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Langedijk J.P.,Janssen Infectious Diseases and Vaccines | Furmanova-Hollenstein P.,Janssen Infectious Diseases and Vaccines | Chaiwatpongsakorn S.,Nationwide Childrens Hospital | Costello H.M.,Nationwide Childrens Hospital | And 10 more authors.
Nature Chemical Biology | Year: 2016

Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in young children and the elderly. Therapeutic small molecules have been developed that bind the RSV F glycoprotein and inhibit membrane fusion, yet their binding sites and molecular mechanisms of action remain largely unknown. Here we show that these inhibitors bind to a three-fold-symmetric pocket within the central cavity of the metastable prefusion conformation of RSV F. Inhibitor binding stabilizes this conformation by tethering two regions that must undergo a structural rearrangement to facilitate membrane fusion. Inhibitor-escape mutations occur in residues that directly contact the inhibitors or are involved in the conformational rearrangements required to accommodate inhibitor binding. Resistant viruses do not propagate as well as wild-type RSV in vitro, indicating a fitness cost for inhibitor escape. Collectively, these findings provide new insight into class I viral fusion proteins and should facilitate development of optimal RSV fusion inhibitors. © 2016 Nature America, Inc. All rights reserved.


Krarup A.,Janssen Infectious Diseases and Vaccines | Truan D.,Janssen Infectious Diseases and Vaccines | Furmanova-Hollenstein P.,Janssen Infectious Diseases and Vaccines | Bogaert L.,Janssen Infectious Diseases and Vaccines | And 6 more authors.
Nature Communications | Year: 2015

Respiratory syncytial virus (RSV) causes acute lower respiratory tract infections and is the leading cause of infant hospitalizations. Recently, a promising vaccine antigen based on the RSV fusion protein (RSV F) stabilized in the native prefusion conformation has been described. Here we report alternative strategies to arrest RSV F in the prefusion conformation based on the prevention of hinge movements in the first refolding region and the elimination of proteolytic exposure of the fusion peptide. A limited number of unique mutations are identified that stabilize the prefusion conformation of RSV F and dramatically increase expression levels. This highly stable prefusion RSV F elicits neutralizing antibodies in cotton rats and induces complete protection against viral challenge. Moreover, the structural and biochemical analysis of the prefusion variants suggests a function for p27, the excised segment that precedes the fusion peptide in the polypeptide chain. © 2015 Macmillan Publishers Limited.


PubMed | Janssen Infectious Diseases and Vaccines, Respiratory Infections Research, The Research Institute at Nationwide Childrens Hospital, Janssen Pharmaceutical and 2 more.
Type: Journal Article | Journal: Nature chemical biology | Year: 2016

Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in young children and the elderly. Therapeutic small molecules have been developed that bind the RSV F glycoprotein and inhibit membrane fusion, yet their binding sites and molecular mechanisms of action remain largely unknown. Here we show that these inhibitors bind to a three-fold-symmetric pocket within the central cavity of the metastable prefusion conformation of RSV F. Inhibitor binding stabilizes this conformation by tethering two regions that must undergo a structural rearrangement to facilitate membrane fusion. Inhibitor-escape mutations occur in residues that directly contact the inhibitors or are involved in the conformational rearrangements required to accommodate inhibitor binding. Resistant viruses do not propagate as well as wild-type RSV in vitro, indicating a fitness cost for inhibitor escape. Collectively, these findings provide new insight into class I viral fusion proteins and should facilitate development of optimal RSV fusion inhibitors.


