Examining the interactions of Galahad™ compound with viruses to develop a novel inactivated influenza A virus vaccine.

Galahad™ is a proanthocyanidin complexed with polysaccharides that inactivates viruses and indicates potential for an innovative approach to making protective vaccines. The polysaccharide portion of Galahad™ consists mainly of arabinan and arabinogalactan. In a seven-day toxicity study in rats, it was not toxic even when tested undiluted. Galahad™ inactivated a wide range of DNA and RNA viruses including adenoviruses, corona viruses such as SARS-CoV-2, and influenza viruses. Electron microscopy studies showed that exposure to Galahad™ caused extensive clumping of virions followed by lack of detection of virions after longer periods of exposure. Based on the viral inactivation data, the hypotheses tested is that Galahad™ inactivation of virus can be used to formulate a protective inactivated virus vaccine. To evaluate this hypothesis, infectious influenza A virus (H5N1, Duck/MN/1525/81) with a titer of 105.7 CCID50/0.1 ml was exposed for 10 min to Galahad™. This treatment caused the infectious virus titer to be reduced to below detectable limits. The Galahad™ -inactivated influenza preparation without adjuvant or preservative was given to BALB/c mice using a variety of routes of administration and dosing regimens. The most protective route of administration and dosing regimen was when mice were given the vaccine twice intranasally, the second dose coming 14 days after the primary vaccine dose. All the mice receiving this vaccine regimen survived the virus challenge while only 20% of the mice receiving placebo survived. This suggests that a Galahad™-inactivated influenza virus vaccine can elicit a protective immune response even without the use of an adjuvant. This technology should be investigated further for its potential to make effective human vaccines.

Inhibition of adenovirus serotype 14 infection by octadecyloxyethyl esters of (S)-[(3-hydroxy-2-phosphonomethoxy)propyl]- nucleosides in vitro.

On September 22, 2008, a physician on Prince of Wales Island, Alaska, notified the Alaska Department of Health and Social Services (ADHSS) of an unusually high number of adult patients with recently diagnosed pneumonia (n = 10), including three persons who required hospitalization and one who died. ADHSS and CDC conducted an investigation to determine the cause and distribution of the outbreak, identify risk factors for hospitalization, and implement control measures. This report summarizes the results of that investigation, which found that the outbreak was caused by adenovirus 14 (Ad14), an emerging adenovirus serotype in the United States that is associated with a higher rate of severe illness compared with other adenoviruses. Among the 46 cases identified in the outbreak from September 1 through October 27, 2008, the most frequently observed characteristics included the following: male (70%), Alaska Native (61%), underlying pulmonary disease (44%), aged > or = 65 years (26%), and current smoker (48%). Patients aged > or = 65 years had a fivefold increased risk for hospitalization. The most commonly reported symptoms were cough (100%), shortness of breath (87%), and fever (74%). Of the 11 hospitalized patients, three required intensive care, and one required mechanical ventilation. One death was reported. Ad14 isolates obtained during the outbreak were identical genetically to those in recent community-acquired outbreaks in the United States which suggests the emergence of a new, and possibly more virulent Ad14 variant. Clinicians should consider Ad14 infection in the differential diagnosis for patients with community-acquired pneumonia, particularly when unexplained clusters of severe respiratory infections are detected.

The Iminosugar UV-4 is a Broad Inhibitor of Influenza A and B Viruses ex Vivo and in Mice.

Iminosugars that are competitive inhibitors of endoplasmic reticulum (ER) α-glucosidases have been demonstrated to have antiviral activity against a diverse set of viruses. A novel iminosugar, UV-4B, has recently been shown to provide protection against lethal infections with dengue and influenza A (H1N1) viruses in mice. In the current study, the breadth of activity of UV-4B against influenza was examined ex vivo and in vivo. Efficacy of UV-4B against influenza A and B viruses was shown in primary human bronchial epithelial cells, a principal target tissue for influenza. Efficacy of UV-4B against influenza A (H1N1 and H3N2 subtypes) and influenza B was demonstrated using multiple lethal mouse models with readouts including mortality and weight loss. Clinical trials are ongoing to demonstrate safety of UV-4B and future studies to evaluate antiviral activity against influenza in humans are planned.

Protease inhibitors targeting coronavirus and filovirus entry.

