Researchers create heat stable vaccines using silica

Researchers at the University of Bath, working with colleagues at the University of Newcastle, have created a technique which can keep vaccines intact at high temperatures by encasing them in silica cages. When a protein in solution is mixed with silica, silicon dioxide binds closely around the protein to match its shape and encases the protein. A major advantage of this method is that it doesn’t require freeze-drying, something that around half of all vaccines won’t survive intact. A powder of ensilicated proteins and the silica cage enveloping the protein means it can be heated to 100°C or stored at 22°C for at least six months with no loss of function.

Once the protein has been encased in silica it can be stored or transported without refrigeration before the silica coat can be removed chemically, leaving the proteins unaffected.

The discovery means that vaccines and other important medicines could be transported much more easily, cheaply and safely, especially to remote areas or places lacking infrastructure where the need is often greatest.

The teams call their method ensilication and hope it will solve the costly and often impractical need for a cold chain to protect protein-based products including vaccines, antibodies and enzymes. The research is published in the journal Scientific Reports. The research team tested the method on three proteins; one from a tetanus vaccine, horse haemoglobin and an enzyme from egg white.

Citation: Chen, Yun-Chu, Tristan Smith, Robert H. Hicks, Aswin Doekhie, Francoise Koumanov, Stephen A. Wells, Karen J. Edler, Jean Van Den Elsen, Geoffrey D. Holman, Kevin J. Marchbank, and Asel Sartbaeva. “Thermal stability, storage and release of proteins with tailored fit in silica.” Scientific Reports 7 (2017): 46568.
Research funding: Royal Society, The Annett Trust
Adapted from press release by the University of Bath.

Current vaccination trials using Zika Purified Inactivated Virus (ZPIV) vaccine

The first of five early stage clinical trials to test the safety and ability of an investigational Zika vaccine candidate called the Zika Purified Inactivated Virus (ZPIV) vaccine to generate an immune system response has begun at the Walter Reed Army Institute of Research (WRAIR) Clinical Trial Center in Silver Spring, Maryland.

The experimental Zika Purified Inactivated Virus (ZPIV) vaccine is based on the same technology WRAIR used in 2009 to successfully develop a vaccine for another flavivirus called Japanese encephalitis. The Zika Purified Inactivated Virus (ZPIV) vaccine contains whole Zika virus particles that have been inactivated, meaning that the virus cannot replicate and cause disease in humans. However, the protein shell of the inactivated virus remains intact so it can be recognized by the immune system and evoke an immune response. The National Institute of Allergy and Infectious Diseases (NIAID) partially supported the preclinical development of the Zika Purified Inactivated Virus (ZPIV) vaccine candidate, including safety testing and non-human primate studies that found that the vaccine induced antibodies that neutralized the virus and protected the animals from disease when they were challenged with Zika virus. WRAIR, NIAID and the Biomedical Advanced Research and Development Authority (BARDA) part of the HHS Office of the Assistant Secretary for Preparedness and Response (ASPR) have established a joint Research Collaboration Agreement to support the development of this vaccine.

Led by WRAIR principal investigator Maj. Leyi Lin, M.D., the new study aims to enroll 75 people ages 18 to 49 years with no prior flavivirus infection. Flaviviruses include Zika virus, yellow fever virus, dengue virus, Japanese encephalitis virus and West Nile virus. Participants will be randomly divided into three groups: the first group (25 participants) will receive two intramuscular injections of the Zika Purified Inactivated Virus (ZPIV)  test vaccine or a placebo (saline) 28 days apart; the other two groups (25 participants each) will receive a two-dose regimen of a Japanese encephalitis virus vaccine or one dose of a yellow fever vaccine before beginning the two-dose Zika Purified Inactivated Virus (ZPIV) vaccine regimen. Investigators chose to administer additional flavivirus vaccines because U.S. service members are often vaccinated against these diseases before deploying to Zika-endemic areas.