PubMed | Janssen Infectious Diseases and Vaccines
Type: | Journal: Nature communications | Year: 2015

Respiratory syncytial virus (RSV) causes acute lower respiratory tract infections and is the leading cause of infant hospitalizations. Recently, a promising vaccine antigen based on the RSV fusion protein (RSV F) stabilized in the native prefusion conformation has been described. Here we report alternative strategies to arrest RSV F in the prefusion conformation based on the prevention of hinge movements in the first refolding region and the elimination of proteolytic exposure of the fusion peptide. A limited number of unique mutations are identified that stabilize the prefusion conformation of RSV F and dramatically increase expression levels. This highly stable prefusion RSV F elicits neutralizing antibodies in cotton rats and induces complete protection against viral challenge. Moreover, the structural and biochemical analysis of the prefusion variants suggests a function for p27, the excised segment that precedes the fusion peptide in the polypeptide chain.


News Article | November 10, 2016
Site: www.eurekalert.org

BOSTON - A study led by researchers at Beth Israel Deaconess Medical Center (BIDMC), in collaboration with scientists at Walter Reed Army Institute of Research (WRAIR), Janssen Vaccines & Prevention B.V., one of the Janssen Pharmaceutical Companies of Johnson & Johnson and Gilead Sciences, Inc., has demonstrated that combining an experimental vaccine with an innate immune stimulant may help lead to viral remission in people living with HIV. In animal trials, the combination decreased levels of viral DNA in peripheral blood and lymph nodes, and improved viral suppression and delayed viral rebound following discontinuation of anti-retroviral therapy (ART). The research team's findings appeared online today in the journal Nature. "The objective of our study was to identify a functional cure for HIV - not to eradicate the virus, but to control it without the need for ART," said lead author Dan Barouch, MD, PhD, Director of the Center for Virology and Vaccine Research at BIDMC. "Current antiretroviral drugs, although they're lifesaving, do not cure HIV. They merely hold it in check. We are trying to develop strategies to achieve ART-free, long-term viral suppression." Typically, vaccines "teach" the body to rid itself of viral invaders by provoking an immune response. However, HIV attacks cells of the immune system. The virus kills the majority of infected immune cells but goes dormant in others. This reservoir of dormant, infected cells, where researchers believe HIV remains hidden during antiretroviral therapy, is the primary reason HIV cannot currently be cured. Barouch and colleagues are working on strategies to draw the virus out of hiding with the goal of eradicating it from the body. "We reasoned that if we can activate the immune cells that might harbor the virus, then the vaccine-induced immune responses might perform better seeking them out and destroying them," said Barouch, who is also a Professor of Medicine at Harvard Medical School. "Indeed, we saw the best results when we combined the vaccine together with the innate immune stimulant." In the two-year long study, researchers monitored the viral loads of 36 rhesus monkeys infected with simian immunodeficiency virus (SIV), a virus similar to HIV that infects non-human primates. After taking suppressive ART drugs for six months, the monkeys were given either the experimental vaccines - an adenovirus serotype 26 vector vaccine and an MVA vector vaccine (Ad26/MVA) - alone, the immune stimulant (an experimental drug that works on a protein of the immune system called TLR-7) alone, or the Ad26/MVA and stimulant combination. A control group received no active treatment. "We found the combination of Ad26/MVA vaccination and TLR7 stimulation proved more effective than either component alone," said Col. Nelson Michael, director of MHRP, who helped design the preclinical study. "This was especially striking in viral load set-point, which impacts the future course of the disease." The experimental vaccine induced a robust immune response, both in magnitude (the number of immune cells generated) and breadth (the number of places on the virus the vaccine can targets). To evaluate the efficacy of the vaccine and the immune stimulant, the researchers discontinued ART in all animals and continued to monitor their viral loads. Animals that received only the vaccine demonstrated some reduction of viral load, but the animals that were given the vaccine/immune stimulant combination showed a reduction in plasma viral RNA levels as well as a 2.5-fold delay of viral rebound compared to controls. All nine animals showed decreased viral loads, and the virus was undetectable in a third of the animals. "If all the animals' viral loads had been undetectable, that would have been a home run," said Barouch. "But the fact that all animals showed a reduction in viral load and three out of nine were undetectable, that's a solid base hit. It's definitely something that we can work from." Study coauthors include Erica Borducchi, Crystal Cabral, Kathryn E. Stephenson, Jinyan Liu, Peter Abbink, David Ng'ang'a, Joseph P. Nkolola, Amanda L. Brinkman, Lauren Peter, Benjamin C. Lee, Jessica Jimenez, David Jetton, Jade Modesir, Shanell Mojta, Abishek Chandrashekar and Katherine Molloy all of BIDMC; Galit Alter of the Ragon Institute of MGH, MIT, and Harvard; Jeff M. Gerold and Alison L. Hill of the Program for Evolutionary Dynamics at Harvard University; Mark G. Lewis, of Bioqual; Maria G. Pau, Hanneke Schuitemaker of Janssen Infectious Diseases and Vaccines; Joseph Hesselgesser and Romas Geleziunas of Gilead Sciences; Jerome H. Kim, Merlin L. Robb and Nelson L. Michael of the U.S. Military HIV Reseach Program, Walter Reed Army Institute of Research. This work was supported by the U.S. Army Medical Research and Material Command and the Military HIV Research Program, Walter Reed Army Institute of Research through its cooperative agreement with the Henry M. Jackson Foundation (W81XWH-11-2-0174). Funding was also provided by the National Institutes of Health (AI096040, AI124377, AI1266030) and the Ragon Institute of MGH, MIT, and Harvard. Beth Israel Deaconess Medical Center is a patient care, teaching and research affiliate of Harvard Medical School and consistently ranks as a national leader among independent hospitals in National Institutes of Health funding. BIDMC is in the community with Beth Israel Deaconess Hospital-Milton, Beth Israel Deaconess Hospital-Needham, Beth Israel Deaconess Hospital-Plymouth, Anna Jaques Hospital, Cambridge Health Alliance, Lawrence General Hospital, MetroWest Medical Center, Signature Healthcare, Beth Israel Deaconess HealthCare, Community Care Alliance and Atrius Health. BIDMC is also clinically affiliated with the Joslin Diabetes Center and Hebrew Rehabilitation Center and is a research partner of Dana-Farber/Harvard Cancer Center and the Jackson Laboratory. BIDMC is the official hospital of the Boston Red Sox. For more information, visit http://www. .