In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coronavirus can use serine proteases localized at the cell surface, for their activation. However, it is currently unclear which protease(s) facilitate viral spread in the infected host. We report here that the cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(E)-4-methylpiperazine-1-carbonyl]amino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. K11777 is already in advanced stages of development for a number of parasitic diseases, such as Chagas disease, and has proven to be safe and effective in a range of animal models. K11777 inhibition of SARS-CoV and Ebola virus entry was observed in the sub-nanomolar range. In order to assess whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized K11777-derivative with that of camostat, an inhibitor of TMPRSS2 and related serine proteases. Employing a pathogenic animal model of SARS-CoV infection, we demonstrated that viral spread and pathogenesis of SARS-CoV is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. Camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. Our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of SARS and potentially MERS, while vinyl sulfone-based inhibitors are excellent lead candidates for Ebola virus therapeutics.

Prophylactic and therapeutic testing of Nicotiana-derived RSV-neutralizing human monoclonal antibodies in the cotton rat model.

Severe lower respiratory tract infection in infants and small children is commonly caused by respiratory syncytial virus (RSV). Palivizumab (Synagis(®)), a humanized IgG1 monoclonal antibody (mAb) approved for RSV immunoprophylaxis in at-risk neonates, is highly effective, but pharmacoeconomic analyses suggest its use may not be cost-effective. Previously described potent RSV neutralizers (human Fab R19 and F2-5; human IgG RF-1 and RF-2) were produced in IgG format in a rapid and inexpensive Nicotiana-based manufacturing system for comparison with palivizumab. Both plant-derived (palivizumab-N) and commercial palivizumab, which is produced in a mouse myeloma cell line, showed protection in prophylactic (p < 0.001 for both mAbs) and therapeutic protocols (p < 0.001 and p < 0.05 respectively). The additional plant-derived human mAbs directed against alternative epitopes displayed neutralizing activity, but conferred less protection in vivo than palivizumab-N or palivizumab. Palivizumab remains one of the most efficacious RSV mAbs described to date. Production in plants may reduce manufacturing costs and improve the pharmacoeconomics of RSV immunoprophylaxis and therapy.

Chimeric severe acute respiratory syndrome coronavirus (SARS-CoV) S glycoprotein and influenza matrix 1 efficiently form virus-like particles (VLPs) that protect mice against challenge with SARS-CoV.

SARS-CoV was the cause of the global pandemic in 2003 that infected over 8000 people in 8 months. Vaccines against SARS are still not available. We developed a novel method to produce high levels of a recombinant SARS virus-like particles (VLPs) vaccine containing the SARS spike (S) protein and the influenza M1 protein using the baculovirus insect cell expression system. These chimeric SARS VLPs have a similar size and morphology to the wild type SARS-CoV. We tested the immunogenicity and protective efficacy of purified chimeric SARS VLPs and full length SARS S protein vaccines in a mouse lethal challenge model. The SARS VLP vaccine, containing 0.8 µg of SARS S protein, completely protected mice from death when administered intramuscular (IM) or intranasal (IN) routes in the absence of an adjuvant. Likewise, the SARS VLP vaccine, containing 4 µg of S protein without adjuvant, reduced lung virus titer to below detectable level, protected mice from weight loss, and elicited a high level of neutralizing antibodies against SARS-CoV. Sf9 cell-produced full length purified SARS S protein was also an effective vaccine against SARS-CoV but only when co-administered IM with aluminum hydroxide. SARS-CoV VLPs are highly immunogenic and induce neutralizing antibodies and provide protection against lethal challenge. Sf9 cell-based VLP vaccines are a potential tool to provide protection against novel pandemic agents.

Single-dose intranasal administration with mDEF201 (adenovirus vectored mouse interferon-alpha) confers protection from mortality in a lethal SARS-CoV BALB/c mouse model.

Interferons (IFNs) are a first line of defense against viral infection. Herein we describe the use of an adenovirus vectored mouse IFN alpha gene (mDEF201) as a prophylactic and treatment countermeasure in a SARS-CoV-infected BALB/c mouse model. Complete survival protection was observed in mice given a single dose of mDEF201 administered intranasally 1, 3, 5, 7, or 14 days prior to lethal SARS-CoV challenge (p<0.001), and body weights of these treated mice were unaffected by the challenge. In addition, low doses of mDEF201 protected lungs in a dose dependent manner as measured by a reduction in gross pathology. Intranasal treatment with mDEF201 ranging from 10(6) to 10(8)PFU significantly protected mice against a lethal SARS-CoV infection in a dose dependent manner up to 12h post infection (p<0.001). The data suggest that mDEF201 is a new class of antiviral agent further development as treatment for SARS-CoV infections.

Targeting Robo4-dependent Slit signaling to survive the cytokine storm in sepsis and influenza.