Additionally, a subgroup of 30 of the participants who receive the two-dose Zika Purified Inactivated Virus (ZPIV) regimen will receive a third dose one year later. All participants in the trial will receive the same Zika Purified Inactivated Virus (ZPIV) vaccine dose at each injection (5 micrograms). A DoD Research Monitor, an independent physician not associated with the protocol, will monitor the conduct of the trial and report any safety issues to the WRAIR Institutional Review Board. Another independent group, the Safety Monitoring Committee, will also monitor participant safety, review data and report any issues to NIAID. As the regulatory sponsor, NIAID ensures the trial follows the study protocol and informs the FDA of any significant adverse events or risks. NIAID also maintains the Investigational New Drug (IND) application (link is external) for the candidate vaccine. The WRAIR study is expected to be completed by fall 2018.

Four additional Phase 1 studies to evaluate the Zika Purified Inactivated Virus (ZPIV) investigational vaccine are expected to launch in the coming months. These include

A trial enrolling 90 adults ages 18-49 years at the Center for Vaccine Development at the Saint Louis University School of Medicine. This site is an NIAID-funded Vaccine and Treatment Evaluation Unit, and Sarah George, M.D., will serve as principal investigator. All participants will receive either two injections of Zika Purified Inactivated Virus (ZPIV) or a placebo 28 days apart. Participants will be randomly assigned to receive either a high, moderate or low dose at both injections to evaluate the optimal dose for use in larger future studies.

A trial enrolling 90 adults ages 21-49 years at the clinical research center CAIMED, part of Ponce Health Sciences University in Puerto Rico. The site is supported by NIAID via a subcontract from the Saint Louis University School of Medicine. This trial will examine the vaccine’s safety and immunogenicity in participants who have already been naturally exposed to dengue virus. Participants will be randomly assigned to receive either a high dose, moderate dose or a placebo. Elizabeth A. Barranco, M.D., will lead the trial.

NIAID’s Vaccine Research Center (VRC) will test the Zika Purified Inactivated Virus (ZPIV) vaccine candidate as a boost vaccination to its DNA Zika vaccine candidate, which entered Phase 1 clinical trials in August. The next part of the study, which will enroll 60 additional participants ages 18-50 years, will take place at the NIH Clinical Center in Bethesda, Maryland, the Center for Vaccine Development at the University of Maryland School of Medicine’s Institute for Global Health in Baltimore, and Emory University in Atlanta. Half of the participants will receive the NIAID Zika virus investigational DNA vaccine followed by a Zika Purified Inactivated Virus (ZPIV) vaccine boost four or 12 weeks later. The remaining participants will receive only two doses of Zika Purified Inactivated Virus (ZPIV) vaccine four or 12 weeks apart. Julie Ledgerwood, D.O., chief of the VRC’s clinical trials program, will serve as principal investigator.

A WRAIR-funded trial enrolling 48 adults ages 18-50 years will be conducted at the Center for Virology and Vaccine Research, part of Beth Israel Deaconess Medical Center and Harvard Medical School in Boston. One group of participants will receive a single dose of the Zika Purified Inactivated Virus (ZPIV)  vaccine and all other participants will receive two doses of the Zika Purified Inactivated Virus (ZPIV) vaccine at varying intervals. Kathryn Stephenson, M.D., M.P.H., of Beth Israel Deaconess Medical Center, will lead the trial.

Scientists with Walter Reed Army Institute of Research, part of the U.S. Department of Defense (DoD), developed the vaccine. The National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH), is co-funding the Phase 1 clinical trial with Walter Reed Army Institute of Research, serving as the regulatory sponsor and providing other support. BARDA is funding the advanced development of the Zika Purified Inactivated Virus (ZPIV) vaccine candidate through a six-year contract with Sanofi Pasteur, which established a collaborative research and development agreement with WRAIR to accelerate further development of the vaccine.

Adapted from press release by NIH and Walter Reed Army Institute of Research

Research finds that better cognitive function is associated with higher physical activity and healthy eating habits (fruits and vegetables)

Findings published this week in the Journal of Public Health reveal that both younger and older Canadian adults who engage in regular physical activity, consume more fruits and vegetables and are normal weight or overweight have overall better cognitive functioning.

