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

Science or Snake Oil: can turmeric really shrink tumours, reduce pain and kill bacteria?

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Turmeric is touted to have many benefits, such as reducing inflammation and preventing cancer. – Photo: http://www.shutterstock.com.au

Gunveen Kaur, Deakin University

Turmeric is a yellow coloured spice widely used in Indian and South East Asian cuisine. It’s prepared from the root of a plant called Curcuma longa and is also used as a natural pigment in the food industry. The Conversation

In the literature, curcumin is reported to be an antioxidant that protects the body against damage from reactive molecules. These are generated in the body as a result of metabolism and cause cell damage (known as free radicals).

It’s also reported to have anti-inflammatory, anti-bacterial and anti-cancer properties, as well as encouraging the death of cells that are dangerous or no longer needed by the body.

Curcumin has been widely studied in relation to numerous ailments, but what does the literature say? Is consuming turmeric beneficial?

For aches and pains

Chronic inflammation has been linked to the development of numerous diseases such as obesity, diabetes, heart disease and cancer. There is some evidence curcumin reduces the levels of certain substances (cytokines) that produce inflammation.

Systematic reviews and meta-analyses, which combine data from several randomised controlled trials (where an intervention is tested against a placebo, while the subjects and those conducting the study don’t know who has received which treatment) support this finding to a certain extent.

A meta-analysis of nine randomised controlled trials showed taking curcumin supplements led to a significant reduction in cytokines that produce inflammation. But the authors claimed these reductions were modest, and it’s unclear if they would actually have a benefit in real life.

These trials were conducted with small sample sizes ranging from 10 to 50 people, which reduces the strength of the evidence. It’s difficult to draw a conclusion on a beneficial dose and how long you should take curcumin, or the population group that can benefit the most from curcumin.

A meta-analysis investigated the effects of turmeric/curcumin on pain levels in joint arthritis patients. The group supplemented with 1000mg of curcumin per day said they had reduced pain compared with the placebo group.

In this study, curcumin was found to be as effective as ibuprofen in terms of reducing pain levels in these patients. But the authors of this meta-analysis themselves suggested that due to small sample size and other methodological issues there is not sufficient evidence to draw definitive conclusions.

For diabetes and heart disease

Curcumin is also thought to be beneficial in preventing insulin resistance (which leads to increased blood sugar), improving high blood sugar and reducing the toxic effects of high blood glucose levels.

But these studies have been conducted in animals and are very few human trials have been conducted in this area.

One study that reported reduction in blood glucose levels in type 2 diabetes patients reports a change in blood glucose from 8.58 to 7.28 millimoles per litre after curcumin supplementation. People with levels above seven are classified as diabetics. So in clinical terms, the change is not that much.

Similarly in relation to heart disease, animal studies show benefits of curcumin supplementation in improving heart health, but there are very few clinical trials conducted in heart disease patients.

Smaller clinical trials looking at ten patients also show benefits of curcumin in reducing serum cholesterol, which is a risk factor for heart disease. But meta-analysis looking at combined effects of different trials does not show these benefits.

For cancer

Curcumin has also been widely studied in relation to its anti-cancer properties. Laboratory and animal studies support this claim, but the evidence for cancer prevention in human trials is lacking.

Although there are some small studies (in 25 cancer patients) that showed reductions in precancerous lesions, and two patients showed shrunken tumors, this small number is not enough to conclude anti-cancerous effects of curcumin.

There is some evidence curcumin lessens the severity of side-effects from radiation therapy such as radiation-induced dermatitis and pneumonitis (inflammation of lungs), but not the cancer itself.

Safety

Research shows not all curcumin we take orally is absorbed. This has led to the use of other things such as lipids (fats) and piperine (found in black pepper), to help it absorb into our system.

High intakes (up to 12 grams a day) of curcumin can cause diarrhoea, skin rash, headaches and yellow-coloured faeces. Looking at the Indian population, they consume about 100mg of curcumin a day, which corresponds to 2 to 2.5 grams of turmeric per day.

But they also consume these amounts over relatively long periods of time (typically their lifespan). There are reports of lower cancer rates in the Indian population and this has been linked to turmeric consumption, but there are no longer term trials proving this link.

It appears that in order to receive benefits from high doses over a short period of time, people are now resorting to injecting turmeric intravenously. There is no evidence to support the benefits of high doses of turmeric or IV injections of turmeric at all.

In fact, at very high doses, curcumin’s predominant activity switches from antioxidant to pro-oxidant, which means rather than preventing cells from damage, it promotes cell damage and has also been reported to cause tumours in rodents.