PubMed | Janssen Research & Development, Janssen Infectious Diseases and Vaccines and Crucell
Type: Journal Article | Journal: Vaccine | Year: 2015

RSV is an important cause of lower respiratory tract infections in children, the elderly and in those with underlying medical conditions. Although the high disease burden indicates an urgent need for a vaccine against RSV, no licensed RSV vaccine is currently available. We developed an RSV vaccine candidate based on the low-seroprevalent human adenovirus serotypes 26 and 35 (Ad26 and Ad35) encoding the RSV fusion (F) gene. Single immunization of mice with either one of these vectors induced high titers of RSV neutralizing antibodies and high levels of F specific interferon-gamma-producing T cells. A Th1-type immune response was indicated by a high IgG2a/IgG1 ratio of RSV-specific antibodies, strong induction of RSV-specific interferon-gamma and tumor necrosis factor-alpha cytokine producing CD8 Tcells, and low RSV-specific CD4 T-cell induction. Both humoral and cellular responses were increased upon a boost with RSV-F expressing heterologous adenovirus vector (Ad35 boost after Ad26 prime or vice versa). Both single immunization and prime-boost immunization of cotton rats induced high and long-lasting RSV neutralizing antibody titers and protective immunity against lung and nasal RSV A2 virus load up to at least 30 weeks after immunization. Cotton rats were also completely protected against challenge with a RSV B strain (B15/97) after heterologous prime-boost immunization. Lungs from vaccinated animals showed minimal damage or inflammatory infiltrates post-challenge, in contrast to animals vaccinated with formalin-inactivated virus. Our results suggest that recombinant human adenoviral Ad26 and Ad35 vectors encoding the RSV F gene have the potential to provide broad and durable protection against RSV in humans, and appear safe to be investigated in infants.