Regular engagement in physical activity and healthy eating has long been associated with a reduced risk for a range of chronic conditions. For older adults, there is a growing body of evidence that exercising may delay the onset of cognitive decline. Similarly, compounds found in fruits and vegetables have been shown to fight illnesses and help maintain healthy processes in the body. Given the increasing rates of inactivity and obesity in the world, researchers are interested in understanding the relationship between clusters of risk factors for cognitive decline, and how lifestyle factors might help prevent or delay it.

Previous studies in Spain and Korea have shown that older adults who eat more fruits and vegetables perform better in mentally stimulating activities than older adults who report eating a lower amount. However, very few studies have investigated the relationships between physical activity and eating fruit and vegetables and the effect it has on the brain for both younger and older adults.

This study examined cross-sectional data from 45,522, 30 years of age and older, participants from the 2012 annual component of the Canadian Community Health Survey. Cognitive function was assessed using a single 6-level question of the Health Utilities Index, which assessed mental processes, such as thinking, memory, and problem solving. Participants were analyzed by their age, level of physical activity, body mass index, and daily intake of fruit and vegetables. Using general linear models and mediation analyses, researchers assessed the relationship between these factors and participants’ overall cognitive function.

The results showed that higher levels of physical activity, eating more fruits and vegetables, and having a BMI in the normal weight (18.5-24.9 kg/m2) or overweight range (25.0-29.9 kg/m2) were each associated with better cognitive function in both younger and older adults. Further, by way of mediation analysis (via the Sobel test), it was determined that higher levels of physical activity may be in part responsible for the relationship between higher daily fruit and vegetable consumption and better cognitive performance.

Dr. Alina Cohen, PhD, explains: “Factors such as adhering to a healthy lifestyle including a diet that is rich in essential nutrients, regular exercise engagement, and having an adequate cardiovascular profile all seem to be effective ways by which to preserve cognitive function and delay cognitive decline.” Further that “It is pertinent that we develop a better understanding of the lifelong behaviors that may contribute to cognitive decline in late life by implementing a life-span approach whereby younger, middle-aged, and older adults are collectively studied, and where lifestyle risk factors are evaluated prior to a diagnosis of dementia.”

Citation: Alina Cohen, Chris I. Ardern, and Joseph Baker
Physical activity mediates the relationship between fruit and vegetable consumption and cognitive functioning: a cross-sectional analysis
Journal of Public Health first published online October 31, 2016
Adapted from press release by Oxford University Press

Polysorbate, a food additive found to be protective against E. coli poisoning

Polysorbate, a safe additive found in everything from ice cream to cosmetics, has been proven to slow the toxic effects of E. coli poisoning. The findings, featured in the current issue of the journal Biofouling, show that polysorbates attack the protective biofilm in which E. coli lives and renders the deadly bacteria harmless, said Chris Waters, Michigan State University associate professor of microbiology and molecular genetics whose laboratory led the research.

Specifically, the team focused on the potent strain isolated from Germany that swept through Europe in 2011, causing thousands of infections and more than 50 deaths. This strain had been previously studied by Waters and Shannon Manning. Having samples of the bacteria at MSU helped the team, led by Rudolph Sloup, MSU microbiology and molecular genetics graduate student, isolate compounds that inhibited biofilms.

“During our animal infection studies, polysorbate 80 had no effect on the numbers of infecting E. coli. This was a little shocking, especially based on how promising our earlier tests had been,” Waters said. “Later, though, our pathology tests showed that polysorbate 80 essentially blocked all toxicity, even though it didn’t reduce the number of bacteria.”

The later confirmation of the successful in vivo experiment using mice models essentially showed that polysorbate 80 strips E. coli of its ability to cause disease allowing the bacteria to pass through the body’s intestinal tract without causing damage. So instead of killing the E. coli like traditional antibiotics, a strategy that works until the E. coli develops resistance to the treatment, this finding suggests an anti-virulence strategy can be quite effective.

Since polysorbate 80 is categorized as a GRAS (generally regarded as safe) compound, it doesn’t require FDA approval to be used as a treatment. Along with its potential for disarming the deadly German E. coli outbreak, polysorbate 80 could potentially help tackle more-common E. coli infections such as traveler’s diarrhea.

The next steps for this research will be to identify how polysorbate 80 inhibits biofilm formation and test its activity in other infection models.

