FMF assay for assessing vaccine generated antibodies in a biomimetic manner

Traditional functional assays such as hemagglutination inhibition (HAI) and micro-neutralization (MN) assays have been routinely used for assessing the vaccine response, since influenza vaccine has been administered in people (1940). Such assays are not always predictive regarding the protection conferred by the influenza vaccine and are not able to monitor neutralization related to stem region of influenza hemagglutinin responsible for virus membrane fusion in the endosomes. In order to study Influenza vaccine response in a more biomimetic manner and overcome the deficiencies of the traditional functional assays, we developed a fluorescent membrane fusion assay (fMF). The assay uses viruses labeled with Octadecyl Rhodmaine B Chloride (R18) to monitor two major neutralization pathways: blocking the attachment of virus to the target cells and blocking of virus membrane fusion in the endosomes. The latter was tested using endosomal acidification inhibitor Bafilomycin a1 which blocked membrane fusion by 85%. Specificity of the assay was tested using two different subtypes of viruses H1N1 (A/Puerto Rico/8/1934 and A/Brisbane/59/2007), and H3N2 virus (A/Aichi/68) with their respective subtype specific stem specific monoclonal antibodies: M145, Aca-1, Aca-2 (H1N1 specific) and Aca-3 (H3N2 specific). Subtype specific mAbs blocked membrane fusion, while a mismatch in virus subtype and the mAb resulted in lack of blocking. We also studied the effect of H1N1 head specific mAb Aca-4, which not only blocked attachment of the virus, but also demonstrated blocking of membrane fusion. Results were validated by testing pre- and post- sera from 2009 seasonal Influenza vaccination and to show that at higher Ab concentration the majority of virus (85%) was blocked from attaching cells, but at lower Ab concentration, where attachment could not be prevented, blocking of membrane fusion was still in effect - up to 50%. Sera screening experiments showed that sera antibodies work beyond just blocking attachment. They also may neutralize the already attached virus by blocking fusion of the viral membrane in the endosomes. The assay has the capacity to monitor blocking of attachment and fusion in a single run. Therefore, it is more representative regarding the natural process of infection and the corresponding neutralization pathways. The assay is unique in terms of assessing stem specific antibodies; stem specific response and its measurement are relevant for the advancement of a universal influenza vaccine

The role of the helper lipid on the DNA transfection efficiency of lipopolyplex formulations.

Multifunctional, lipopolyplex formulations comprising a mixture of cationic liposomes and cationic, receptor-targeting peptides have potential use in gene therapy applications. Lipopolyplex formulations described here are typically far more efficient transfection agents than binary lipoplex or polyplex formulations. It has been shown previously that the peptide component mediates both DNA packaging and targeting of the nanoparticle while in this report we investigate the contribution of the lipid component. We hypothesised that the lipid components synergise with the peptides in the transfection process by promoting endosomal escape after lipid bilayer fusion. Lipopolyplexes were prepared with cationic liposomes comprising DOTAP with either neutral lipid DOPE or DOPC. DOPE promotes fusogenic, inverted hexagonal lipid structures while DOPC promotes more stable laminar structures. Lipopolyplexes containing DOPE showed substantially higher transfection efficiency than those formulated with DOPC, both in vitro and in vivo. DOPE-containing lipopolyplexes showed rapid endosomal trafficking and nuclear accumulation of DNA while DOPC-containing formulations remained within the late endo-lysosomal compartments. These findings are consistent with previous finding for the role of DOPE in lipoplexes and support the hypothesis regarding the function of the lipid components in lipopolyplexes. These findings will help to inform future lipopolyplex design, strategies and clinical development processes

Replication Cycle and Molecular Biology of the West Nile Virus

West Nile virus (WNV) is a member of the genus Flavivirus in the family Flaviviridae. Flaviviruses replicate in the cytoplasm of infected cells and modify the host cell environment. Although much has been learned about virion structure and virion-endosomal membrane fusion, the cell receptor(s) used have not been definitively identified and little is known about the early stages of the virus replication cycle. Members of the genus Flavivirus differ from members of the two other genera of the family by the lack of a genomic internal ribosomal entry sequence and the creation of invaginations in the ER membrane rather than double-membrane vesicles that are used as the sites of exponential genome synthesis. The WNV genome 3\u27 and 5\u27 sequences that form the long distance RNA-RNA interaction required for minus strand initiation have been identified and contact sites on the 5\u27 RNA stem loop for NS5 have been mapped. Structures obtained for many of the viral proteins have provided information relevant to their functions. Viral nonstructural protein interactions are complex and some may occur only in infected cells. Although interactions between many cellular proteins and virus components have been identified, the functions of most of these interactions have not been delineated

SARS-CoV-2 spreads through cell-to-cell transmission

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein, we provide evidence that SARS-CoV-2 spreads through cell-cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than is SARS-CoV spike, which reflects, in part, their differential cell-cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While angiotensin-converting enzyme 2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccinee sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis

Reactivation from latency displays HIV particle budding at plasma membrane, accompanying CD44 upregulation and recruitment

<p>Abstract</p> <p>Background</p> <p>It has been accepted that HIV buds from the cell surface in T lymphocytes, whereas in macrophages it buds into intracellular endosomes. Recent studies, on the other hand, suggest that HIV preferentially buds from the cell surface even in monocytic cells. However, most studies are based on observations in acutely infected cells and little is known about HIV budding concomitant with reactivation from latency. Such studies would provide a better understanding of a reservoir for HIV.</p> <p>Results</p> <p>We observed HIV budding in latently infected T lymphocytic and monocytic cell lines following TNF-α stimulation and examined the upregulation of host factors that may be involved in particle production. Electron microscopy analysis revealed that reactivation of latently infected J1.1 cells (latently infected Jurkat cells with HIV-1) and U1 cells (latently infected U937 cells with HIV-1) displayed HIV particle budding predominantly at the plasma membrane, a morphology that is similar to particle budding in acutely infected Jurkat and U937 cells. When mRNA expression levels were quantified by qRT-PCR, we found that particle production from reactivated J1.1 and U1 cells was accompanied by CD44 upregulation. This upregulation was similarly observed when Jurkat and U937 cells were acutely infected with HIV-1 but not when just stimulated with TNF-α, suggesting that CD44 upregulation was linked with HIV production but not with cell stimulation. The molecules in endocytic pathways such as CD63 and HRS were also upregulated when U1 cells were reactivated and U937 cells were acutely infected with HIV-1. Confocal microscopy revealed that these upregulated host molecules were recruited to and accumulated at the sites where mature particles were formed at the plasma membrane.</p> <p>Conclusion</p> <p>Our study indicates that HIV particles are budded at the plasma membrane upon reactivation from latency, a morphology that is similar to particle budding in acute infection. Our data also suggest that HIV expression may lead to the upregulation of certain host cell molecules that are recruited to sites of particle assembly, possibly coordinating particle production.</p

Cryo-EM Structures of Eastern Equine Encephalitis Virus Reveal Mechanisms of Virus Disassembly and Antibody Neutralization

Alphaviruses are enveloped pathogens that cause arthritis and encephalitis. Here, we report a 4.4-Å cryoelectron microscopy (cryo-EM) structure of eastern equine encephalitis virus (EEEV), an alphavirus that causes fatal encephalitis in humans. Our analysis provides insights into viral entry into host cells. The envelope protein E2 showed a binding site for the cellular attachment factor heparan sulfate. The presence of a cryptic E2 glycan suggests how EEEV escapes surveillance by lectin-expressing myeloid lineage cells, which are sentinels of the immune system. A mechanism for nucleocapsid core release and disassembly upon viral entry was inferred based on pH changes and capsid dissociation from envelope proteins. The EEEV capsid structure showed a viral RNA genome binding site adjacent to a ribosome binding site for viral genome translation following genome release. Using five Fab-EEEV complexes derived from neutralizing antibodies, our investigation provides insights into EEEV host cell interactions and protective epitopes relevant to vaccine design

Edible bird's nest (EBN) is a potential natural product against influenza virus infection

Edible bird’s nest (EBN) is an emergent industry in Malaysia. In 2016, EBN worth RM 1.2 billion with the total production of 228-tonne metrics. EBN is salivary secretion of swiftlets (Aerodramus spp.) that contained various nutritive values. Back in Tang Dynasty, it had been recognized as a natural product with broad medicinal effects. Recently, various scientific studies have been done to elucidate the medicinal properties of this precious food. For the past 10 years, antiviral effects of EBN had been explained via in-vitro and in-vivo well-designed researches, in which generally EBN is exerting a good alternative food for prophylactic and therapeutic agent against Influenza A virus infection in the laboratory setting. The limited study had been done to identify the bioactive ingredient of EBN that have antiviral properties. Nevertheless, based on the previous nutritional studies, some contents of EBN have been hypothesised to serve as an antiviral agent, and comprehensive study is required to explicate those claimed. The aim of this paper is to review on the recent discovery pertaining to the potential antiviral effect of EBN in the cell culture and animal model studies

Dengue virus entry and trafficking: Perspectives as antiviral target for prevention and therapy

Dengue virus (DENV) is the etiological agent of the most important human viral infection transmitted by mosquitoes in the world. In spite of the serious health threat that dengue represents, at present there are no vaccine or antiviral agents available and treatment of patients consists of supportive therapy. This review will focus on the process of DENV entry into the host cell as a potential target for antiviral therapy. The recent advances in the knowledge of viral and cellular molecules and mechanisms involved in binding, internalization and trafficking of DENV into the host cell until virion uncoating are discussed, together with an overview of the strategies and compounds evaluated for development of antiviral agents targeted to DENV entry.Fil: Castilla, Viviana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Piccini, Luana Érica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Damonte, Elsa Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentin