• Dr. Jon Gentsch
• Dr. Judy Lew
• Dr. Helen Cicirello
• Dr. Vivek Jain
• Dr. Baoming Jiang
• Dr. Umesh Parashar
• Dr. Jacqueline Tate
• Ms. Melissa Arvay

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