The big test for malaria vaccine RTS,S

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In 2015, Africa accounted for 92% of global deaths and 90% of all malaria cases.

Beginning next year, the World Health Organisation will begin pilot tests of the injectable malaria vaccine RTS,S (or Mosquirix) on 750,000 children aged 5-17 months in Ghana, Kenya and Malawi. The vaccine has been successfully put through a Phase III trial, in which the drug is tested for safety and efficacy. Any decision on wider use will be taken based on the results of the pilot tests in the three countries. If the vaccine does indeed prove to be ready for large-scale use, it will be a milestone in the fight against malaria. Although the number of cases globally and in the African region came down by 21% between 2010 and 2015, in 2015 itself the number of deaths worldwide on account of the disease was as high as 429,000. According to WHO estimates, Africa accounted for 92% of these deaths, and 90% of the 212 million new cases that year. In such a scenario, even a vaccine with limited benefits could yield a substantial improvement. The vaccine, given in four doses, protects against Plasmodium falciparum, which is the most prevalent malaria parasite in Africa. The three countries have been chosen as they have settings with moderate-to-high transmission of malaria and already have in place malaria control programmes such as the use of bed-nets, rapid diagnostic tests and combination therapy. Each country is to decide where precisely to run the pilots.

The first three doses of the vaccine will be administered with a minimum interval of one month between each dose, followed by the fourth dose 15 to 18 months after the third dose. The first dose will be administered at about five months of age and the third dose has to be completed by nine months of age. While the drop-out rate increases as the number of doses increases, the biggest challenge is the fourth dose, which warrants a new immunisation contact to be made 15 to 18 months after the last dose. In Phase III trials, the efficacy of the vaccine was around 30% when children received all the four doses; the vaccine also reduced the most severe cases by a third. But there was a significant drop in these benefits when children did not receive the fourth dose. Given the low protection efficacy of the vaccine even in tightly controlled clinical settings, the pilot tests will be useful in evaluating the likelihood of replicating the immunisation schedule in the context of routine health-care settings. Also, the extent to which the vaccine reduces the all-cause mortality has to be evaluated as this was not “adequately addressed” during the trial. There is, specifically, a need to ascertain if excess cases of meningitis and cerebral malaria seen during the trials are causally related to the vaccination. Unlike other vaccines, the less-than-optimum protection offered by this vaccine would mean that existing malaria intervention measures will have to be used in conjunction to reduce the incidence of the disease.

Published in The Hindu on April 28, 2017

Zika vaccine Phase I trial to begin next month in India

measuring microcephaly. Photo WHO

The Phase I trial will not involve pregnant women.

Bharat Biotech International Ltd in Hyderabad will start a Phase I clinical trial of Zika virus vaccine (MR 766) in two centres in India next month. The inactivated  vaccine being tested in humans will be an African Zika virus strain. “We have already got the approval from DCGI [Drug Controller General of India] in March to carry out the Phase I clinical trial,” says Dr. Krishna Ella, Chairman and Managing Director of Bharat Biotech.

It will be a randomised, placebo-controlled, double-blind trial involving 48 adults, both men and women (who are not pregnant). Each volunteer will receive two doses of the vaccine on days 0 and 30 and followed-up for a year for both safety and immune responses. Three different dose ranges (2.5, 5 and 10 microgram) will be tested.

There will be three arms in the trial and each arm will get one of the three different dose ranges. There will be 16 subjects per arm.

The Phase I human clinical trial is being initiated based on the promising results seen in animals trials. In the animal trials, two doses of the vaccine made using an African Zika virus strain conferred 100% protection against mortality and disease in mice. The protection was the same when the mice were infected with an Asian and an African Zika virus strains.

While the viral load was “undetectable” in the case of vaccinated mice, the amount of virus in unvaccinated mice shot up four days after being infected. All the unvaccinated mice died eight days after infection by the African strain of the virus and 12 days after infection by the Asian strain.

Published in The Hindu on April 18, 2017

Bharat Biotech’s Zika virus vaccine confers 100% protection in mice

zika-microcephaly - Photo WHO

The vaccine made using an African strain confers 100% protection against infection and mortality caused by Asian and African Zika virus strains.

The Hyderabad-based Bharat Biotech’s killed (inactivated) Zika virus vaccine using an African strain (MR 766) has shown 100% efficacy against mortality and disease in animal studies. A ‘killed virus vaccine’ or ‘inactivated vaccine’ contains virus that has been grown in culture and then killed using physical or chemical processes. The whole virus was used for developing the vaccine.