The innate immune system provides a first line of defense against invading pathogens by releasing multiple inflammatory cytokines, such as interleukin-1beta and tumor necrosis factor-alpha, which directly combat the infectious agent and recruit additional immune responses. This exuberant cytokine release paradoxically injures the host by triggering leakage from capillaries, tissue edema, organ failure, and shock. Current medical therapies target individual pathogens with antimicrobial agents or directly either blunt or boost the host's immune system. We explored a third approach: activating with the soluble ligand Slit an endothelium-specific, Robo4-dependent signaling pathway that strengthens the vascular barrier, diminishing deleterious aspects of the host's response to the pathogen-induced cytokine storm. This approach reduced vascular permeability in the lung and other organs and increased survival in animal models of bacterial endotoxin exposure, polymicrobial sepsis, and H5N1 influenza. Thus, enhancing the resilience of the host vascular system to the host's innate immune response may provide a therapeutic strategy for treating multiple infectious agents.

Induction of interferon-gamma-inducible protein 10 by SARS-CoV infection, interferon alfacon 1 and interferon inducer in human bronchial epithelial Calu-3 cells and BALB/c mice.

BACKGROUND: The pathogenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) is poorly understood. Several mechanisms involving both direct effects on target cells and indirect effects via the immune system might exist. SARS-CoV has been shown in vitro to induce changes of cytokines and chemokines in various human and animal cells. We previously reported that interferon (IFN) alfacon-1 was more active against SARS-CoV infection in human bronchial epithelial Calu-3 cells than in African green monkey kidney epithelial cells on day 3 post-infection. METHODS: In the current study, we first evaluated the efficacy of IFN-alfacon 1 in Calu-3 cells during the first 7 days of virus infection. We then used the two-antibody sandwich ELISA method to detect IFN-gamma-inducible protein 10 (IP-10). We further evaluated the efficacy of antivirals directed against SARS-CoV infection in BALB/c mice. RESULTS: A potent, prolonged inhibition of SARS-CoV replication in Calu-3 cells with IFN-alfacon 1 was observed. Furthermore, IP-10, an IFN-inducible leukocyte chemoattractant, was detected in Calu-3 cells after SARS-CoV infection. Interestingly, IP-10 expression was shown to be significantly increased when SARS-CoV-infected Calu-3 cells were treated with IFN alfacon-1. IP-10 expression was detected in the lungs of SARS-CoV-infected BALB/c mice. Significantly high levels of mouse IP-10 in BALB/c mice was also detected when SARS-CoV-infected mice were treated with the interferon inducer, polyriboinosinic-polyribocytidylic acid stabilized with poly-L-lysine and carboxymethyl cellulose (poly IC:LC). Treatment with poly IC:LC by intranasal route were effective in protecting mice against a lethal infection with mouse-adapted SARS-CoV and reduced the viral lung titres. CONCLUSIONS: Our data might provide an important insight into the mechanism of pathogenesis of SARS-CoV and these properties might be therapeutically advantageous.

Broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family Coronaviridae.

Viruses of the family Coronaviridae have recently emerged through zoonotic transmission to become serious human pathogens. The pathogenic agent responsible for severe acute respiratory syndrome (SARS), the SARS coronavirus (SARS-CoV), is a member of this large family of positive-strand RNA viruses that cause a spectrum of disease in humans, other mammals, and birds. Since the publicized outbreaks of SARS in China and Canada in 2002-2003, significant efforts successfully identified the causative agent, host cell receptor(s), and many of the pathogenic mechanisms underlying SARS. With this greater understanding of SARS-CoV biology, many researchers have sought to identify agents for the treatment of SARS. Here we report the utility of the potent antiviral protein griffithsin (GRFT) in the prevention of SARS-CoV infection both in vitro and in vivo. We also show that GRFT specifically binds to the SARS-CoV spike glycoprotein and inhibits viral entry. In addition, we report the activity of GRFT against a variety of additional coronaviruses that infect humans, other mammals, and birds. Finally, we show that GRFT treatment has a positive effect on morbidity and mortality in a lethal infection model using a mouse-adapted SARS-CoV and also specifically inhibits deleterious aspects of the host immunological response to SARS infection in mammals.

Antiviral activities and phosphorylation of 5-halo-2'-deoxyuridines and N-methanocarbathymidine in cells infected with vaccinia virus.