PubMed | U.S. Army, Janssen Infectious Diseases and Vaccines, Massachusetts Institute of Technology, Gilead Sciences and 3 more.
Type: Journal Article | Journal: Nature | Year: 2016

The development of immunologic interventions that can target the viral reservoir in HIV-1-infected individuals is a major goal of HIV-1 research. However, little evidence exists that the viral reservoir can be sufficiently targeted to improve virologic control following discontinuation of antiretroviral therapy. Here we show that therapeutic vaccination with Ad26/MVA (recombinant adenovirus serotype 26 (Ad26) prime, modified vaccinia Ankara (MVA) boost) and stimulation of TLR7 (Toll-like receptor 7) improves virologic control and delays viral rebound following discontinuation of antiretroviral therapy in SIV-infected rhesus monkeys that began antiretroviral therapy during acute infection. Therapeutic vaccination with Ad26/MVA resulted in a marked increase in the magnitude and breadth of SIV-specific cellular immune responses in virologically suppressed, SIV-infected monkeys. TLR7 agonist administration led to innate immune stimulation and cellular immune activation. The combination of Ad26/MVA vaccination and TLR7 stimulation resulted in decreased levels of viral DNA in lymph nodes and peripheral blood, and improved virologic control and delayed viral rebound following discontinuation of antiretroviral therapy. The breadth of cellular immune responses correlated inversely with set point viral loads and correlated directly with time to viral rebound. These data demonstrate the potential of therapeutic vaccination combined with innate immune stimulation as a strategy aimed at a functional cure for HIV-1 infection.


Ader-Ebert N.,University of Bern | Khosravi M.,University of Bern | Herren M.,University of Bern | Avila M.,University of Bern | And 7 more authors.
PLoS Pathogens | Year: 2015

Despite large vaccination campaigns, measles virus (MeV) and canine distemper virus (CDV) cause major morbidity and mortality in humans and animals, respectively. The MeV and CDV cell entry system relies on two interacting envelope glycoproteins: the attachment protein (H), consisting of stalk and head domains, co-operates with the fusion protein (F) to mediate membrane fusion. However, how receptor-binding by the H-protein leads to F-triggering is not fully understood. Here, we report that an anti-CDV-H monoclonal antibody (mAb-1347), which targets the linear H-stalk segment 126-133, potently inhibits membrane fusion without interfering with H receptor-binding or F-interaction. Rather, mAb-1347 blocked the F-triggering function of H-proteins regardless of the presence or absence of the head domains. Remarkably, mAb-1347 binding to headless CDV H, as well as standard and engineered bioactive stalk-elongated CDV H-constructs treated with cells expressing the SLAM receptor, was enhanced. Despite proper cell surface expression, fusion promotion by most H-stalk mutants harboring alanine substitutions in the 126-138 “spacer” section was substantially impaired, consistent with deficient receptor-induced mAb-1347 binding enhancement. However, a previously reported F-triggering defective H-I98A variant still exhibited the receptor-induced “head-stalk” rearrangement. Collectively, our data spotlight a distinct mechanism for morbillivirus membrane fusion activation: prior to receptor contact, at least one of the morbillivirus H-head domains interacts with the membrane-distal “spacer” domain in the H-stalk, leaving the F-binding site located further membrane-proximal in the stalk fully accessible. This “head-to-spacer” interaction conformationally stabilizes H in an auto-repressed state, which enables intracellular H-stalk/F engagement while preventing the inherent H-stalk’s bioactivity that may prematurely activate F. Receptor-contact disrupts the “head-to-spacer” interaction, which subsequently “unlocks” the stalk, allowing it to rearrange and trigger F. Overall, our study reveals essential mechanistic requirements governing the activation of the morbillivirus membrane fusion cascade and spotlights the H-stalk “spacer” microdomain as a possible drug target for antiviral therapy.

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