Two doses (5 and 10 microgram) of the vaccine given through intramuscular route on days 0 and 21 to mice were found to protect the animals against Zika virus seven days after the second vaccination. The vaccine was found to confer 100% protection against infection caused by an Asian Zika virus strain as well as by the African Zika virus strain.

All the animals that were not vaccinated died eight days after infection by the African strain of the virus and 12 days after infection by the Asian strain. All the mice that did not receive the vaccine showed progressive morbidity before succumbing to infection.

While all the animals that received the vaccine exhibited “undetectable” viral load, the amount of virus present in animals that did not receive the vaccine peaked four days after being infected with either the African or Asian Zika virus strain. The results of the study were published in the journal Scientific Reports.

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The AG129 mouse is highly immunocompromised.

“The vaccine was developed using the African strain of the virus. It is important to prove that the vaccine developed with the African strain also protects against Zika infection caused by the contemporary Asian strains of Zika virus. Importing the contemporary Asian strains into the country was difficult, and hence the vaccine challenge studies with Asian strain had to be outsourced to a CRO in the U.S.,” says Dr. K. Sumathy from Bharat Biotech and the first author of the paper.

A particular kind of mouse — AG129 — which is highly immunocompromised and hence highly susceptible to virus infection was used for studying the protection conferred by the vaccine against Zika virus, disease pathogenesis and mortality. All the AG129 animals that received the vaccine showed 100% protection against the virus, demonstrating the superior efficacy of the vaccine.

Immunogenicity studied

Additionally, the level of immune response induced by the vaccine was also studied using another kind of mouse model — Balb/c mice. Unlike the AG129 mice, this mouse model is immunocompetent and elicits full spectrum of immune response. Animals that received the vaccine developed Zika neutralising antibodies on day 14 after the first dose and a week after the second dose. When the animals were infected with Zika virus post-vaccination, the virus in the vaccinated animals was “undetectable”, while 72-96 hours after infection it peaked in animals that did not receive the vaccine.

“In both the mice models, the vaccine-induced protective immunity against virus challenge was observed,” says Dr. Sumathy. “The vaccine was made only with the African virus strain, but the vaccinated mice was challenged [infected] with both the African and the Asian strains. Our vaccine offered equivalent protection against challenge with both the African and the Asian strains of Zika virus.”

Though 5 and 10 microgram of the vaccine were tested, the amount of antibodies elicited by the higher dose was “not significantly” higher than that elicited by 5 microgram of the vaccine, says Dr. Sumathy. Vaccination protected the animals against Zika virus and disease up to 14 and 20 days after being challenged with the virus.

Passive immunisation

Bharat Biotech also carried out passive immunization studies to show that the Zika vaccine-induced antibodies confer protection against the virus in mice that were exposed to the virus. Rabbits were vaccinated with the vaccine and the vaccine-induced antibodies were given to mice. While no virus was detected in mice 24-144 hours after passive immunisation, the viral load peaked 72-96 hours in mice that did not receive the vaccine-induced antibodies.

“Our study shows that the choice of Zika virus strain may not be a limiting factor in vaccine development,” she says.

Published in The Hindu on April 17, 2017

TB vaccine trial on adults begins in June in India

VPM 1002 TB vaccine

The candidate vaccine VPM1002 will be tested in India.

In June this year, the Pune-based Serum Institute of India Pvt Limited will begin a Phase II/III vaccine trial for tuberculosis using a novel, recombinant BCG (bacillus Calmette-Guérin) vaccine. The double-blind, placebo-controlled, randomised trial will be carried out on 2,000 adults who have been successfully treated (and cured) for TB. While 1,000 adults will receive the vaccine, the remaining volunteers will receive a placebo. A single dose of the vaccine will be administered and the volunteers will be followed-up for a year. The trial will be conducted in 15-17 centres across India.

The new TB vaccine (VPM1002) that will be tested is based on the BCG vaccine that is currently being used but is more powerful and efficacious as it contains a gene that is better recognised by the immune system.

“Adults who have completed TB treatment will be first screened and enrolled if found eligible 2-4 weeks after completion of TB treatment,” says Dr. Prasad S. Kulkarni, Medical Director at Serum Institute. “Traces of the drugs may be present in the body for two weeks after completion of the treatment. Since the vaccine contains live, weakened bacteria, the drugs can kill them if given earlier than two weeks after completing the treatment.”

The vaccine will be first administered in 200 volunteers and safety of the vaccine will be tested. “If there are no safety concerns then the trial will continue in the remaining 1,800 volunteers,” he says.

