Llama nanobodies: A breakthrough in building HIV immunity

A research team at Georgia State University has developed tiny, potent molecules that are capable of targeting hidden strains of HIV. The source? Antibody genes from llama DNA.

The research, led by Assistant Professor of Biology Jianliang Xu, uses llama-derived nanobodies to broadly neutralize numerous strains of HIV-1, the most common form of the virus. A new study from this team has been published in the journal Advanced Science.

"This virus has evolved a way to escape our immune system. Conventional antibodies are bulky, so it's difficult for them to find and attack the virus' surface," Xu explained. "These new antibodies can do this in an easier way."

Scientists in pursuit of effective HIV treatment and prevention have been working with animals in the camelid family -- like llamas -- for about 15 years. That's because the shape and features of their antibodies make them nimbler and more effective at identifying and neutralizing foreign objects, like the HIV virus.

This new research presents a widely applicable method to enhance the performance of nanobodies. Nanobodies are engineered antibody fragments that are about one-tenth the size of a conventional antibody. They are derived from flexible, Y-shaped heavy chain-only antibodies -- made up of two heavy chains -- which are more effective at fighting certain viruses than conventional antibodies with light chains.

The nanobodies are derived from flexible, Y-shaped antibodies made up of heavy-chain peptides which may be more effective at fighting certain viruses.

For the study, researchers immunized llamas with a specially designed protein which results in the production of neutralizing nanobodies. Xu and his team then identified nanobodies that can target vulnerable sites on the virus. When the team engineered the nanobodies into a triple tandem format -- by repeating short lengths of DNA -- the resulting nanobodies demonstrated remarkable effectiveness, neutralizing 96 percent of a diverse panel of HIV-1 strains.

Further analysis uncovered that these nanobodies mimic the recognition of the CD4 receptor -- a key player in HIV infection. To enhance their potency, the nanobodies were fused with a broadly neutralizing antibody (bNAb), resulting in a new antibody with unprecedented neutralizing abilities.

"Instead of developing a cocktail of antibodies, now we can make a single molecule that can neutralize HIV," Xu said. "We are working with a broadly neutralizing nanobody that can neutralize over 90 percent of the circulating HIV strains, and when we combine that with another bNAb which also neutralizes some 90 percent, together, they can neutralize close to 100 percent."

Xu began this research at the National Institutes of Health Vaccine Research Center in Bethesda, Md., where he collaborated with a team of more than 30 scientists. The team included Peter Kwong, professor of biochemistry and molecular biophysics at Columbia University and co-author of the study. Since coming to Georgia State in 2023, Xu has been mentoring Payton Chan, a Ph.D. candidate at Georgia State. Together, they are working to expand these potential remedies.

Chan said she is excited about the prospects of the innovative research.

"These nanobodies are the best and most potently neutralizing antibodies to date, which I think is very promising for the future of HIV therapeutics and antibody research," Chan said. "I hope one day there will be approval of these nanobodies for the treatment of HIV."

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Researchers create total synthesis of HIV replication inhibitor

Having control over how a dish is cooked is always a good idea. Taking a hint from the kitchen, scientists appear to have discovered a way to produce a true structure of the rare but naturally-occurring anti-HIV compound Lancilactone C from start to finish.

Its non-cytotoxicity in mammals could make this triterpenoid an ideal candidate for treating AIDS if its biological activity were clear -- and if only it were abundant in nature.

Now, a research group at Kyoto University has succeeded in creating a domino-like synthesis of Lancilactone C's unique seven-membered ring structure.

"Our synthetic method revealed that the proposed structure of Lancilactone C was initially incorrect," says Chihiro Tsukano of Kyoto University's Graduate School of Agriculture. "But we successfully derived its true structure from our spectral data and understanding of its biosynthesis."

In addition to this revelation, Tsukano realized that the electrocyclization -- a rearrangement reaction in organic chemistry -- used in the total synthesis also occurs in biosynthesis. Ironically, it remains a mystery of whether the proposed structure containing an unsaturated seven-membered ring might exist in nature as an analog -- or equivalent compound -- and how it might affect the expression of biological activity.

Tsukano's team utilized the domino-like reaction to enable the total synthesis of lancilactones and related triterpenoids. This outcome has inspired the team to further their research in optimizing compound structures, leading to possible development of novel antivirals.

The endless loop of required medication and multi-drug therapies often correlates with a lower quality of life for economically burdened patients.

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Research offers clues for potential widespread HIV cure in people

New research from Oregon Health & Science University is helping explain why at least five people have become HIV-free after receiving a stem cell transplant. The study's insights may bring scientists closer to developing what they hope will become a widespread cure for the virus that causes AIDS, which has infected about 38 million people worldwide.

Published today in the journal Immunity, the OHSU-led study describes how two nonhuman primates were cured of the monkey form of HIV after receiving a stem cell transplant. It also reveals that two circumstances must co-exist for a cure to occur and documents the order in which HIV is cleared from the body -- details that can inform efforts to make this cure applicable to more people.