Although curcumin is showing some encouraging effects in reducing markers of inflammation in humans, the majority of the pharmacological effects of curcumin are in lab studies or animal experiments. Until there are more high quality randomised controlled trials conducted to confirm the benefits of curcumin or turmeric, it’s best to consume turmeric orally as a spice as part of a healthy, nutritious diet.

Gunveen Kaur, Lecturer In Nutritional Sciences, Deakin University

(This article was originally published on The Conversation. Read the original article.)

IISc team unravels how vitamin C helps kill bacteria

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Prof. Dipankar Chatterji and Kirtimaan Syal found vitamin C was inhibiting (p)ppGpp synthesis.

That vitamin C, an anti-oxidant agent, boosts and strengthens immunity is well known. Its ability to speed-up recovery from tuberculosis and impede the TB causing bacteria from causing disease, and even kill the bacteria in culture at high concentration are also known. Now, a study by a team of researcher at the Indian Institute of Science (IISc), Bengaluru has found the molecular mechanism by which vitamin C impedes and even kills Mycobacterium smegmatis, non-pathogenic bacteria that belongs to the same genus as the TB causing mycobacteria. The results were published in the journal FEMS Microbiology Letters.

During times of stress or hostile conditions, such as increased temperature and presence of antibiotics, bacteria tend to come together and form a biofilm to protect themselves. The stress response pathway is crucial for bacteria to survive during hostile conditions. So blocking this pathway is a sure way of killing the bacteria.

In mycobacterium, the (p)ppGpp (Guanosine pentaphospahte or Guanosine tetraphosphate) is a key molecule in the stress response pathway. The (p)ppGpp is synthesised by Rel protein, which in turn is made by the Rel gene.

The team led by Prof. Dipankar Chatterji from the Molecular Biophysics Unit at IISc looked at the effects of vitamin C on the stress response pathway. “We chose vitamin C because its structure is similar to (p)ppGpp,” says Prof. Chatterji. “So we hypothesised that vitamin C should be competing to bind to the Rel enzyme and inhibiting (p)ppGpp synthesis.”

To test their hypothesis, the researchers conducted experiments using M. smegmatis; M. smegmatis is used as a model organism for TB causing Mycobacterium tuberculosis.

In vitro studies showed “significant” inhibition of (p)ppGpp synthesis in the presence of vitamin C. The inhibition level was seen to be increasing as the vitamin C concentration increased. More the vitamin C concentration the greater the possibility of vitamin C binding to the Rel enzyme, thus inhibiting (p)ppGpp synthesis. At about 10 mM concentration, the synthesis of (p)ppGpp was completely inhibited.

The binding of vitamin C to the Rel enzyme is weak and this explains why high concentration of vitamin C is needed to inhibit (p)ppGpp synthesis.

“Using Mycobacterial cells we found that 1 mM of vitamin C produced 50% inhibition in (p)ppGpp synthesis. Vitamin C is able to get inside cells and inhibit (p)ppGpp synthesis,” says Kirtimaan Syal from IISc and the first author of the paper.

When 2 mM of vitamin C was added, “significant” defect in biofilm formation was seen. There was more than 50% reduction in viability of cells in a matter of four days when M. smegmatis was treated with 2mM of vitamin C. The viability of cells reduced even further with time, raising the possibility of therapeutic implications.

“This suggests that vitamin C can act as a precursor for more potential inhibitors; it can be chemically modified into more potential derivatives,” they write. “Vitamin C is natural, and it can form one of the nutrient-based treatments of the disease. Vitamin C is water soluble and has no toxic effect,” says Dr. Syal.

“We are trying to synthesise derivatives of vitamin C to enhance inhibition of (p)ppGpp synthesis even at lower concentration,” Dr. Syal says.

Published in The Hindu on April 22, 2017

Why do we see H1N1 cases every year?

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The Michigan species circulating in India has killed 160 people.

The spread of influenza virus declines when the temperature shoots up. But this year, despite the summer temperature crossing 40 degrees Celsius in some parts of the country, the number of H1N1 cases and occasional deaths have not stopped. Since its first appearance in 2009-2010, influenza A H1N1 has come to stay and has become a part of the seasonal flu virus; H3N2 and Influenza B are the other two to become the seasonal flu in India.

According to the World Health Organisation (WHO), since December 2016, H1N1, H3N2 and Influenza B have been circulating in India. During September 2016-February 2017, H3N2 has been predominant in most countries, with only “low levels” of the H1N1 viruses circulating in the northern hemisphere, says the WHO. In a written reply in the Lok Sabha, Minister of State for Health Anupriya Patel said H1N1 had claimed 160 lives in the country between January 1 and March 26 and 6,062 cases had been registered. The highest number of deaths, 63, was reported from Maharashtra, she said. Last year, between January and December 31, 265 people had died and 1,786 cases were reported.