The safety of the vaccine has already been tested in two Phase I trials — 80 adults in Germany (2009) and 24 adults in South Africa (2010) — and one Phase 2a trial in South Africa in 2012 in 48 newborn infants who have not been exposed to HIV. “These trials have confirmed the safety of the vaccine and sufficient strengthening of the immune system,” says Umesh Shaligram, Director-R&D, Serum Institute.

The results of the Phase 2a trial in newborns in South Africa published in February this year in the journal Clinical and Vaccine Immunology has confirmed the safety of the vaccine. “The VPM1002 is a safe, well-tolerated, and immunogenic vaccine in newborn infants, confirming results from previous trials in adults,” the paper says.

BCG - WHO

The currently used BCG vaccine causes problems in HIV positive babies.

A Phase 2b trial on 416 newborns who have either been exposed or not exposed to HIV is currently under way in South Africa. “Results of the Phase 2b trial will be known in August-September this year. So far there have been no safety concerns,” Dr. Kulkarni says.

While the currently used BCG vaccine causes BCG-related disease in HIV-positive babies (due to reduced immunity), the recombinant version is expected to be safe in babies exposed to HIV.

Serum Institute is also planning to start next year a Phase III trial in newborns in India.

The recombinant vaccine was developed by a team led by Stefan H.E. Kaufmann, the founding director of the Max Planck Institute for Infection Biology, Berlin. The Max Planck Institute holds the patent and has licensed the vaccine to VPM; VPM, in turn, has out licensed it to Serum Institute.

In February last year Prof. Kaufmann told me: “The vaccine being tested is intended to replace the current BCG vaccine and will be administered to young children to protect them against tuberculosis. Adults may also be able to benefit from it later.”

Published in The Hindu on March 29, 2017

India begins caged trials of GM mosquitoes to control dengue, chikungunya

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Laboratory studies using GM mosquitoes have been carried out in India since 2012

Outdoor caged trials to demonstrate the efficiency of genetically modified mosquitoes to suppress wild female Aedes aegypti mosquito populations that causes dengue, chinkungunya and Zika were launched on January 23 in Dawalwadi, Badnapur in  Maharashtra’s Jalna district.

Based on the results of the caged trials, which use the Release of Insects carrying Dominant Lethal genes (RIDL) technology, and permission from Indian regulatory authorities, Gangabishan Bhikulal Investment and Trading Limited (GBIT) and Oxitec, plan to conduct open field trials in the country.

The announcement was made on January 23 after the field cage facility was inaugurated by Dr. Soumya Swaminathan, Director General of the Indian Council of Medical Research.

Laboratory-based studies have already been carried out in India since 2012 by GBIT and Oxitec and these studies have demonstrated the compatibility of Aedes aegypti mosquitoes. “The efficiency to kill offspring was over 99% and male mosquitoes imported from the U.K were able to mate with locally available wild female mosquitoes and the longevity of imported mosquitoes was the same as the wild ones,” says Dr. Shaibal Dasgupta, Project Leader, GBIT, Delhi.

Oxitec’s technology uses genetically modified male Aedes aegypti mosquitoes that carry a dominant lethal gene. When male GM mosquitoes mate with wild female mosquitoes the lethal gene is passed on to offspring. The lethal gene in the offspring produces a protein that stops their cells from functioning normally and prevents other genes essential for survival from turning on. This causes the mosquito larvae to die before reaching adulthood.

Since male mosquitoes do not bite humans, the release of genetically modified males will not increase the risk of dengue, chinkungunya and Zika.

mosquito-larvae“The caged trials, which will last 50-55 weeks, will study the efficiency of vector suppression and mating capacity,” Dr. Dasgupta says. “Surveillance [to undertake open field studies] to gather data on predominance of Aedes mosquitoes in the wild has already started. The open field studies, which will be of one-year duration, will be conducted in two villages and two control villages in close vicinity of the caged study area in Jalna.”

“It is a promising technology and India must certainly look at new vector control methods,” says Dr. Swaminathan. “From studies carried out in other countries we know the safety is beyond doubt, but efficiency has to be proved, especially in big cities and towns.”

“There are practical problems of raising large number of mosquitoes needed for vector control — 100-150 [GM] mosquitoes are needed per person for months together. So some innovation in breeding techniques is needed,” says Dr. Swaminathan.

