Twenty plus Years of Data Demonstrating Virus Filtration as an Effective and Robust Step for Large Virus Removal.

Virus filtration has been demonstrated to be an effective and robust dedicated viral clearance step that is used in biopharmaceutical manufacturing processes. Here we present virus filtration data from a multicompany collaboration with data compiled from WuXi Advanced Therapies' and Charles River Laboratories' internal viral clearance databases spanning more than 25 years. The data were sorted by virus removal and type and then further subdivided into murine leukemia virus only, pseudorabies virus only, and reovirus type 3 only categories to allow for analyses of viral clearance results. A total of 2311 virus filtrations were analyzed, composed of 1516 murine leukemia virus, 385 pseudorabies virus, and 410 reovirus type 3 virus filtrations. These data provide clear evidence that will help supplement both internal and industry-wide initiatives focused on using prior knowledge for the creation of modular claims for small virus retentive filters and allow better allocations of resources typically spent on potentially unnecessary studies.

Interaction between nectin-1 and the human natural killer cell receptor CD96.

Regulation of Natural Killer (NK) cell activity is achieved by the integration of both activating and inhibitory signals acquired at the immunological synapse with potential target cells. NK cells express paired receptors from the immunoglobulin family which share common ligands from the nectin family of adhesion molecules. The activating receptor CD226 (DNAM-1) binds to nectin-2 and CD155, which are also recognized by the inhibitory receptor TIGIT. The third receptor in this family is CD96, which is less well characterized and may have different functions in human and mouse models. Human CD96 interacts with CD155 and ligation of this receptor activates NK cells, while in mice the presence of CD96 correlates with decreased NK cell activation. Mouse CD96 also binds nectin-1, but the effect of this interaction has not yet been determined. Here we show that human nectin-1 directly interacts with CD96 in vitro. The binding site for CD96 is located on the nectin-1 V-domain, which comprises a canonical interface that is shared by nectins to promote cell adhesion. The affinity of nectin-1 for CD96 is lower than for other nectins such as nectin-3 and nectin-1 itself. However, the affinity of nectin-1 for CD96 is similar to its affinity for herpes simplex virus glycoprotein D (HSV gD), which binds the nectin-1 V-domain during virus entry. The affinity of human CD96 for nectin-1 is lower than for its known activating ligand CD155. We also found that human erythroleukemia K562 cells, which are commonly used as susceptible targets to assess NK cell cytotoxicity did not express nectin-1 on their surface and were resistant to HSV infection. When expressed in K562 cells, nectin-1-GFP accumulated at cell contacts and allowed HSV entry. Furthermore, overexpression of nectin-1-GFP led to an increased susceptibility of K562 cells to NK-92 cell cytotoxicity.

Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination.

Zika virus (ZIKV) has recently emerged as a pandemic associated with severe neuropathology in newborns and adults. There are no ZIKV-specific treatments or preventatives. Therefore, the development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins. Here we demonstrate that a single low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) encoding the pre-membrane and envelope glycoproteins of a strain from the ZIKV outbreak in 2013 elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 µg of nucleoside-modified ZIKV mRNA-LNP protected mice against ZIKV challenges at 2 weeks or 5 months after vaccination, and a single dose of 50 µg was sufficient to protect non-human primates against a challenge at 5 weeks after vaccination. These data demonstrate that nucleoside-modified mRNA-LNP elicits rapid and durable protective immunity and therefore represents a new and promising vaccine candidate for the global fight against ZIKV.

Epigallocatechin-3-gallate rapidly remodels PAP85-120, SEM1(45-107), and SEM2(49-107) seminal amyloid fibrils.

Semen harbors amyloid fibrils formed by proteolytic fragments of prostatic acid phosphatase (PAP248-286 and PAP85-120) and semenogelins (SEM1 and SEM2) that potently enhance HIV infectivity. Amyloid but not soluble forms of these peptides enhance HIV infection. Thus, agents that remodel these amyloid fibrils could prevent HIV transmission. Here, we confirm that the green tea polyphenol, epigallocatechin-3-gallate (EGCG), slowly remodels fibrils formed by PAP248-286 termed SEVI (semen derived enhancer of viral infection) and also exerts a direct anti-viral effect. We elucidate for the first time that EGCG remodels PAP85-120, SEM1(45-107), and SEM2(49-107) fibrils more rapidly than SEVI fibrils. We establish EGCG as the first small molecule that can remodel all four classes of seminal amyloid. The combined anti-amyloid and anti-viral properties of EGCG could have utility in preventing HIV transmission.

Repurposing Hsp104 to Antagonize Seminal Amyloid and Counter HIV Infection.

Naturally occurring proteolytic fragments of prostatic acid phosphatase (PAP248-286 and PAP85-120) and semenogelins (SEM1 and SEM2) form amyloid fibrils in seminal fluid, which capture HIV virions and promote infection. For example, PAP248-286 fibrils, termed SEVI (semen-derived enhancer of viral infection), can potentiate HIV infection by several orders of magnitude. Here, we design three disruptive technologies to rapidly antagonize seminal amyloid by repurposing Hsp104, an amyloid-remodeling nanomachine from yeast. First, Hsp104 and an enhanced engineered variant, Hsp104(A503V), directly remodel SEVI and PAP85-120 fibrils into non-amyloid forms. Second, we elucidate catalytically inactive Hsp104 scaffolds that do not remodel amyloid structure, but cluster SEVI, PAP85-120, and SEM1(45-107) fibrils into larger assemblies. Third, we modify Hsp104 to interact with the chambered protease ClpP, which enables coupled remodeling and degradation to irreversibly clear SEVI and PAP85-120 fibrils. Each strategy diminished the ability of seminal amyloid to promote HIV infection, and could have therapeutic utility.