Have any steps been taken?

With over 32,000 people infected and nearly 2,000 killed in 2015, H1N1 highlighted how ill-prepared the country was in preventing the spread of an infectious disease and managing it. Influenza infection causes significant morbidity and mortality, though the number of people infected with H1N1 and the number of people who die are fewer than dengue.

What India needs is a national policy for influenza immunisation. While pregnant mothers, children aged below five and young people with asthma, cardiovascular disease, diabetes and high blood pressure are at a greater risk, there is no hard qualitative and quantitative data on the vulnerable population. In the absence of information on who is most susceptible to H1N1 infection and very likely to die, framing a national policy will be harder and take a long time.

But until a national policy for influenza immunisation is in place, individuals, particularly those who are highly vulnerable, should get vaccinated and practise safe health measures such as correct cough etiquette (not coughing into our fingers but at elbow), staying at home if infected, and not sharing towels with others.

Healthcare workers who handle high-risk patients should particularly get vaccinated. While it may be prudent to get all health workers vaccinated, a study by researchers from Christian Medical College, Vellore, showed that healthcare workers are more likely to get infected in the community than in healthcare settings.

Different circulating strain

The Pune-based National Institute of Virology has sequenced the whole genome of H1N1 and has not found any critical mutation responsible for the spread or increased mortality. While the California strain had been circulating across the world since the 2009 pandemic, during 2016 California strain and Michigan strain were circulating in India. However, this year, the H1N1 surveillance revealed that the Michigan strain was circulating, with no sign of the California strain.

Vaccination to prevent infection

On March 2, the WHO flu vaccine advisory group recommended the composition of influenza virus vaccines for use in the 2017-2018 northern hemisphere influenza season. It announced that the Michigan strain replaced the California strain in the northern hemisphere.

Based on the WHO recommendation, the Pune-based Serum Institute of India has started making influenza vaccine using the Michigan strain, but the vaccine is yet to reach the market. According to an official of Serum Institute, the vaccine containing the California strain will be protective, to a large extent, against the H1N1 strain now circulating in India.

Published in The Hindu on April 22, 2017

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

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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

IISc’s potent molecules show promise for TB therapy

SEM photo of Mycobacterium tuberculosis bacteria. - Photo NIAD-Optimized

The two molecules were able to prevent biofilm formation and even disrupt biofilms that had formed.

Scientists at the Indian Institute of Science (IISc) Bengaluru have developed two new, potent molecules that can severely impact the survival of mycobacteria, including Mycobacterium tuberculosis that causes TB. The results were published in the journal Antimicrobial Agents and Chemotherapy.

Unlike most antibiotics that target the bacterial metabolism by aiming at the cellular components, the novel molecules inhibit the stress response pathway of mycobacteria. The stress response pathway is crucial for bacteria to survive during hostile conditions such as lack of nutrients and the presence of antibiotics, to name a few. So any inhibition of this pathway will lead to its death.

The master regulator of stress pathway in the case of mycobacteria is (p)ppGpp (Guanosine pentaphospahte or Guanosine tetraphosphate). Though a molecule that inhibits the (p)ppGpp formation has already been synthesised, the efficacy is not much. “Very high concentration of Relacin molecule is needed to inhibit the pathway and, therefore, the efficacy is low. So we synthesised two new molecules — acetylated compound (AC compound) and acetylated benzoylated compound (AB compound) — by bringing about a modification in the base of the Relacin molecule,” says Prof. Dipankar Chatterji from the Division of Biological Sciences at IISc and the corresponding author of the paper.

“We found both the molecules to be very good inhibitors of stress response. The two compounds affected the rate of synthesis of (p)ppGpp and also reduced the cell survival,” he says. Laboratory studies showed that the two molecules were not toxic to human cells and were able to penetrate the human lung epithelial cells.

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Inhibiting (p)ppGpp synthesis would target the survival of the bacteria, says Dr. Kirtimaan Syal.

“We found our compounds were targeting the Rel gene. The Rel gene makes Rel protein, which in turn synthesises (p)ppGpp. When the Rel gene is knocked out, the long-term survival of Mycobacterium smegmatis decreases,” says Prof. Chatterji.

“The Alarmone molecule “(p)ppgpp”, a modified nucleotide, is ubiquitous in bacteria and absent in humans. Inhibiting (p)ppgpp synthesis would specifically target the survival of bacteria without having any effects on humans,” says Dr. Kirtimaan Syal from the Division of Biological Sciences, IISc and the first author of the paper.