In the case of the RIDL technology, the genetically altered male mosquitoes have to be released in large numbers at regular intervals. Only then can they compete with the wild normal male insects for mating. Since the larvae die before reaching adulthood, the technology is a “self-limiting approach (the genetic modifications are not perpetuated in wild populations)”, notes a piece published in The Lancet (February 1, 2016).

The first open field trial by Oxitec was carried out in 2010 in the Caribbean island of Grahnd Cayman. The trial was successful in reducing the mosquito population by 80%. Similar trials have since been carried out in west Panama, Malaysia and more recently in Brazil with at least 90% reduction in vector population.

Another alternative

“India is looking at another alternative. We are about to sign a memorandum of understanding next month with Monash University for vector control using Wolbachia-infected A. aegypti mosquitoes,” she says. Pilot studies of mosquitoes harbouring the Wolbachia bacterium are being conducted in Colombia, Brazil, Australia, Vietnam and Indonesia to help control the spread of dengue virus.

Vector control using Wolbachia-infected A. aegypti mosquitoes is achieved by using the life-shortening bacteria strain in both male and female mosquitoes. Uninfected wild females embryos fertilised by Wolbachia-infected males fail to develop, while embryos from infected females fertilised by infected or uninfected wild males survive. As Wolbachia is maternally inherited, the bacteria are passed on to offspring immaterial of which male mosquito the female mosquito mated with. Dengue, Zika or chikunguya viruses cannot replicate when mosquitoes have Wolbachia and hence cannot be transmitted to humans when the mosquito bites.

Unlike the RIDL technology, a salient feature of Wolbachia is that it is self-sustainable, making it a very low-cost intervention to mosquito vector control. The downside is that the release of even a single female mosquito infected with Wolbachia bacteria could “potentially lead to the alien Wolbachia spreading in the target population” says a June 2013 report in the journal Pathogens and Global Health.

Published in The Hindu on January 25, 2017

Indian researchers enhance BCG vaccine efficacy using anti-leprosy drug

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The TB bacteria load was 50 times less in mice that got the vaccine and anti-leprosy drug than mice that got only the vaccine. Dhiraj Kumar Singh (left), Dr. Anand Ranganathan (middle) and Prof. Gobardhan Das

Indian researchers have been able to bring about more than 50-fold improvement in the efficacy of the commonly used TB vaccine — Bacillu Calmette Guerin (BCG) — by giving mice anti-leprosy drug (clofazimine) for a month along with a single dose of the vaccine. The duration of protection lasted till the end of the trial protocol period of 120 days. Results were published on August 29, 2016 in The Journal of Infectious Diseases.

“But the mice vaccinated with BCG will remain equally protected if just two doses of anti-leprosy drug are given on the day of vaccination and on day seven. This is because the drug has a long half-life of 28 days in mice,” says Prof. Gobardhan Das, the corresponding author of the paper from the Special Centre for Molecular Medicine, Jawaharlal Nehru University (JNU), Delhi.

To test the efficacy of the novel strategy, the BCG vaccinated mice that were co-treated with anti-leprosy drugs for a month were exposed to TB bacteria. “Sixty days after infection, there was more than 100-fold reduction in the TB bacterial load in the lungs of the mice that got both the vaccine and drug compared with mice that did not the vaccine and drug,” says Dhiraj Kumar Singh, a co-author of the paper from JNU. “The bacterial load was more than 50 times lesser in mice that got the vaccine and drug compared with mice that got only the vaccine.”

“This is an exciting advance over Prof Das’ earlier work on the limited efficacy of the BCG vaccine,” says Prof. Anand Ranganathan, a co-author in the paper from the Special Centre for Molecular Medicine, JNU. “It should be noted that a new anti-TB vaccine is unfortunately still a good decade or two away. Given this, an improvement in the existing vaccine, through administration of an existing drug, is as good as it can get.”

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The anti-leprosy drug increases the amount of central memory T cells in mice.

BCG efficacy 

The BCG vaccine efficacy is critically dependent on the generation of long-lasting memory cells called the central memory T (Tcm) cells. The Tcm cells, in turn, generate effector memory T (Tem) cells that kill the TB bacteria. Though BCG has more than 80 per cent efficacy in protecting children from meningitis and military TB, the vaccine’s efficacy diminishes with time, particularly in TB endemic regions.

This is because people in TB endemic countries are continuously exposed to TB bacteria as every third person harbours them. With regular exposure to TB bacteria, the Tem cells that fight the bacteria get used up and the pool of Tcm cells that get converted to Tem cells eventually get exhausted, thereby rendering the host to TB infection, explains Singh. If the Tcm cells are much higher in number to start with then they can convert to Tem cells for a longer period of time and produce a much rapid and stronger response against TB bacteria and protect the individual from TB infection for an extended period.