BACKGROUND: The antipoxviral activities and phosphorylation of N-methanocarbathymidine ([N]-MCT) and four 5-halo-2'-deoxyuridines, namely 5-fluoro-(FdU), 5-chloro-(CldU), 5-bromo-(BrdU), and 5-iodo-(IdU) derivatives, were explored. METHODS: Antiviral activities and nucleoside metabolism were determined in C127I mouse, LLC-MK2 monkey, and A549 human cells infected with thymidine-kinase-containing and -deficient (TK+ and TK-) vaccinia (WR strain) viruses. RESULTS: The antiviral potencies of CldU, BrdU and IdU were increased 16-26-fold in LLC-MK2 cells infected with TK+ compared with TK- virus infections, but enhancement of activity was much less in the other cell lines. (N)-MCT was nearly equally active against TK+ and TK- viruses in the three cell lines. Antiviral activity of FdU was associated with cytotoxicity. Uninfected and infected cells metabolized compounds to mono-, di- and triphosphates. The thymidine, BrdU and IdU triphosphate levels were higher in C127I and LLC-MK2 cells infected with TK+ than with TK- virus. (N)-MCT monophosphate levels were much higher in TK+ virus-infected cells, but without corresponding increases in (N)-MCT triphosphate. Furthermore, TK+ virus infections did not appreciably alter (N)-MCT triphosphate levels in other mouse (L929), monkey (MA-104 and Vero) and human cell lines (A549). Antiviral potency of the compounds was greater in C127I than in LLC-MK2 cells, yet lower intracellular triphosphate levels were found in C127I cells. CONCLUSION: We conclude that viral TK plays an important role in increasing the antiviral potencies of these compounds in some cell lines, but minimally in others. These findings may have implications in treating infected animals with compounds that are dependent upon poxvirus TK for their activation, because viral TK activity may vary greatly due to cell type.

Is the anti-psychotic, 10-(3-(dimethylamino)propyl)phenothiazine (promazine), a potential drug with which to treat SARS infections? Lack of efficacy of promazine on SARS-CoV replication in a mouse model.

Phenothiazine and derivatives were tested for inhibition of SARS-CoV replication. Phenothiazine slightly inhibited SARS-CoV replication in a neutral red (NR) uptake assay. Adding a propylamino group to give promazine reduced virus yields (VYR assay) with an EC(90)=8.3+/-2.8 microM, but without selectivity. Various substitutions in the basic phenothiazine structure did not promote efficacy. Phenazine ethosulfate was the most potent compound by VYR assay (EC(90)=6.1+/-4.3 microM). All compounds were toxic (IC(50)=6.6-74.5 microM) except for phenoxathiin (IC(50)=858+/-208 microM) and 10-(alpha-diethylamino-propionyl) phenothiazine.HCl (IC(50)=195+/-71.2 microM). Consequently, none were selective inhibitors of SARS-CoV replication (SI values <1-3.3 microM). These data portended the poor efficacy of promazine in a SARS-CoV mouse lung replication model. Intraperitoneal treatment with promazine using a prophylactic (-4h)/therapeutic regimen of 1, 10, or 50mg/(kg day) did not reduce virus lung titers at day 3, yet prolonged virus replication to 14 days. Similar therapeutic promazine doses were not efficacious. Thus, promazine did not affect SARS-CoV replication in vitro or in vivo, nor were any other phenothiazines efficacious in reducing virus replication. Therefore, treating SARS infections with compounds like promazine is not warranted.

Interferon alfacon 1 inhibits SARS-CoV infection in human bronchial epithelial Calu-3 cells.

The primary targets for SARS-CoV infection are the epithelial cells in the respiratory and intestinal tract. The angiotensin-converting enzyme 2 (ACE-2) has been identified as a functional receptor for SARS-CoV. ACE-2 has been shown to be expressed at the apical domain of polarized Calu-3 cells. In this report, interferon alfacon 1 was examined for inhibitory activities against SARS-CoV on human lung carcinoma epithelial Calu-3 cell line and the other three African green monkey kidney epithelial cell lines. Interferon alfacon 1 demonstrated significant antiviral activity in neutral red uptake assay and virus yield reduction assay. The data might provide an important insight into the mechanism of pathogenesis of SARS-CoV allowing further development of antiviral therapies for treating SARS infections.

Intranasal Protollin-formulated recombinant SARS S-protein elicits respiratory and serum neutralizing antibodies and protection in mice.

The feasibility of developing a prophylactic vaccine against SARS was assessed by comparing the immune responses elicited by immunizing mice with a recombinant SARS spike glycoprotein (S-protein) formulated with different adjuvants, given by different routes. In both young and aged mice, an intranasal Protollin-formulated S-protein vaccine elicited high levels of antigen-specific IgG in serum, comparable to those elicited by an intramuscular Alum-adsorbed S-protein vaccine. Serum antibodies were shown to be virus neutralizing. Intranasal immunization of young mice with the Protollin-formulated vaccine elicited significant levels of antigen-specific lung IgA in contrast to mice immunized with the intramuscular vaccine in which no antigen-specific lung IgA was detected. Following live virus challenge of aged mice, no virus was detected in the lungs of intranasally immunized mice, in contrast to intramuscularly immunized mice whose lung virus titers were comparable to those observed in control mice.

Neutralizing viruses in suspensions by copper oxide-based filters.

We report the capacity of copper oxide-containing filters to reduce infectious titers of a panel of viruses spiked into culture media. Enveloped, nonenveloped, RNA, and DNA viruses were affected, suggesting the possibility of using copper oxide-containing devices to deactivate a wide spectrum of infectious viruses found in filterable suspensions.

Inhibitors of measles virus.

Measles virus (MV) infections have been almost eradicated in some industrialized nations. However, MV continues to cause severe disease and mortality in the world and is responsible for clusters of exogenous-borne disease in essentially disease-free countries. Because of the ebb and flow of immunization campaigns, especially in the poverty-stricken and war-torn Third World, and the ominous potential for severe disease and mortality, it is vital that research for discovery of therapeutic countermeasures should continue. To that end, a number of compounds have been evaluated for efficacy in vitro and in animal models, and several therapeutic modalities have been tested in the clinic. The only current therapies used in the clinic include ribavirin administered orally or intravenously, alone or in combination with immune serum globulin; these therapies have demonstrated variable efficacy. Therefore, drug discovery efforts have been launched to supplement the existing treatments for MV infections. Antisense molecules, adenosine and guanosine nucleosides, including ring-expanded 'fat' nucleoside analogues, brassinosteroids, coumarins, peptide inhibitors, modulators of cholesterol synthesis and a variety of natural products have been screened for efficacy and toxicity both in vitro and in animals. However, none of these agents has gone into human clinical trials and most will not merit further development due to toxicity concerns and/or low potency. Thus, further research is needed to develop more potent and less toxic drugs that could be used for treating MV infections to supplement the existing MV vaccine campaigns.

Inhibition of severe acute respiratory syndrome-associated coronavirus (SARSCoV) by calpain inhibitors and beta-D-N4-hydroxycytidine.

We evaluated two types of compounds for efficacy in inhibiting SARSCoV replication in vitro: calpain inhibitors (a class of cellular cysteine proteinases) and a number of nucleoside analogues. Cytopathic effect reduction assays visually determined with spectrophotometric verification by neutral red (NR) uptake assay were used to evaluate cytotoxicity and antiviral potency of the compounds. Significantly inhibitory compounds were then evaluated in virus yield reduction assays. Two calpain inhibitors, Val-Leu-CHO (calpain inhibitor VI) and Z-Val-Phe-Ala-CHO (calpain inhibitor III), were the most potent inhibitors of SARSCoV. By virus yield reduction assay, calpain inhibitor VI had a 90% effective concentration (EC90) of 3 microM and calpain inhibitor III had an EC90 of 15 microM. Beta-D-N4-hydroxycytidine was the most selective nucleoside analogue inhibitor with an EC90 of 6 microM by virus yield reduction assay. These compounds or analogues warrant further evaluation as potential therapies for treating SARS or could be used as lead compounds for discovery of more potent SARSCoV inhibitors.

An orally bioavailable oxime ether capsid binder with potent activity against human rhinovirus.

A series of capsid-binding compounds was screened against human rhinovirus (HRV) using a CPE based assay. The ethyl oxime ether 14 was found to have outstanding anti-HRV activity (median IC(50) 4.75 ng/mL), and unlike the equivalent ethyl ester compound 3 (Pirodavir), it has good oral bioavailability, making it a promising development candidate. Compound 14 illustrates that an oxime ether group can act as a metabolically stable bioisostere for an ester functionality.

Interferon-alpha. Amarillo Biosciences.

Amarillo Biosciences is developing low-dose oral interferon-alpha (IFNalpha; Veldona) as a potential treatment for primary Sjøgren's syndrome, oral mucositis in cancer patients, hepatitis B and C virus (HBV and HCV) infections, and bone marrow disorders. The company is also developing a topical formulation of IFNalpha for the potential treatment of genital warts. The product is registered in Ghana for the treatment of HBV infection. In October 2001, Amarillo licensed rights to its low-dose oral IFNalpha to Atrix Laboratories for the Orphan indications of oral papillomavirus and Behçet's disease.