Earlier studies have shown that when the rel gene is deleted, the long-term survival ability under stress was lost; the M. tuberculosis bacteria was unable to persist in mice and unable to form tubercle lesions in guinea pigs.

“The major reason for prolonged treatment of TB is the bacterium’s ability to persist in dormant form, which is tolerant to most antibiotics used in the treatment regimen. So inhibition of (p)ppGpp-mediated persistence could help in shortening the treatment regime, dealing with the emergence of multiple drug resistance and treatment of chronic infections, Dr. Syal says.

Inhibiting biofilm

Under hostile conditions, bacteria tend to form biofilms, which protect the bacteria from stress and induce tolerance to antibiotics. Recent studies have shown that tuberculosis bacteria that cannot form a biofilm cannot survive inside the host. Evidences have shown that at the time of infection, the M. tuberculosis display a biofilm-like phenotype and this helps the bacteria to survive inside the host.

Studies carried out by the researchers showed that both the molecules were able to inhibit biofilm formation by M. tuberculosis and M. smegmatis and also disrupt the already formed biofilm. “The biofilm formed by TB bacteria is very dangerous. The ability of the molecules to destroy the biofilm and even prevent its formation is a very important achievement,” says Prof. Chatterji.

Since there are very few antibiotics that target the stress response pathway of the bacteria, the two molecules offer great promise. “The next step is to test the molecules on animals. We have not thought about it. It will also be interesting to see if the bacteria develop resistance against these molecules,” Prof. Chatterji says.

Published in The Hindu on April 15, 2017

IGIB team enhances the efficiency of DNA delivery into the skin for treating skin disorders

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Dr. Munia Ganguli (left) and Dr. Manik Vij have improved DNA penetration into skin by pretreating the skin with silicone oil. – Photo: Lavanya Lokhande.

By pretreating the skin with silicone oil, a team of researchers led by Dr. Munia Ganguli from the Delhi-based Institute of Genomics and Integrative Biology (CSIR-IGIB) has been successful in delivering plasmid DNA into the skin with greater efficiency and without destroying the integrity of the skin. Unlike other enhancers currently being used, preliminary studies show that silicone oil did not get into the skin nor cause any harm. Enhancing the ability of the plasmid DNA, packaged as a nanometer-sized complex with a peptide, to penetrate the skin will go a long way in efficiently delivering drugs for skin disorders. The results were published in the journal Molecular Therapy.

“Topical application of silicone oil on the skin prior to applying the DNA-peptide (which acts as a carrier of DNA) complex allows the DNA to reach the lower part of the epidermal layer of the skin; a little bit of DNA gets into the dermis as well,” says Dr. Ganguli, the corresponding author of the paper.

The skin with its three layers — stratum corneum (top layer), the epidermis (middle layer) and dermis (inner layer) — acts as a tough barrier for the entry of any foreign substance. Since the top layer of the skin is rich in lipids it becomes particularly difficult for the DNA (which is water-loving or hydrophilic) to penetrate it.

Only 30% of cells have the DNA complex when the skin is not pretreated with silicone oil. It increases to 45% once the skin is pretreated. “Silicone oil forms an occlusive layer which prevents water loss from the skin and keeps it well hydrated. The rise in hydration pressure, in turn, opens up many porous pathways for entry of the DNA complexes into the skin,” says Dr. Manika Vij from CSIR-IGIB and the first author of the paper. Besides increased hydration, there are also minor changes in the lipid and protein organisation in the skin.

The use of another enhancer (sodium laureth sulfate-phenyl piperazine — SLA-PP) in place of silicone oil also improves DNA penetration but it was found to damage the skin and was highly toxic to the skin cells; when applied on cell lines, plenty of cells died after 24 hours.

The researchers used hairless mice (the absence of hair follicles makes the skin more closely comparable to human skin) to test the penetration of DNA into the skin. Since the DNA is labelled with fluorescein, it was possible to measure the amount of nanocomplexes that got into the skin by measuring the fluorescence. Other tests revealed that topical application of silicone oil does not damage the integrity of the skin or damage the tissues.

Other potential applications

Talking about potential use of the DNA nanocomplexes along with silicone oil, Dr. Vij says: “In the DNA we can put any gene that encodes for any specific therapeutic protein. This way we can address several skin diseases.”

The researchers are planning to test the ability of the peptide-DNA complexes to cross the skin and enter the blood. “If it does, then it increases the potential to address diseases of other organs,” Dr. Vij says. “We are yet to carry out studies to see if the DNA gets into the blood circulation or gets locally degraded in the skin cells.”

Published in The Hindu on April 14, 2017