"Five patients have already demonstrated that HIV can be cured," said the study's lead researcher, Jonah Sacha, Ph.D., a professor at OHSU's Oregon National Primate Research Center and Vaccine and Gene Therapy Institute.

"This study is helping us home in on the mechanisms involved in making that cure happen," Sacha continued. "We hope our discoveries will help to make this cure work for anyone, and ideally through a single injection instead of a stem cell transplant."

The first known case of HIV being cured through a stem cell transplant was reported in 2009. A man who was living with HIV was also diagnosed with acute myeloid leukemia, a type of cancer, and underwent a stem cell transplant in Berlin, Germany. Stem cell transplants, which are also called bone marrow transplants, are used to treat some forms of cancer. Known as the Berlin patient, he received donated stem cells from someone with a mutated CCR5 gene, which normally codes for a receptor on the surface of white blood cells that HIV uses to infect new cells. A CCR5 mutation makes it difficult for the virus to infect cells, and can make people resistant to HIV. Since the Berlin patient, four more people have been similarly cured.

This study was conducted with a species of nonhuman primate known as Mauritian cynomolgus macaques, which the research team previously demonstrated can successfully receive stem cell transplants. While all of the study's eight subjects had HIV, four of them underwent a transplant with stem cells from HIV-negative donors, and the other half served as the study's controls and went without transplants.

Of the four that received transplants, two were cured of HIV after successfully being treated for graft-versus-host disease, which is commonly associated with stem cell transplants.

Other researchers have tried to cure nonhuman primates of HIV using similar methods, but this study marks the first time that HIV-cured research animals have survived long term. Both remain alive and HIV-free today, about four years after transplantation. Sacha attributes their survival to exceptional care from Oregon National Primate Research Center veterinarians and the support of two study coauthors, OHSU clinicians who care for people who undergo stem cell transplants: Richard T. Maziarz, M.D., and Gabrielle Meyers, M.D.

"These results highlight the power of linking human clinical studies with pre-clinical macaque experiments to answer questions that would be almost impossible to do otherwise, as well as demonstrate a path forward to curing human disease," said Maziarz, a professor of medicine in the OHSU School of Medicine and medical director of the adult blood and marrow stem cell transplant and cellular therapy programs in the OHSU Knight Cancer Institute.

The how behind the cure

Although Sacha said it was gratifying to confirm stem cell transplantation cured the nonhuman primates, he and his fellow scientists also wanted to understand how it worked. While evaluating samples from the subjects, the scientists determined there were two different, but equally important, ways they beat HIV.

First, the transplanted donor stem cells helped kill the recipients' HIV-infected cells by recognizing them as foreign invaders and attacking them, similar to the process of graft-versus-leukemia that can cure people of cancer.

Second, in the two subjects that were not cured, the virus managed to jump into the transplanted donor cells. A subsequent experiment verified that HIV was able to infect the donor cells while they were attacking HIV. This led the researchers to determine that stopping HIV from using the CCR5 receptor to infect donor cells is also needed for a cure to occur.

The researchers also discovered that HIV was cleared from the subjects' bodies in a series of steps. First, the scientists saw that HIV was no longer detectable in blood circulating in their arms and legs. Next, they couldn't find HIV in lymph nodes, or lumps of immune tissue that contain white blood cells and fight infection. Lymph nodes in the limbs were the first to be HIV-free, followed by lymph nodes in the abdomen.

The step-wise fashion by which the scientists observed HIV being cleared could help physicians as they evaluate the effectiveness of potential HIV cures. For example, clinicians could focus on analyzing blood collected from both peripheral veins and lymph nodes. This knowledge may also help explain why some patients who have received transplants initially have appeared to be cured, but HIV was later detected. Sacha hypothesizes that those patients may have had a small reservoir of HIV in their abdominal lymph nodes that enabled the virus to persist and spread again throughout the body.

Sacha and colleagues continue to study the two nonhuman primates cured of HIV. Next, they plan to dig deeper into their immune responses, including identifying all of the specific immune cells involved and which specific cells or molecules were targeted by the immune system.

This research is supported by the National Institutes of Health (grants AI112433, AI129703, P51 OD011092) and the Foundation for AIDS Research (grant 108832), and the Foundation for AIDS Immune Research. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

In our interest of ensuring the integrity of our research and as part of our commitment to public transparency, OHSU actively regulates, tracks and manages relationships that our researchers may hold with entities outside of OHSU. In regard to this research, Dr. Sacha has a significant financial interest in CytoDyn, a company that may have a commercial interest in the results of this research and technology. Review?details of OHSU's conflict of interest program?to find out more about how we manage these business relationships.

All research involving animal subjects at OHSU must be reviewed and approved by the university's?Institutional Animal Care and Use Committee (IACUC). The IACUC's priority is to ensure the health and safety of animal research subjects. The IACUC also reviews procedures to ensure the health and safety of the people who work with the animals. No live animal work may be conducted at OHSU without IACUC approval.

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Successful cure of HIV infection after stem cell transplantation, study suggests

Haematopoietic stem cell transplantation for the treatment of severe blood cancers is the only medical intervention that has cured two people living with HIV in the past. An international group of physicians and researchers from Germany, the Netherlands, France, Spain, and the United States has now identified another case in which HIV infection has been shown to be cured in the same way. In a study published this week in Nature Medicine, in which DZIF scientists from Hamburg and Cologne played a leading role, the successful healing process of this third patient was for the first time characterised in great detail virologically and immunologically over a time span of ten years.

An infection with the human immunodeficiency virus (HIV) was previously considered incurable. The reason for this is that the virus "sleeps" in the genome of infected cells for long periods of time, making it invisible and inaccessible to both the immune system and antiviral drugs. The "Düsseldorf patient," a 53-year-old man, is now the third person in the world to be completely cured of the HI virus by a stem cell transplant.

The patient, treated at the University Hospital Düsseldorf for his HIV infection, had received a stem cell transplant due to a blood cancer. As in the cases of the first two patients named "Berlin" and "London," the Düsseldorf patient received stem cells from a healthy donor whose genome contains a mutation in the gene for the HIV-1 co-receptor CCR5. This mutation makes it impossible for most HI viruses to enter human CD4+ T-lymphocytes, their major target cells.

Following transplantation, the patient was carefully monitored virologically and immunologically for almost ten years. Using a variety of sensitive techniques, the researchers analysed the patient's blood and tissue samples to closely monitor immune responses to HIV and the continued presence or even replication of the virus. Already shortly after transplantation and over the entire course of the study years, neither replicating virus nor antibodies or reactive immune cells against HIV were detected. More than four years ago, the antiviral therapy against HIV was discontinued. Ten years after transplantation and four years after the end of anti-HIV therapy, the Düsseldorf patient could be declared cured by the international research consortium.

"This case of curing a chronic HIV infection by stem cell transplantation shows that HIV can in principle be cured," says Prof. Julian Schulze zur Wiesch, DZIF scientist at the University Medical Center Hamburg-Eppendorf and one of the study leads. "In particular, the results of this study are also enormously important for further research into a cure for HIV for the vast majority of people living with HIV for whom stem cell transplantation is not an option."

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Scientists reveal encouraging findings in first-in-human clinical trial evaluating HIV vaccine approach

While scientists have struggled in the past to create an effective vaccine against HIV, a novel vaccine design strategy being pursued by researchers at Scripps Research, IAVI, Fred Hutchinson Cancer Center (Fred Hutch) and the National Institutes of Health, National Institute of Allergy and Infectious Diseases (NIAID) Vaccine Research Center (VRC) shows new promise, according to data from a first-in-human clinical trial.

In a paper published in Science on December 2, 2022, the scientists reveal critical new insights into their novel vaccine strategy, which involves a stepwise approach to producing antibodies capable of targeting a wide range of HIV variants.

"The data we are publishing in Science demonstrates for the first time that one can design a vaccine that elicits made-to-order antibodies in humans. We specified in advance certain molecular properties of the antibodies that we wanted to elicit, and the results of this trial show that our vaccine antigen consistently induced precisely those types of antibodies," says co-senior author William Schief, PhD, a professor and immunologist at Scripps Research and executive director of vaccine design at IAVI's Neutralizing Antibody Center, whose laboratory developed the vaccine antigen. "We believe this vaccine design strategy will be essential to make an HIV vaccine and may help the field create vaccines for other difficult pathogens."

The Phase 1 trial, known as IAVI G001, tested the first stage in a multi-stage HIV vaccine regimen the researchers are developing. The trial results show that the vaccine had a favorable safety profile and induced the targeted response in 97% of people who were vaccinated. Importantly, the Science study also provides a detailed immunological analysis of the vaccine responses.

"HIV represents an area of dire unmet need across the world, which is what makes the findings from our Phase 1 clinical trial so encouraging," says Mark Feinberg, MD, PhD, president and CEO of IAVI. "Through the close-knit collaboration of many different scientists, disciplines and institutions, we are that much closer to designing an effective vaccine that could help end the HIV pandemic."

Priming the Immune System

Broadly neutralizing antibodies (bnAbs) are a rare type of antibody that can fight and protect against many different variants of a virus -- including HIV. This is why scientists have tried to develop an HIV vaccine that induces bnAbs, but thus far without success.

The researchers in the study are using a strategy known as 'germline targeting' to eventually produce bnAbs that can protect against HIV. The first step of germline targeting involves stimulating the rare immune cells -- known as bnAb-precursor B cells -- that can eventually evolve into the cells that produce the bnAbs needed to block the virus. To accomplish this first step, the researchers designed a customized molecule -- known as an immunogen -- that would "prime" the immune system and elicit responses from these rare bnAb-precursor cells.

The overarching goal of the IAVI G001 trial was to determine if the vaccine had an acceptable safety profile and could induce responses from these bnAb-precursor B cells.

"Through extensive safety and tolerability monitoring during the trial, we showed the vaccine had a favorable safety profile, while still inducing the necessary target cells," says study author Dagna Laufer, MD, vice president and head of clinical development at IAVI. "This represents a large step forward in developing an HIV vaccine that is both safe and effective."

To determine if the targeted bnAb-precursor B cells were induced, the researchers carried out a sophisticated analytical process.

"The workflow of multidimensional immunological analyses has taken clinical trial evaluation to the next level," says co-senior author Adrian B. McDermott, PhD, former chief of the Vaccine Immunology Program at the NIAID VRC. "In evaluating these important immunological factors, we helped show why the vaccine antigen was able to induce the targeted response in 97% of vaccine recipients."

IAVI G001 was sponsored by IAVI and took place at two sites: George Washington University (GWU) in Washington, D.C., and Fred Hutch in Seattle, enrolling 48 healthy adult volunteers. Participants received either a placebo or two doses of the vaccine antigen, eOD-GT8 60mer, along with an adjuvant developed by the pharmaceutical company GSK. Julie McElrath, MD, PhD, co-senior author, senior vice president and director of Fred Hutch's Vaccine and Infectious Disease Division, and David Diemert, MD, professor of medicine at GWU School of Medicine and Health Sciences, were lead investigators at the trial sites.

A Deeper Immunological Dive

The study also carefully examined the properties of the antibodies and B cells induced by the vaccine antigen, in what Schief likens to "looking under the car hood" to understand how the immune system operated in response to the vaccine. One analysis showed that the vaccine antigen first stimulated an average of 30 to 65 different bnAb precursors per person vaccinated, and then caused those cells to multiply. This helped explain why the vaccine induced the desired response in almost all participants.

Other analyses delved into the specific mutations the bnAb-precursor B cells acquired over time and how tightly they bound to the vaccine antigen. These investigations showed that that after each dose of the vaccine, the bnAb-precursor B cells gained affinity and continued along favorable maturation pathways.

One concern for this type of vaccine approach is the notion of "competitors" -- in other words, the B cells induced by the vaccine antigen that are not bnAb precursors. The researchers extensively studied the "competitor" responses, and the results were very encouraging. Although the majority of the B cells triggered by vaccination were, in fact, "competitors," these undesired B cells could not match the binding strength of the desired bnAb precursors and did not seem to impede maturation of the bnAb-precursor responses.

"These findings were very encouraging, as they indicated that immunogen design principles we used could be applied to many different epitopes, whether for HIV or even other pathogens," adds Schief.

With these promising data in hand spanning both safety and immune responses, the researchers will continue to iterate and design boosting immunogens that could eventually induce the desired bnAbs and provide protection against the virus. These findings also come shortly after two additionalstudies in Immunity published in September 2022, which helped validate the germline-targeting approach for vaccinating against HIV.

"Working together with IAVI, Scripps Research, the VRC, GWU, additional investigators at Fred Hutch and many others, this trial and additional analyses will help inform design of the remaining stages of a candidate HIV vaccine regimen -- while also enabling others in the field to develop vaccine strategies for additional viruses," says McElrath of Fred Hutch.

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New study reveals why HIV remains in human tissue even after antiretroviral therapy

Thanks to antiretroviral therapy, HIV infection is no longer the life sentence it once was. But despite the effectiveness of drugs to manage and treat the virus, it can never be fully eliminated from the human body, lingering in some cells deep in different human tissues where it goes unnoticed by the immune system.

Now, new research by University of Alberta immunologist Shokrollah Elahi reveals a possible answer to the mystery of why infected people can't get rid of HIV altogether.

Elahi and his team found that in HIV patients, killer T cells -- a type of white blood cells responsible for identifying and destroying cells infected with viruses -- have very little to none of a protein called CD73.

Because CD73 is responsible for migration and cell movement into the tissue, the lack of the protein compromises the ability of killer T cells to find and eliminate HIV-infected cells, explained Elahi.

"This mechanism explains one potential reason for why HIV stays in human tissues forever," he said, adding that the research also shows the complexity of HIV infection.

"This provides us the opportunity to come up with potential new treatments that would help killer T cells migrate better to gain access to the infected cells in different tissues."

After identifying the role of CD73 -- a three-year project -- Elahi turned his focus to understanding potential causes for the drastic reduction. He found it is partly due to the chronic inflammation that is common among people living with HIV.

"Following extensive studies, we discovered that chronic inflammation results in increased levels of a type of RNA found in cells and in blood, called microRNAs," he explained. "These are very small types of RNA that can bind to messenger RNAs to block them from making CD73 protein. We found this was causing the CD73 gene to be suppressed."

The team's discovery also helps explain why people with HIV have a lower risk of developing multiple sclerosis, Elahi noted.

"Our findings suggest that reduced or eliminated CD73 can be beneficial in HIV-infected individuals to protect them against MS. Therefore, targeting CD73 could be a novel potential therapeutic marker for MS patients."