Citation: Sloup, Rudolph E., Roberto J. Cieza, David B. Needle, Robert B. Abramovitch, Alfredo G. Torres, and Christopher M. Waters. “Polysorbates prevent biofilm formation and pathogenesis of Escherichia coli O104: H4.” Biofouling 32, no. 9 (2016): 1131-1140.
Research funding: National Institutes of Health
Adapted from press release by Michigan State University

Scientists find why it is difficult to create vaccine for Hepatitis C virus

Researchers have been trying for decades to develop a vaccine against the globally endemic hepatitis C virus (HCV). Now scientists at The Scripps Research Institute (TSRI) have discovered one reason why success has so far been elusive. Using a sophisticated array of techniques for mapping tiny molecular structures, the TSRI scientists analyzed a lab-made version of a key viral protein, which has been employed in some candidate hepatitis C virus vaccines to induce the body’s antibody response to the virus. The researchers found that the part of this protein meant as the prime target of the vaccine is surprisingly flexible. Presenting a wide variety of shapes to the immune system, it thus likely elicits a wide variety of antibodies, most of which cannot block viral infection. The report, published online ahead of print by the Proceedings of the National Academy of Sciences the week of October 24, 2016.

Key protein on hepatitis C virus, E2, is exceptionally flexible,
 helping to explain why scientists have had difficulty targeting
it with a vaccine. Credit: Christina Corbaci and Leopold Kong

The Law and Wilson laboratories have been working together in recent years to study hepatitis C virus structure for clues to successful vaccine design. In 2013, for example, the team successfully mapped the atomic structure of the viral envelope protein E2, including the site where it binds to surface receptors on liver cells. Because this receptor-binding site on E2 is crucial to hepatitis C virus’s ability to infect its hosts, it has an amino-acid sequence that is relatively invariant from strain to strain. The receptor-binding site is also relatively accessible to antibodies, and indeed many of the antibodies that have been found to neutralize a broad set of hepatitis C virus strains do so by targeting this site.

For all these reasons, hepatitis C virus’s receptor-binding site has been considered an excellent target for a vaccine. But although candidate hepatitis C virus vaccines mimicking the E2 protein have elicited high levels of antibodies against the receptor-binding site, these antibody responses–in both animal models and human clinical trials–have not been very effective at preventing hepatitis C virus infection of liver cells in laboratory assays.

To understand why, the Law and Wilson laboratories teamed up with TSRI Associate Professor Andrew Ward and used electron microscopy and several other advanced structural analysis tools to take a closer look at hepatitis C virus’s E2 protein, in particular the dynamics of its receptor binding site. Their investigations focused on the “recombinant” form of the E2 protein, produced in the lab and therefore isolated from the rest of the virus. Recombinant E2 is a prime candidate for hepatitis C virus vaccine design and is much easier to purify and study than E2 from whole virus particles.

One finding was that recombinant E2, probably due to its many strong disulfide bonds, has great structural stability, with an unusually high melting point of 85°C. However, the TSRI scientists also found evidence that, within this highly buttressed construction, the receptor binding site portion is extraordinarily loose and flexible in the recombinant protein. “It adopts a very wide range of conformations,” said study first author Leopold Kong, of TSRI at the time of the study, now at the National Institutes of Health.

Prior studies have shown that hepatitis C virus’s receptor binding site adopts a narrow range of conformations (shapes) when bound by virus-neutralizing antibodies. A vaccine that elicited high levels of antibodies against only these key conformations would in principle provide effective protection. But this study suggests that the E2 protein used in candidate vaccines displays far too many other binding-site conformations–and thus elicits antibodies that mostly do nothing to stop the actual virus.

Law and Wilson and their colleagues plan to follow up by studying E2 and its receptor binding site as they are presented on the surface of the actual virus. They also plan to design a new version of E2 or even an entirely different scaffold protein, on which the receptor binding site is stabilized in conformations that will elicit virus-neutralizing antibodies.