Tinkering with protection

This is precisely what the team led by Prof. Das achieved. The researchers nearly doubled the size of  central memory T (Tcm) cell pool by administering anti-leprosy drug (5 mg/kg body weight) to mice that were already vaccinated with BCG. “Initially after vaccination both Tcm and Tem cells are produced.  Because we don’t want Tem cells, we try to make most of the cells into Tcm cells. This is achieved by administering the anti-leprosy drug on the same day of BCG vaccination.  Since the drug blocks the potassium channel of Tem cells, the production of Tem cells is slowed down or inhibited and the cells are pushed to become Tcm cells. This leads to an increase in Tcm pool,” says Prof. Das.

“Our study suggests that clofazimine [anti-leprosy drug] treatment enhances the pool of Tcm cells induced by the BCG vaccine, and these cells have the potential to continuously replace Tem cells at the site of infection,” they write. “Therefore, clofazimine might function as an effective immune modulator to enhance the efficacy of BCG vaccination in humans.”

“Adults who have already been vaccinated with BCG can be given another dose of BCG along with clofazimine anti-leprosy drug. Since mice were protected for 3-4 months, we can conservatively say that the vaccine protection could last for 40 years in humans,” says Prof. Das.

The next step is to test the novel strategy in monkeys and then in humans. “I would like to carry out the test in monkeys if I get funding,” Prof. Das says.

Published in The Hindu on August 30, 2016

Indian researchers use a novel drug to inhibit skin cancer in mice

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Dr. Gopal Kundu (left) and Dhiraj Kumar found 90 per cent of lung metastasis getting cleared by the drug at the end of 27 days.

Researchers at the Pune-based National Centre for Cell Science (NCCS) have been successful in isolating cancer stem cells that cause skin cancer. They have also been able to demonstrate that a compound (Andrographolide) isolated and purified from a herb (Andrographis paniculata) that is found in India to be effective in inhibiting tumour growth — both skin cancer and lung metastasis — in mice. The results were published on July 29, 2016 in the Journal of Investigative Dermatology.

Stem cell properties

“We used multiple markers, including CD133, to isolate and characterise the cancer stem cells in mice and human-specific skin cancer cell lines,” says Dr. Gopal C. Kundu, the senior author of the paper from NCCS. The CD133 positive cells exhibit stem cell properties in various cancers.

To establish that CD133 positive cancer stem cells cause cancer, the researchers injected the cells subcutaneously into mice. All the animals that were injected with CD133 positive cancer stem cells developed skin cancer (melanoma). “The CD133 positive cancer stem cells developed tumour faster than non-cancer stem cells,” he says. The CD133 positive cancer stem cells also produced lung metastasis in a different set of mice that received the cells through intravenous injection.

The drug can eliminate the drug resistant melanoma stem cells that are responsible for tumour relapse and mortality.To test the efficacy of Andrographolide drug candidate, which is being tested in several clinical trials, in inhibiting tumour growth, the researchers injected the compound into the tumour bearing mice. Two different drug dosages were used. The compound at 50 mg/kg body weight dosage was able to inhibit primary tumour (melanoma) growth by about two-third at the end of 24 days. In the case of a higher dosage of 150 mg/kg body weight, the compound was found to inhibit melanoma growth by more than two-third at the end of 24 days.

“Usually patients with malignant skin cancer die because of lung metastasis,” Dr. Kundu says. So the researchers tested the efficacy of the compound in inhibiting lung metastasis by injecting the drug directly into the mice which were previously injected with melanoma cancer stem cells intravenously. “At the end of 27 days, the drug cleared the lung metastasis by about 90 per cent,” he says.

“Substantial experimental evidences from the current study indicate that the compound [Andrographolide] has therapeutic potential for the management of cancer stem-cell mediated melanoma growth and lung metastasis,” Dr. Kundu says.

The ability of the compound to inhibit stem cell mediated-tumour growth and lung metastasis becomes significant as CD133 positive cells show enhanced ability to flush out cancer drugs from inside the cells thereby increasing the probability of tumour relapse. In short, Andrographolide can eliminate the drug resistant CD133 positive melanoma stem cells that are mainly responsible for tumour relapse and mortality.

Further studies are required to test the drug’s ability to inhibit skin cancer and lung metastasis.

Published in The Hindu on August 12, 2016