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Immune system-stimulating nanoparticle could lead to more powerful vaccines

A common strategy to make vaccines more powerful is to deliver them along with an adjuvant -- a compound that stimulates the immune system to produce a stronger response.

Researchers from MIT, the La Jolla Institute for Immunology, and other institutions have now designed a new nanoparticle adjuvant that may be more potent than others now in use. Studies in mice showed that it significantly improved antibody production following vaccination against HIV, diphtheria, and influenza.

"We started looking at this particular formulation and found that it was incredibly potent, better than almost anything else we had tried," says Darrell Irvine, the Underwood-Prescott Professor with appointments in MIT's departments of Biological Engineering and Materials Science and Engineering; an associate director of MIT's Koch Institute for Integrative Cancer Research; and a member of the Ragon Institute of MGH, MIT, and Harvard.

The researchers now hope to incorporate the adjuvant into an HIV vaccine that is currently being tested in clinical trials, in hopes of improving its performance.

Irvine and Shane Crotty, a professor at the Center for Infectious Disease and Vaccine Research at the La Jolla Institute for Immunology, are the senior authors of the study, which appears today in Science Immunology. The lead authors of the paper are Murillo Silva, a former MIT postdoc, and Yu Kato, a staff scientist at the La Jolla Institute.

More powerful vaccines

Although the idea of using adjuvants to boost vaccine effectiveness has been around for decades, there are only a handful of FDA-approved vaccine adjuvants. One is aluminum hydroxide, an aluminum salt that induces inflammation, and another is an oil and water emulsion that is used in flu vaccines. A few years ago, the FDA approved an adjuvant based on saponin, a compound derived from the bark of the Chilean soapbark tree.

Saponin formulated in liposomes is now used as an adjuvant in the shingles vaccine, and saponins are also being used in a cage-like nanoparticle called an immunostimulatory complex (ISCOM) in a Covid-19 vaccine that is currently in clinical trials.

Researchers have shown that saponins promote inflammatory immune responses and stimulate antibody production, but how they do that is unclear. In the new study, the MIT and La Jolla team wanted to figure out how the adjuvant exerts its effects, and to see if they could make it more potent.

They designed a new type of adjuvant that is similar to the ISCOM adjuvant but also incorporates a molecule called MPLA, which is a toll-like receptor agonist. When these molecules bind to toll-like receptors on immune cells, they promote inflammation. The researchers call their new adjuvant SMNP (saponin/MPLA nanoparticles).

"We expected that this could be interesting because saponin and toll-like receptor agonists are both adjuvants that have been studied separately and shown to be very effective," Irvine says.

The researchers tested the adjuvant by injecting it into mice along with a few different antigens, or fragments of viral proteins. These included two HIV antigens, as well as diphtheria and influenza antigens. They compared the adjuvant to several other approved adjuvants and found that the new saponin-based nanoparticle elicited a stronger antibody response than any of the others.

One of the HIV antigens that they used is an HIV envelope protein nanoparticle, which presents many copies of the gp120 antigen that is present on the HIV viral surface. This antigen recently completed initial testing in phase 1 clinical trials. Irvine and Crotty are part of the Consortium for HIV/AIDS Vaccine Development at the Scripps Research Institute, which ran that trial. The researchers now hope to develop a way to manufacture the new adjuvant at large scale so it can be tested along with an HIV envelope trimer in another clinical trial beginning next year. Clinical trials that combine envelope trimers with the traditional vaccine adjuvant aluminum hydroxide are also underway.

"Aluminum hydroxide is safe but not particularly potent, so we hope that (the new adjuvant) would be an interesting alternative to elicit neutralizing antibody responses in people," Irvine says.

Rapid flow

When vaccines are injected into the arm, they travel through lymph vessels to the lymph nodes, where they encounter and activate B cells. The research team found that the new adjuvant speeds up the flow of lymph to the nodes, helping the antigen to get there before it starts to break down. It does this in part by stimulating immune cells called mast cells, which previously were not known to be involved in vaccine responses.

"Getting to the lymph nodes quickly is useful because once you inject the antigen, it starts slowly breaking down. The sooner a B cell can see that antigen, the more likely it's fully intact, so that B cells are targeting the structure as it will be present on the native virus," Irvine says.

Additionally, once the vaccine reaches the lymph nodes, the adjuvant causes a layer of cells called macrophages, which act as a barrier, to die off quickly, making it easier for the antigen to get into the nodes.

Another way that the adjuvant helps boost immune responses is by activating inflammatory cytokines that drive a stronger response. The TLR agonist that the researchers included in the adjuvant is believed to amplify that cytokine response, but the exact mechanism for that is not known yet.

This kind of adjuvant could also be useful for any other kind of subunit vaccine, which consists of fragments of viral proteins or other molecules. In addition to their work on HIV vaccines, the researchers are also working on a potential Covid-19 vaccine, along with J. Christopher Love's lab at the Koch Institute. The new adjuvant also appears to help stimulate T cell activity, which could make it useful as a component of cancer vaccines, which aim to stimulate the body's own T cells to attack tumors.