Citation: Structural flexibility at a major conserved antibody target on hepatitis C virus E2 antigen.
Authors: Leopold Kong, et., al.
Journal: Proceedings of National Academy of Sciences
Research funding: National Institutes of Health, Skaggs Institute for Chemical Biology
Adapted from press release by The Scripps Research Institute

Study reveals potential new treatment target Nuak1 kinase to prevent accumulation of protein in early Alzheimer’s disease

Taking a pill that prevents the accumulation of toxic molecules in the brain might someday help prevent or delay Alzheimer’s disease, according to scientists at Baylor College of Medicine, Texas Children’s Hospital and Johns Hopkins University School of Medicine.

The study, published today in Cell Press journal Neuron, took a three-pronged approach to help subdue early events that occur in the brain long before symptoms of Alzheimer’s disease are evident. The scientists were able to prevent those early events and the subsequent development of brain pathology in experimental animal models in the lab.

Brain section from mouse carrying the dementia-causing P301S mutation
in human tau shows accumulation of tau neurofibrillary tangles (in dark brown,
left). When Nuak1 levels are decreased by 50 percent (P301S/Nuak1+/-; right),
fewer tau tangles accumulate. Credit: The Zoghbi lab/Baylor College of Medicine

“Scientists in the field have been focusing mostly on the final stages of Alzheimer’s disease,” said first author Dr. Cristian Lasagna-Reeves, postdoctoral fellow in the Zoghbi lab. “Here we tried to find clues about what is happening at the very early stages of the illness, before clinical irreversible symptoms appear, with the intention of preventing or reducing those early events that lead to devastating changes in the brain decades later.”

The scientists reasoned that if they could find ways to prevent or reduce tau accumulation in the brain, they would uncover new possibilities for developing drug treatments for these diseases.
Cells control the amount of their proteins with other proteins called enzymes. To find which enzymes affect tau accumulation, the scientists systematically inhibited enzymes called kinases.  “We inhibited about 600 kinases one by one and found one, called Nuak1, whose inhibition resulted in reduced levels of tau,” said Zoghbi, who is also an investigator at the Howard Hughes Medical Institute.

The scientists screened the enzymes in two different systems, cultured human cells and the laboratory fruit fly. Screening in the fruit fly allowed the scientists to assess the effects of inhibiting the enzymes in a functional nervous system in a living organism.

“We found one enzyme, Nuak1, whose inhibition consistently resulted in lower levels of tau in both human cells and fruit flies,” said Zoghbi. “Then we took this result to a mouse model of Alzheimer’s disease and hoped that the results would hold, and they did. Inhibiting Nuak1 improved the behavior of the mice and prevented brain degeneration.”  Brain section from mouse carrying the dementia-causing P301S mutation in human tau shows accumulation of tau neurofibrillary tangles (in dark brown, left). When Nuak1 levels are decreased by 50% (P301S/Nuak1+/-; right), fewer tau tangles accumulate.

 “Confirming in three independent systems – human cells, the fruit fly and the mouse – that Nuak1 inhibition results in reduced levels of tau and prevents brain abnormalities induced by tau accumulation, has convinced us that Nuak1 is a reliable potential target for drugs to prevent diseases such as Alzheimer’s,” said Zoghbi. “The next step is to develop drugs that will inhibit Nuak1 in hope that one day would be able to lower tau levels with low toxicity in individuals at risk for dementia due to tau accumulation.”

Publication: Reduction of Nuak1 Decreases Tau and Reverses Phenotypes in a Tauopathy Mouse Model.Authors: Cristian A. Lasagna-Reeves et. al.,
Journal: Neuron
Research funding: This work was supported by the Howard Hughes Medical Institute, the Robert A. and Renee E. Belfer Family Foundation, the Hamill Foundation, the Chapman Foundation, and the National Institutes of Health. Support also was provided by the Texas Alzheimer’s Research and Care Consortium-Investigator Grant Program, the Darrel K. Royal Foundation grant, the Canadian Institutes of Health Research Fellowship, the Mass Spectrometry-Proteomics Core Laboratory (MS-PCL) and the confocal microscopy, neuroconnectivity and mouse behavioral cores of the Baylor College of Medicine Intellectual and Developmental Disabilities Research Center , the Johns Hopkins University Morris Udall Parkinson’s Disease Center of Excellence and Alzheimer Disease Research Center.
Adapted from press release by Baylor College of Medicine.