A molecular tweezer antagonizes seminal amyloids and HIV infection.

Semen is the main vector for HIV transmission and contains amyloid fibrils that enhance viral infection. Available microbicides that target viral components have proven largely ineffective in preventing sexual virus transmission. In this study, we establish that CLR01, a 'molecular tweezer' specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils. Moreover, CLR01 abrogates semen-mediated enhancement of viral infection by preventing the formation of virion-amyloid complexes and by directly disrupting the membrane integrity of HIV and other enveloped viruses. We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism. CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself. These combined anti-amyloid and antiviral activities make CLR01 a promising topical microbicide for blocking infection by HIV and other sexually transmitted viruses.

Broad-spectrum antibiotic or G-CSF as potential countermeasures for impaired control of bacterial infection associated with an SPE exposure during spaceflight.

A major risk for astronauts during prolonged space flight is infection as a result of the combined effects of microgravity, situational and confinement stress, alterations in food intake, altered circadian rhythm, and radiation that can significantly impair the immune system and the body's defense systems. We previously reported a massive increase in morbidity with a decrease in the ability to control a bacterial challenge when mice were maintained under hindlimb suspension (HS) conditions and exposed to solar particle event (SPE)-like radiation. HS and SPE-like radiation treatment alone resulted in a borderline significant increase in morbidity. Therefore, development and testing of countermeasures that can be used during extended space missions in the setting of exposure to SPE radiation becomes a serious need. In the present study, we investigated the efficacy of enrofloxacin (an orally bioavailable antibiotic) and Granulocyte colony stimulating factor (G-CSF) (Neulasta) on enhancing resistance to Pseudomonas aeruginosa infection in mice subjected to HS and SPE-like radiation. The results revealed that treatment with enrofloxacin or G-CSF enhanced bacterial clearance and significantly decreased morbidity and mortality in challenged mice exposed to suspension and radiation. These results establish that antibiotics, such as enrofloxacin, and G-CSF could be effective countermeasures to decrease the risk of bacterial infections after exposure to SPE radiation during extended space flight, thereby reducing both the risk to the crew and the danger of mission failure.

Hindlimb suspension and SPE-like radiation impairs clearance of bacterial infections.

A major risk of extended space travel is the combined effects of weightlessness and radiation exposure on the immune system. In this study, we used the hindlimb suspension model of microgravity that includes the other space stressors, situational and confinement stress and alterations in food intake, and solar particle event (SPE)-like radiation to measure the combined effects on the ability to control bacterial infections. A massive increase in morbidity and decrease in the ability to control bacterial growth was observed using 2 different types of bacteria delivered by systemic and pulmonary routes in 3 different strains of mice. These data suggest that an astronaut exposed to a strong SPE during extended space travel is at increased risk for the development of infections that could potentially be severe and interfere with mission success and astronaut health.

Phosphorothioate 2' deoxyribose oligomers as microbicides that inhibit human immunodeficiency virus type 1 (HIV-1) infection and block Toll-like receptor 7 (TLR7) and TLR9 triggering by HIV-1.

Topical microbicides may prove to be an important strategy for preventing human immunodeficiency virus type 1 (HIV-1) transmission. We examined the safety and efficacy of sequence-nonspecific phosphorothioate 2' deoxyribose oligomers as potential novel microbicides. A short, 13-mer poly(T) phosphorothioate oligodeoxynucleotide (OPB-T) significantly inhibited infection of primary peripheral blood mononuclear cells (PBMC) by high-titer HIV-1(Ba-L) and simian immunodeficiency virus mac251 (SIV(mac251)). Continuous exposure of human vaginal and foreskin tissue explants to OPB-T showed no toxicity. An abasic 14-mer phosphorothioate 2' deoxyribose backbone (PDB) demonstrated enhanced anti-HIV-1 activity relative to OPB-T and other homo-oligodeoxynucleotide analogs. When PDB was used to pretreat HIV-1, PDB was effective against R5 and X4 isolates at a half-maximal inhibitory concentration (IC(50)) of <1 µM in both PBMC and P4-R5 MAGI cell infections. PDB also reduced HIV-1 infectivity following the binding of virus to target cells. This novel topical microbicide candidate exhibited an excellent in vitro safety profile in human PBMC and endocervical epithelial cells. PDB also retained activity in hydroxyethylcellulose gel at pH 4.4 and after transition to a neutral pH and was stable in this formulation for 30 days at room temperature. Furthermore, the compound displayed potent antiviral activity following incubation with a Lactobacillus strain derived from normal vaginal flora. Most importantly, PDB can inhibit HIV-1-induced alpha interferon production. Phosphorothioate 2' deoxyribose oligomers may therefore be promising microbicide candidates that inhibit HIV-1 infection and also dampen the inflammation which is critical for the initial spread of the virus.