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New imaging method reveals HIV's sugary shield in unprecedented detail

 Scientists from Scripps Research and Los Alamos National Laboratory have devised a method for mapping in unprecedented detail the thickets of slippery sugar molecules that help shield HIV from the immune system.

Mapping these shields will give researchers a more complete understanding of why antibodies react to some spots on the virus but not others, and may shape the design of new vaccines that target the most vulnerable and accessible sites on HIV and other viruses.

The sugar molecules, or "glycans," are loose and stringy, and function as shields because they are difficult for antibodies to grip and block access to the protein surface. The shields form on the outermost spike proteins of HIV and many other viruses, including SARS-CoV-2, the coronavirus that causes COVID-19, because these viruses have evolved sites on their spike proteins where glycan molecules -- normally abundant in cells -- will automatically attach.

"We now have a way to capture the full structures of these constantly fluctuating glycan shields, which to a great extent determine where antibodies can and can't bind to a virus such as HIV," says the study's lead author Zachary Berndsen, PhD, a postdoctoral research associate in the structural biology lab of Scripps Research Professor Andrew Ward, PhD.

The same wavy flexibility that makes these sugary molecules resistant to antibodies has made them impossible for researchers to capture with traditional atomic-scale imaging. In the new study, which appears in the Proceedings of the National Academy of Sciences, the scientists developed techniques that, for the first time, allow these elusive molecules to be mapped in great detail on the surface of the HIV spike protein, known as "Env."

The Scripps Research team collaborated with the lab of Gnana Gnanakaran, PhD, staff scientist at Los Alamos National Laboratory, which is equipped with high-performance computing resources that enabled fresh approaches for modeling the glycans.

The researchers combined an atomic-scale imaging method called cryo-electron microscopy (cryo-EM) with sophisticated computer modeling and a molecule-identifying technique called site-specific mass spectrometry. Cryo-EM relies on averaging tens or hundreds of thousands of individual snapshots to create a clear image, thus highly flexible molecules like glycans will appear only as a blur, if they show up at all.

But by integrating cryo-EM with the other technologies, the researchers were able to recover this lost glycan signal and use it to map sites of vulnerability on the surface of Env.

"This is the first time that cryo-EM has been used along with computational modeling to describe the viral shield structure in atomic detail," says Srirupa Chakraborty, PhD, co-lead author and post-doctoral researcher in the Gnanakaran lab at Los Alamos National Laboratory.

The new combined approach revealed the glycans' structure and dynamic nature in extreme detail and helped the team better understand how these complex dynamics affect the features observed in the cryo-EM maps. From this wealth of information, the team observed that individual glycans do not just wiggle around randomly on the spike protein's surface, as once was thought, but instead clump together in tufts and thickets.

"There are chunks of glycans that seem to move and interact together," Berndsen says. "In between these glycan microdomains is where antibodies apparently have the opportunity to bind."

Experimental HIV vaccines rely on modified, lab-made Env proteins to elicit antibody responses. In principle, these vaccines' effectiveness depends in part on the positioning and extent of the shielding glycans on these lab-made viral proteins. Therefore, Berndsen and colleagues applied their method to map the glycans on a modified HIV Env protein, BG505 SOSIP.664, which is used in an HIV vaccine currently being evaluated in clinical trials.

"We found spots on the surface of this protein that normally would be covered with glycans but weren't -- and that may explain why antibody responses to that site have been noted in vaccination trials," Berndsen says.

That finding, and others in the study, showed that Env's glycan shield can vary depending on what type of cell is being used to produce it. In HIV's infections of humans, the virus uses human immune cells as factories to replicate its proteins. But viral proteins used to make vaccines normally are produced in other types of mammalian cells.

In another surprise discovery, the team observed that when they used enzymes to slowly remove glycans from HIV Env, the entire protein began to fall apart. Berndsen and colleagues suspect that Env's glycan shield, which has been considered merely a defense against antibodies, may also have a role in managing Env's shape and stability, keeping it poised for infection.

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Breakthrough study on molecular interactions could improve development of new medicines

A first-of-its-kind study on molecular interactions by biomedical engineers in the University of Minnesota's College of Science and Engineering will make it easier and more efficient for scientists to develop new medicines and other therapies for diseases such as cancer, HIV and autoimmune diseases.

The study resulted in a mathematical framework that simulates the effects of the key parameters that control interactions between molecules that have multiple binding sites, as is the case for many medicines. Researchers plan to use this computational model to develop a web-based app that other researchers can use to speed the development of new therapies for diseases.

The research is published in the Proceedings of the National Academy of Sciences (PNAS).

"The big advance with this study is that usually researchers use a trial-and-error experimental method in the lab for studying these kinds of molecular interactions, but here we developed a mathematical model where we know the parameters so we can make accurate predictions using a computer," said Casim Sarkar, a University of Minnesota biomedical engineering associate professor and senior author of the study. "This computational model will make research much more efficient and could accelerate the creation of new therapies for many kinds of diseases."

The research team studied three main parameters of molecular interactions -- binding strength of each site, rigidity of the linkages between the sites, and the size of the linkage arrays. They looked at how these three parameters can be "dialed up" or "dialed down" to control how molecule chains with two or three binding sites interact with one another. The team then confirmed their model predictions in lab experiments.

"At a fundamental level, many diseases can be traced to a molecule not binding correctly," said Wesley Errington, a University of Minnesota biomedical engineering postdoctoral researcher and lead author of the study. "By understanding how we can manipulate these 'dials' that control molecular behavior, we have developed a new programming language that can be used to predict how molecules will bind."

The need for a mathematical framework to decode this programming language is highlighted by the researchers' finding that, even when the interacting molecule chains have just three binding sites each, there are a total of 78 unique binding configurations, most of which cannot be experimentally observed. By dialing the parameters in this new mathematical model, researchers can quickly understand how these different binding configurations are affected, and tune them for a wide range of biological and medical applications.

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By targeting flu-enabling protein, antibody may protect against wide-ranging strains

Influenza virus illustration

A nationwide team of researchers has found an antibody that protects mice against a wide range of potentially lethal influenza viruses, advancing efforts to design of a universal vaccine that could either treat or protect people against all strains of the virus.

The study, which Scripps Research conducted jointly with Washington University School of Medicine in St. Louis and Icahn School of Medicine at Mount Sinai in New York, points to a new approach to tackle severe cases of the flu, including pandemics. The research is published in the Oct. 25 issue of Science.

Scripps Research's Ian Wilson, DPhil, one of three senior co-authors, says the antibody at the center of the study binds to a protein called neuraminidase, which is essential for the flu virus to replicate in the body.

The protein, located on the surface of the virus, enables infected host cells to release the virus so it can spread to other cells. Tamiflu, the most widely used drug for severe flu infection, works by inactivating neuraminidase. However, many forms of neuraminidase exist, depending on the flu strain, and such drugs aren't always effective -- particularly as resistance to the drugs is developing.

"There are many strains of influenza virus that circulate so every year we have to design and produce a new vaccine to match the most common strains of that year," says co-senior author Ali Ellebedy, PhD, an assistant professor of pathology and immunology at Washington University. "Now imagine if we could have one vaccine that protected against all influenza strains, including human, swine and other highly lethal avian influenza viruses. This antibody could be the key to design of a truly universal vaccine."

Ellebedy discovered the antibody -- an immune molecule that recognizes and attaches to a foreign molecule -- in blood taken from a patient hospitalized with flu at Barnes-Jewish Hospital in St. Louis in the winter of 2017.

Ellebedy was working on a study analyzing the immune response to flu infection in humans in collaboration with the Washington University Emergency Care and Research Core, which was sending him blood samples from consenting flu patients. He quickly noticed that a particular blood sample was unusual: In addition to containing antibodies against hemagglutinin, the major protein on the surface of the virus, it contained other antibodies that were clearly targeting something else.

"At the time we were just starting, and I was setting up my lab so we didn't have the tools to look at what else the antibodies could be targeting," says Ellebedy, an assistant professor of medicine and of molecular microbiology.

He sent three of the antibodies to co-senior author Florian Krammer, PhD, a microbiology professor at the Icahn School of Medicine at Mount Sinai. An expert on neuraminidase, Krammer tested the antibodies against his extensive library of neuraminidase proteins. At least one of the three antibodies blocked neuraminidase activity in all known types of neuraminidase in flu viruses, representing a variety of human and nonhuman strains.

"The breadth of the antibodies really came as a surprise to us," says Krammer. "Typically, anti-neuraminidase antibodies can be broad within a subtype, like H1N1, but an antibody with potent activity across subtypes was unheard of. At first, we did not believe our results. Especially the ability of the antibodies to cross between influenza A and influenza B viruses is just mind-boggling. It is amazing what the human immune system is capable of if presented with the right antigens."

To find out whether the antibodies could be used to treat severe cases of flu, Krammer and colleagues tested them in mice that were given a lethal dose of influenza virus. All three antibodies were effective against many strains, and one antibody, called "1G01," protected against all 12 strains tested, which included all three groups of human flu virus as well as avian and other nonhuman strains.

"All the mice survived, even if they were given the antibody 72 hours after infection," Ellebedy says. "They definitely got sick and lost weight, but we still saved them. It was remarkable. It made us think that you might be able to use this antibody in an intensive care scenario when you have someone sick with flu and it's too late to use Tamiflu."

Tamiflu must be administered within 24 hours of symptoms. A drug that could be used later would help many people diagnosed after the Tamiflu window has closed. But before the researchers could even think of designing such a drug based on the antibody, they needed to understand how it was interfering with neuraminidase.

They turned to Scripps Research's Wilson, known globally for his work as a structural biologist. Wilson is Chair of the Institute's Department of Integrative Structural and Computational Biology, and has made numerous seminal findings that have shaped efforts to develop universal vaccines for flu and other complex viruses such as HIV.

Wilson and Xueyong Zhu, PhD, a staff scientist in Wilson's lab, mapped the structures of the antibodies while they were bound to neuraminidase. They found that the antibodies each had a loop that slid inside the active site of neuraminidase like a stick between gears. The loops prevented neuraminidase from releasing new virus particles from the surface of cells, thereby breaking the cycle of viral production in host cells.

"We were surprised at how these antibodies managed to insert a single loop into the conserved active site without contacting the surrounding hypervariable regions, thereby achieving much greater breadth against the neuraminidase of different influenza viruses than we have seen before," Wilson says.

The structures showed that the antibodies provide such broad protection because they target the conserved residues in the active site of the neuraminidase protein. That site stays much the same across distantly related flu strains because even minor changes could abolish the protein's ability to do its job, thereby preventing the virus from replicating.

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Drug reverses signs of liver disease in people living with HIV

Researchers at the National Institutes of Health and their colleagues at Massachusetts General Hospital (MGH) in Boston report that the injectable hormone tesamorelin reduces liver fat and prevents liver fibrosis (scarring) in people living with HIV. The study was conducted by the National Institute of Allergy and Infectious Diseases (NIAID) and the National Cancer Institute, both parts of NIH. The findings were published online today in The Lancet HIV.

"Many people living with HIV have overcome significant obstacles to live longer, healthier lives, though many still experience liver disease," said NIAID Director Anthony S. Fauci, M.D. "It is encouraging that tesamorelin, a drug already approved to treat other complications of HIV, may be effective in addressing non-alcoholic fatty liver disease."

Non-alcoholic fatty liver disease, or NAFLD, frequently occurs alongside HIV, affecting as many as 25% of people living with HIV in the developed world. However, no effective treatments currently exist to treat the condition, which is a risk factor for progressive liver disease and liver cancer. Investigators led by Colleen M. Hadigan, M.D., senior research physician in NIAID's Laboratory of Immunoregulation, and Steven K. Grinspoon, M.D., Chief of the Metabolism Unit at MGH, tested whether tesamorelin could decrease liver fat in men and women living with both HIV and NAFLD. Among the participants enrolled, 43% had at least mild fibrosis, and 33% met the diagnostic criteria for a more severe subset of NAFLD called nonalcoholic steatohepatitis (NASH). Thirty-one participants were randomized to receive daily 2-mg injections of tesamorelin, and 30 were randomized to receive identical-looking injections containing a placebo. Researchers provided nutritional counseling to all participants, as well as training in self-administering the daily injections. Researchers then compared measures of liver health in both groups at baseline and 12 months.

After one year, participants receiving tesamorelin had better liver health than those receiving placebo, as defined by reduction in hepatic fat fraction (HFF) -- the ratio of fat to other tissue in the liver. The healthy range for HFF is less than 5%. Thirty-five percent of study participants receiving tesamorelin achieved a normal HFF, while only 4% of those on placebo reached that range with nutritional advice alone. Overall, tesamorelin was well-tolerated and reduced participants' HFF by an absolute difference of 4.1% (corresponding to a 37% relative reduction from the beginning of the study). While nine participants receiving placebo experienced onset or worsening of fibrosis, only two participants in the tesamorelin group experienced the same. Additionally, levels of several blood markers associated with inflammation and liver damage -- including the enzyme alanine aminotransferase (ALT) -- decreased more among those taking tesamorelin compared to those on a placebo, particularly among those with increased levels at the beginning of the study.

Given these positive results, investigators suggest expanding the indication for tesamorelin to include people living with HIV who have been diagnosed with NAFLD. They also recommend additional research to determine if tesamorelin could contribute to long-term protection against serious liver disease in people without HIV.

"Our hope is that this intervention may help people living with HIV, as well as benefit HIV-negative people with liver abnormalities," said Dr. Hadigan. "Further research may inform us of the potential long-term benefits of this approach and develop formulations that can benefit everyone with liver disease, regardless of HIV status."

Egrifta (tesamorelin) was approved in 2010 by the U.S. Food and Drug Administration to reduce excess abdominal fat in HIV patients with lipodystrophy -- a complication characterized by an abnormal distribution of body fat initially associated with older classes of HIV medications. The most commonly reported side effects in previous clinical trials evaluating Egrifta included joint pain (arthralgia), skin redness and rash at the injection site (erythema and pruritis), stomach pain, swelling, and muscle pain (myalgia). Worsening blood sugar control occurred more often in trial participants treated with Egrifta than with placebo.

"Because tesamorelin proved effective in treating abnormal fat build-up in the abdomens of people in the context of HIV and related medication use, we hypothesized that the drug might also reduce fat that accrues in the liver and causes damage in a similar population," said Dr. Grinspoon.

While liver disease is often associated with heavy alcohol use, NAFLD occurs when excess fat builds up in the liver without alcohol as a contributing factor. This condition may progress to liver damage, cirrhosis or cancer that could be life-threatening and necessitate liver transplantation.

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