Partial Listing of Experimental Viral Models Utilized in the Evaluation of the Proposed Antiviral Prophylactic Nasal Gel.
Viral infections are a significant cause of morbidity and mortality in humans throughout the world. However, modern medicine has a very limited ability to prevent viral diseases. While traditional vaccination strategies have been highly successful against a subset of viruses, the antigenic variation of viruses as well as the shear number of viral pathogens has limited the efficacy of this approach.
This observation is exemplified by the finding that while most common respiratory infections are caused by Rhinoviruses, Coronaviruses, Adenoviruses and Orthomyxoviruses, a number of other viral families are also frequently implicated. Indeed, over 300 serologically distinct viruses are known to cause the pathology associated with the
However, the safe, low cost, low technology, and non-toxic bioengineering of the terminally differentiated nasal pharyngeal epithelial host cells may provide a radically new antiviral prophylactic approach that gives rise to a transient, broad-spectrum, prophylaxis against virally transmitted respiratory infections (Figure 1). [ McCoy & Scott, 2005, Sutton & Scott, 2010] This polymer-based technology is derivative of the polymer-based “immunocamouflage” technology of blood cells being actively developed within the Canadian Blood Services to reduce the risk of transfusion reactions and alloimmunization to donor red blood cells. [Scott et al., 1997, Scott & Murad, 1998, Murad et al., 1999a , Murad et al., 1999b, Bradley et al., 2001, Bradley et al., 2002, Bradley & Scott, 2007, Rossi et al., 2010b]
As schematically shown in Figure 1, the non-toxic bioengineering of the nasal cavity attenuates or prevents viral respiratory infections at the primary site of infection - the nasopharyngeal cell surface of the upper respiratory tract. Surprisingly to some, the primary mode of viral entry in respiratory diseases is via accidental inoculation of the nasal passage via contaminated hands. As demonstrated in Figure 1A, the initial inoculum (1) is typically
small but, upon successful invasion, replicates and produces progeny virus (2) that invade adjacent cells (3) producing further progeny that may remain in the upper respiratory tract or may progress to the lower respiratory tract (4). [Winther, 2011] However, as shown in Figure 1B, the application of the activated mPEG-gel within the nasal cavity covalently modifies the terminally differentiated epithelial cells producing a physical and charge neutralization barrier preventing viral recognition of known and unknown viral receptors. Consequent to this camouflage, the successful tissue invasion by the initial viral inoculum (1) is significantly reduced and few progeny virus (2) are produced. Subsequent replication cycles (3) are also reduced decreasing the severity or onset of disease in both the upper and lower (4) respiratory tracts. The relative efficacy of the mPEG-antiviral barrier is denoted by the intensity of the blue shading and decreases with distance from the nostril opening.
The antiviral effects of grafted polymer occurs at the epithelial cell membrane-environment interface (Figure 1C). The efficacy of the grafted polymer is dependent upon the size/topography of the viral receptor and the size (molecular weight; m.w.) of the polymer. The size of the polymer governs the Flory radii (
In contrast to traditional vaccine approaches, this novel intranasal antiviral prophylactic provides immediate, albeit transient, protection against a broad spectrum of respiratory viral pathogens. It is these pathogens that can, and do, create massive healthcare emergencies in
2. PEGylation: Inhibition of virus-receptor recognition and binding
Our earlier studies (
Of these polymers, mPEG is the best characterized and is synthesized from poly(ethylene glycol) [HO-(CH2CH2O)n-CH2CH2OH]. [Roberts et al., 2002] The first –OH group is used to covalently attach the PEG-moiety to a linker compound that in turn is used to covalently modify cell membrane proteins. Because the second terminal -OH group of PEG confers some residual chemical reactivity, this is replaced by a –CH3 moiety, to form activated mPEG: CH3-(CH2CH2O)n-CH2CH2-
Importantly, viral binding to its host cell is highly analogous to receptor-ligand interactions. For viral infections to occur, viruses must bind to receptor(s) located on the cell surface (Figure 1C). Hence the immunocamouflage of either the virus or host cell would theoretically inhibit viral invasion and subsequent disease due to both charge camouflage and the steric hindrance resulting from the molecular intra-chain flexibility and rapid mobility of the heavily hydrated PEG chains (Figure 3). Moreover, the global camouflage of the cell surface effectively masks both known and unknown viral receptors resulting in a nonspecific, non-immunological, broad-spectrum antiviral effect. [McCoy & Scott, 2005, Sutton & Scott, 2010]
The biophysical basis of this antiviral effect is schematically shown in Figures 3-4. The PEG layer obscures the inherent electrical charge associated with surface proteins since the charged molecules become buried beneath the viscous, hydrated, neutral PEG layer. [Szleifer, 1997, Satulovsky et al., 2000, Bradley et al., 2002, Bradley & Scott, 2004, Le & Scott, 2010] As schematically shown in Figure 3, the surface of a generic cell may have a net negative charge due to the proteins and carbohydrates present on the cell surface. The
positively charged counter-ions from a bulk aqueous solution migrate and interact with the surface to neutralize the surface charge. This creates a Surface Potential Gradient, with the electric potential being the highest at the surface and decreasing with the distance away from the surface. The Shear Plane (SP) is defined as the region around the surface, where counter-ions behave as if they are physically attached to the cell and roughly approximates a neutral net charge. Polymer grafting alters the location of the shear plane relative to the membrane surface and this change is directly influenced by the size and density of the grafted polymer. As demonstrated in Figure 3A, a low molecular weight polymer (
The highly malleable nature of mPEG polymers results in a broad range of possibilities of enhancing its antiviral effects via the use of both linear or branched molecules over an extraordinarily wide range of molecular weights and grafting densities (Figure 2). Of biologic importance, the absolute effects arising from both the migration of the SP within the Surface Potential Gradient and/or the steric hindrance of viral attachment need only be minor as the non-covalent forces that mediate receptor-ligand interactions are relatively weak and easily disrupted by the biophysical changes mediated by the grafted polymer (Figure 4). Thus, the bioengineering of the nasal pharyngeal epithelial cells with an mPEG nasal gel may provide significant opportunities to attenuate or block viral invasion of the initial viral inoculum, as well as any progeny, thereby reducing the both disease progression and severity.
A critical concern of this approach is the safety (acute and chronic) of the polymer. PEG is generally viewed by the US FDA as a safe compound and is widely used in food, cosmetic and pharmaceutical formulations. Previous studies have demonstrated a lack of toxicity of PEG with polymer lengths greater than 400 Da. Human experiments dating from the late 1940’s demonstrated both orally and intravenously administer PEG (
3. Broad spectrum antiviral prophylaxis
Virally mediated respiratory diseases remain a critical problem for humans despite all our advances in pharmacology and vaccine development over the last 150 years. [Spector, 1995] This is in large part due to the shear number of pathogens (>300 serologically distinct viruses) capable of causing the respiratory pathology associated with the
Importantly, for the intervention to be effective, it does not have to inactivate 100% of a viral threat. Rather, such prophylactic intervention must reduce the viral exposure to sub-infective levels and/or inhibit person-to-person transmission. As shown in Figure 5, all viruses have a threshold infective level. This may range from 1-2 virions per person for an extremely contagious (but not necessarily lethal) virus to several hundred or thousand viral particles in order to cause disease. The biologic objective of an activated mPEG-Gel would be to reduce the successful invasions of a respiratory virus to a level below the infectious dose necessary to cause disease. Moreover, when viewed in the context of ‘
With these key concepts in mind, we have been pursuing the functional utility and formulation of an anti-viral prophylactic polymer gel against a broad range of viruses
However, our studies suggest that the application of the activated polymer gel within the nasal cavity could prove to be a highly effective antiviral prophylactic (Figure 1B). To further explore this prophylactic approach, experimental viruses were chosen to include both enveloped and non-enveloped viruses, receptor and fusion mediated viral invasion, large and small viruses and to be representative of known human respiratory viruses (
|Naked Icosahedral Capsid||70-90 nm||Receptor Mediated Endocytosis||mCAR||4.6 nm|
|Enveloped Helical Capsid||80-160 nm||Fusion||Not identified - other family members use APN||13.5nm (APN)|
|Theiler’s Murine Encephalo-myelitis Virus
|Naked Icosahedral Capsid||28-30 nm||Receptor Mediated Endocytosis||Not identified - other family members use ICAM-1||18.7 nm (ICAM-1)|
|Respiratory Syncytial Virus
|Enveloped Helical Capsid||150-300 nm||Fusion||Not Identified||Unknown|
|Simian Virus 40
|Naked Icosahedral Capsid||45-55 nm||Receptor Mediated Endocytosis||MHC-1||7 nm|
|Murine Homologue of Coxsackie and Adenovirus Receptor (mCAR); Aminopeptidase N (APN); Intracellular Adhesion Molecule-1 (ICAM-1); and Major Histocompatibility Molecule-1 (MHC-1). SV40 is not a respiratory virus but is a well characterized experimental model suitable for experimental study. Receptor size references as follows: MHC-1 [Bjorkman
3.1. Antiviral nasal gel
To determine if an activated mPEG-gel could inhibit viral invasion and proliferation,
To determine if viruses were amenable to PEGylation by the chemistry noted in Figure 2, an analysis of envelope and capsid proteins was done. The amino acid composition and sequence of the envelope and capsid proteins suggested that all viruses had suitable targets for lysine based grafting. For example, analysis of a number of human RSV isolates demonstrate that both the F and G capsid glycoproteins are lysine rich and provide an excellent substrate for direct viral modification with mPEG. Infection assays confirmed this analysis. These experiments demonstrated that direct covalent modification of the virion by mPEG resulted in an almost complete abrogation of viral infection and proliferation [Sutton & Scott, 2010]. However some significant variability in the efficacy of small and large polymers was noted. As shown in Figure 7A, at low grafting concentrations (≤ 2 mM) small molecular weight mPEG demonstrated superiority over large polymers when modifying the virus (RSV) directly. At these low virus:mPEG ratios the improved efficacy of the 2 kDa polymer may be partially due to the distribution of virus specific proteins within the viral envelope. On the RSV virion the G (attachment) and F (fusion) proteins form glycoprotein spikes that are 6-10 nm apart and extent 11-20 nm from the virion surface. Thus, given the distance between glycoprotein spikes on the RSV virion, grafting of 2 kDa mPEG will likely result in the direct modification of a greater number of glycoprotein spikes. Grafting of 20 kDa mPEG will likely result in fewer spikes being modified as the initially bound mPEG will exclude other polymer chains from grafting to spikes in close proximity. At higher grafting concentrations, the self-exclusion effect of grafted mPEG is partially overcome resulting in high levels of surface modification and prevention of viral binding to the host cell. Importantly, soluble mPEG (
While the utility of direct viral modification might be beneficial in blood banking environments bereft of modern viral detection methodology, this application would have little or no practical value in the prevention of respiratory disease. To this end, the
In contrast to Figure 7A, small chain polymers were completely ineffective when used to directly modify the RSV host cell – even at very high grafting concentrations (Figure 7B). This suggests that the unknown membrane receptor for RSV extends well away from the membrane surface and the 2 kDa polymer is unable to effectively camouflage it. However, cellular grafting of small polymers may provide partial protection against some viruses. As shown in Figure 8, low molecular weight mPEG (3.4 kDa) when grafted to host cells does provide anti-viral protection over multiple logs of viral challenge when using SV40. Even at very high levels SV40 challenge (
The observed relationship between polymer molecular weight and the physical size of the grafting target was consistently observed across multiple viral models. Further analysis of the relationship was examined using a latex bead – plasma adsorption model to which mPEGs of various molecular weights were grafted (Figure 9). Using small latex beads (1.2 µm), short chain polymers were more effective as noted by the decreased amount of adsorbed fluorescently labeled plasma protein. In contrast, with large 8 µm latex beads (or 8µm RBC) large chain polymers are most effective at preventing the adsorption of the fluorescently labeled plasma. In agreement with this finding, PEGylation of viruses (0.02-200 µm) also demonstrate that short chain polymers (2 kDa) are more effective at preventing viral invasion and infection while large chain polymers provide superior protection when grafted to host cells (
To determine if a nasal gel would adequately cover the tissue of interest,
Indeed, studies using a pigmented nasal gel demonstrated that a gel can provide protection to both the upper and lower respiratory tract in a Guinea Pig model of respiratory Syncytial Virus (RSV). Dissection of the Guinea Pig nasal cavity demonstrated uniform staining of the cavity surface. Moreover, as shown in Figure 10, application of a PEG-based gel containing India Ink resulted in significant protection even within the lung tissue.
To date, we have demonstrated that the covalent grafting of mPEG to virus itself or to the host/target cell results in a highly effective broad-spectrum antiviral prophylaxis. Consequent to the immunocamouflage of the host cell, viral entry and propagation by members the
3.2. Alternative preventative or therapeutics approaches
Current methods to prevent respiratory viral disease range from homeopathic drug treatments (
Potential pharmacological means of preventing viral infections are to block either viral entry, or proliferation, within the host cell. However again there are no United States FDA-approved
Thus, patients typically turn to over-the-counter (OTC) cough and cold remedies which focus on the relief of symptoms associated with the common cold. Currently in North America alone an estimated $3-6 billion (US) is spent on symptom relief. A few OTC compounds do attempt to prevent or, after infection, attenuate viral infection. These commercial products include compounds such as ZICAM Nasal Gel (active ingredient: zinc gluconate) and ColdFX (active ingredient: proprietary natural extract containing poly-furanosyl-pyranosyl-saccharides in a concentration of greater than 80%). [Hirt et al., 2000, McElhaney et al., 2006] For drug efficacy, ZICAM must be applied intranasally every 2-4 hours. However, Zicam’s proven mode of action is highly specific and, in laboratory studies, only prevents infection by those rhinoviruses that use ICAM1 as the viral receptor. [Bella & Rossmann, 1999] Hence, it is completely ineffective against the majority of cold viruses that do not use ICAM1 (
Other experimental approaches have also been investigated but are also are highly specific to single viral strains and/or families. For example, two other experimental methods of preventing rhinoviral infection have been studied which, like ZICAM, targets the rhinovirus:ICAM-1 interaction. These are the intranasal application of soluble Intracellular Adhesion Molecule (sICAM) or anti-ICAM-1 antibodies (rhinovirus receptor murine monoclonal antibody; RRMA). Like ZICAM, these experimental drugs require intranasal application every 2-4 hours. [Marlin et al., 1990, Huguenel et al., 1997, Turner et al., 1999] Both of these drugs do demonstrate some
Thus, the antiviral prophylactic polymer nasal gel illustrated here represents the only broad spectrum antiviral prophylactic agent described to date. Application of the activated nasal gel is uncomplicated and requires a minimal (~3-5 minutes) application time to effectively camouflages known and unknown viral receptors. After application, the individual then simply “blows their nose” into a tissue to remove any residual carrier fluid. Current data suggests that a single daily application provides maximal protection and retains significant efficacy for up to 60 hours. A brief comparison of a cross sample of the above approaches with our proposed antiviral nasal gel is shown in Table 2A/B.
|Lack of Inherent
|Longevity of Protection by a Single Application||
||2-4 Hr*||2-4 Hr*||2-4 Hr*||**|
a Anti-ICAM Antibody; +/- Highly Strain Specific; * Only against Rhinoviruses utilizing ICAM-1 as viral receptor; ** Protection if generated, can last weeks to years.
3.3. Non-respiratory antiviral applications
Are there other applications for an antiviral prophylactic gel? Humans are beset by a host of viral pathogens and common to all of these pathogens is the need to gain entry into its target cell in order to proliferate. Not all viruses or modes of entry (
In both male-female and male-male transmission, the HIV virus typically enters the host via Langerhans cells, a resident epidermal dendritic cell, dermal dendritic cells, and/or resident CD4+ lymphocytes all of which are present within the mucosal and sub-epithelial tissues of the anogenital region (Figure 11A). Viral entry is gained primarily via two receptors located on the surface of the cell: CD195 (CCR5) and CD184 (CXCR4). [Piguet & Steinman, 2007] If dendritic (antigen presenting) cells capture the virus, it is either endocytosed or can be transferred directly to CD4+ CD195+ T lymphocyte via an infection synapse. Proliferation of the virus within T cells is followed by infection of additional lymphocytes via either CD184 or CD195. Importantly, all of these events are analogous to cellular invasion by respiratory viruses and amendable to disruption by grafted polymer. In a model analogous to spermicide or lubricant, an activated polymer gel could be applied either vaginally or anally prior to at-risk behavior creating an antiviral barrier on the epithelial cell surfaces of the mucosal tissue (Figure 11B) and preventing viral binding via the biophysical mechanisms already described. As shown in Figure 12, proof-of-concept studies clearly demonstrate that grafting of mPEG to CD184 and CD195 positive cells inhibits recognition of these markers by high affinity antibodies.
Because the risk of HIV transmission per single interaction is relatively low (estimated to be 0.04% for female-to-male; 0.08% for male-to-female; and 1.7% for receptive anal intercourse; Boily et al., 2009), the application of an effective activated mPEG-based mucosally applied antiviral gel would further reduce the risk of transmission. Moreover, PEG itself is a lubricant and would reduce the risk of traumatic tissue injury and microabrasions – known risk factors in HIV transmission. The lubricant effect of the mPEG-gel would be of particular importance with regards to anal transmission as the anal epithelium ranges from a multi- to single-layer epithelial lining prone to tearing (in contrast to the vagina which is a multicellular stratified squamous epithelium and resistant to traumatic injury). This simple anatomical difference in large part underlies the differential transmission risk associated with anal intercourse. Finally, the immunocamouflage of the infected dendritic cells and macrophages would also make the cells less prone to activation by other pathogens due to the camouflage of key surface receptors. In the absence of activation, HIV proliferation within infected cells is greatly diminished decreasing viral shedding.
Indeed, there are several topical microbiocidal gels in clinical trials that aim to prevent against HIV infection either non-specifically (surfactants and acidifying agents) or specifically (viral entry and reverse transcriptase inhibitors). Some surfactants, such as nonoxynol-9, experimentally were shown to result in viral disruption but also exerted toxicity to host cells. Further clinical trials of nonoxynol-9 in HIV endemic areas actually demonstrated increased risk of HIV infection with its use. [Rustomjee et al., 1999, Van Damme et al., 2002, Herold et al., 2011] These studies suggested that the surfactant-induced mucosal injury appeared to mimic the presence of microabrasions already known to increase HIV transmission. Other surfactants such as sodium lauryl sulfate (invisible condom) disrupt enveloped and non-enveloped viruses and are associated with decreased (relative to nonoxynol-9) mucosal toxicity. [Howett & Kuhl, 2005, Mbopi-Keou et al., 2010] In contrast to surfactants, the mPEG antiviral gel is of low toxicity to cells while simultaneously providing significant camouflage of the mucosal surface thereby preventing or decreasing the initial viral interaction with resident dendritic cells and T lymphocytes.
The safe, low cost, low technology, non-toxic, and transient bioengineering of the terminally differentiated nasal pharyngeal epithelial host cells may provide a radically new antiviral prophylactic approach. Moreover, this approach may be applicable to other non-respiratory viruses in which a polymer gel can be applied. This antiviral protection arises from the ability of the grafted polymer to directly impede cellular invasion and subsequent proliferation thus abrogating the disease process at its initial stage. Moreover, this technology is effective against: 1) enveloped and non-enveloped viruses; 2) receptor-mediated and fusiogenic viruses; 3) small and large viruses; 4) DNA and RNA viruses; and 5) viruses with known and unknown viral receptors.
The envisioned use of this prophylactic technology is via a simple intranasal gel application in at risk individuals in acute, high transmissibility, environments (
Application of the activated nasal gel is surprisingly uncomplicated and requires a minimal (~3-5 minutes) application time to the host (
Respiratory pathogens can, and do, create massive healthcare emergencies in at risk populations. The development of this inexpensive, easy to use intranasal antiviral gel would be of significant health and economic benefit to both the at risk individual and to governmental and health agencies addressing respiratory disease crises. Importantly, the term ‘at risk’ is relative and may range from environmental or political upheavals to more mundane environments such as air travel and visiting grandchildren.
Bella J. Rossmann M. G. 1999Review: rhinoviruses and their ICAM receptors. 128(1), 69-74.
Bjorkman P. J. Saper M. A. Samraoui B. Bennett W. S. Strominger J. L. Wiley D. C. 1987Structure of the human class I histocompatibility antigen, HLA-A2. 329(6139), 506-512.
Block K. I. Mead M. N. 2003Immune system effects of echinacea, ginseng, and astragalus: a review. 2(3), 247-267.
Boily M. C. Baggaley R. F. Wang L. Masse B. White R. G. Hayes R. J. Alary M. 2009Heterosexual risk of HIV-1 infection per sexual act: systematic review and meta-analysis of observational studies. 9(2), 118-129.
Bradley A. J. Murad K. L. Regan K. L. Scott M. D. 2002Biophysical consequences of linker chemistry and polymer size on stealth erythrocytes: size does matter. 1561(2), 147-158.
Bradley A. J. Scott M. D. 2004Separation and purification of methoxypoly(ethylene glycol) grafted red blood cells via two-phase partitioning. 807(1), 163-168.
Bradley A. J. Scott M. D. 2007Immune complex binding by immunocamouflaged [poly(ethylene glycol)-grafted] erythrocytes. 82(11), 970-975.
Bradley A. J. Test S. T. Murad K. L. Mitsuyoshi J. Scott M. D. 2001Interactions of IgM ABO antibodies and complement with methoxy-PEG-modified human RBCs. 41(10), 1225-1233.
Chen A. M. Scott M. D. 2001Current and future applications of immunological attenuation via pegylation of cells and tissue. 15(12), 833-847.
Chen A. M. Scott M. D. 2003Immunocamouflage: prevention of transfusion-induced graft-versus-host disease via polymer grafting of donor cells. 67(2), 626-636.
Chen A. M. Scott M. D. 2006Comparative analysis of polymer and linker chemistries on the efficacy of immunocamouflage of murine leukocytes. 34(3), 305-322.
Cutler B. Justman J. 2008Vaginal microbicides and the prevention of HIV transmission. 8(11), 685-697.
Damodaran V. B. Fee C. J. Ruckh T. Popat K. C. 2010Conformational studies of covalently grafted poly(ethylene glycol) on modified solid matrices using X-ray photoelectron spectroscopy. 26(10), 7299-7306.
Garg S. Goldman D. Krumme M. Rohan L. C. Smoot S. Friend D. R. 2010Advances in development, scale-up and manufacturing of microbicide gels, films, and tablets. 88 Suppl 1S 19 29.
Hayden F. G. Gwaltney J. M. J. Colonno R. J. 1988Modification of experimental rhinovirus colds by receptor blockade. 9(4), 233-247.
He Y. Chipman P. R. Howitt J. Bator C. M. Whitt M. A. Baker T. S. Kuhn R. J. Anderson C. W. Freimuth P. Rossmann M. G. 2001Interaction of coxsackievirus B3 with the full length coxsackievirus-adenovirus receptor. 8(10), 874-878.
Herold B. C. Mesquita P. M. Madan R. P. Keller M. J. 2011Female Genital Tract Secretions and Semen Impact the Development of Microbicides for the Prevention of HIV and Other Sexually Transmitted Infections. 65(3), 325-333.
Heuberger M. Drobek T. Spencer N. D. 2005Interaction forces and morphology of a protein-resistant poly(ethylene glycol) layer. 88(1), 495-504.
Hirt M. Nobel S. Barron E. 2000Zinc nasal gel for the treatment of common cold symptoms: a double-blind, placebo-controlled trial. 79(10), 778-80, 782.
Howett M. K. Kuhl J. P. 2005Microbicides for prevention of transmission of sexually transmitted diseases. 11(29), 3731-3746.
Huguenel E. D. Cohn D. Dockum D. P. Greve J. M. Fournel M. A. Hammond L. Irwin R. Mahoney J. Mc Clelland A. Muchmore E. Ohlin A. C. Scuderi P. 1997Prevention of rhinovirus infection in chimpanzees by soluble intercellular adhesion molecule-1. 155(4), 1206-1210.
Hussain M. M. Tranum-Jensen J. Noren O. Sjostrom H. Christiansen K. 1981Reconstitution of purified amphiphilic pig intestinal microvillus aminopeptidase. Mode of membrane insertion and morphology. 199(1), 179-186.
Jefferson T. Jones M. Doshi P. Del Mar C. 2009Neuraminidase inhibitors for preventing and treating influenza in healthy adults: systematic review and meta-analysis. 339b5106.
Jun C. D. Carman C. V. Redick S. D. Shimaoka M. Erickson H. P. Springer T. A. 2001Ultrastructure and function of dimeric, soluble intercellular adhesion molecule-1 (ICAM-1). 276(31), 29019-29027.
Kandel R. Hartshorn K. L. 2001Prophylaxis and treatment of influenza virus infection. 15(5), 303-323.
Katriel G. Stone L. 2010Pandemic dynamics and the breakdown of herd immunity. 5(3), e9565.
Kravetz H. M. Knight V. Chanock R. M. Morris J. A. J. O. H. N. S. O. N. K. M. R. I. F. K. I. N. D. D. U. T. Z. J. P. 1961Respiratory syncytial virus. III. Production of illness and clinical observations in adult volunteers. 176657 663.
Le Y. Scott M. D. 2010Immunocamouflage: the biophysical basis of immunoprotection by grafted methoxypoly(ethylene glycol) [mpeg]. 62631 2641.
Lim J. H. Davis G. E. Wang Z. Li V. Wu Y. Rue T. C. Storm D. R. 2009Zicam-induced damage to mouse and human nasal tissue. 4(10), e7647.
Marlin S. D. Staunton D. E. Springer T. A. Stratowa C. Sommergruber W. Merluzzi V. J. 1990A soluble form of intercellular adhesion molecule-1 inhibits rhinovirus infection. 344(6261), 70-72.
Mbopi-Keou F. X. Trottier S. Omar R. F. Nkele N. N. Fokoua S. Mbu E. R. Domingo M. C. Giguere J. F. Piret J. Mwatha A. Masse B. Bergeron M. G. 2010A randomized, double-blind, placebo-controlled Phase II extended safety study of two Invisible Condom formulations in Cameroonian women. 81(1), 79-85.
Mc Coy L. L. Scott M. D. 2005Broad spectrum antiviral prophylaxis: Inhibition of viral infection by polymer grafting with methoxypoly(ethylene glycol). In (T. PF, ed.), Wiley & Sons, Hoboken, NJ, 379 395.
Mc Elhaney J. E. Goel V. Toane B. Hooten J. Shan J. J. 2006Efficacy of COLD-fX in the prevention of respiratory symptoms in community-dwelling adults: a randomized, double-blinded, placebo controlled trial. 12(2), 153-157.
Mi L. Z. Grey M. J. Nishida N. Walz T. Lu C. Springer T. A. 2008Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs. 47(39), 10314-10323.
Miller S. C. Delorme D. Shan J. J. 2009CVT-E002 stimulates the immune system and extends the life span of mice bearing a tumor of viral origin. 7(4), 127-136.
Murad K. L. Gosselin E. J. Eaton J. W. Scott M. D. 1999aStealth cells: prevention of major histocompatibility complex class II-mediated T-cell activation by cell surface modification. 94(6), 2135-2141.
Murad K. L. Mahany K. L. Brugnara C. Kuypers F. A. Eaton J. W. Scott M. D. 1999bStructural and functional consequences of antigenic modulation of red blood cells with methoxypoly(ethylene glycol). 93(6), 2121-2127.
Murata Y. Falsey A. R. 2007Respiratory syncytial virus infection in adults. 12(4 Pt B), 659 670.
(2009) Over-the-counter medications: Zicam nasal products may cause loss of sense of smell. 273
Paulke-Korinek M. Kundi M. Rendi-Wagner P. de Martin A. Eder G. Schmidle-Loss B. Vecsei A. Kollaritsch H. 2011Herd immunity after two years of the universal mass vaccination program against rotavirus gastroenteritis in Austria. Vaccine,
Piguet V. Steinman R. M. 2007The interaction of HIV with dendritic cells: outcomes and pathways. 28(11), 503-510.
Roberts M. J. Bentley M. D. Harris J. M. 2002Chemistry for peptide and protein PEGylation. 54(4), 459-476.
Rossi N. A. Constantinescu I. Brooks D. E. Scott M. D. Kizhakkedathu J. N. 2010aEnhanced cell surface polymer grafting in concentrated and nonreactive aqueous polymer solutions. 132(10), 3423-3430.
Rossi N. A. Constantinescu I. Kainthan R. K. Brooks D. E. Scott M. D. Kizhakkedathu J. N. 2010bRed blood cell membrane grafting of multi-functional hyperbranched polyglycerols. 31(14), 4167-4178.
Rustomjee R. Abdool Karim. Q. Abdool Karim. S. S. Laga M. Stein Z. 1999Phase 1 trial of nonoxynol-9 film among sex workers in South Africa. 13(12), 1511-1515.
Satulovsky J. Carignano M. A. Szleifer I. 2000Kinetic and thermodynamic control of protein adsorption. 97(16), 9037-9041.
Scott M. D. Chen A. M. 2004Beyond the red cell: pegylation of other blood cells and tissues. 11(1), 40-46.
Scott M. D. Murad K. L. 1998Cellular camouflage: fooling the immune system with polymers. 4(6), 423-438.
Scott M. D. Murad K. L. Koumpouras F. Talbot M. Eaton J. W. 1997Chemical camouflage of antigenic determinants: stealth erythrocytes. 94(14), 7566-7571.
Shaffer C. B. Critchfield F. H. 1947The absorption and excretion of the solid polyethylene glycols ("Carbowax" compounds). 36152 157.
Smyth H. E. Carpenter C. P. Shaffer C. B. 1947The toxicity of high molecular weight polyethylene glycols; chronic oral and parental administration. 36157 160.
Spector S. L. 1995The common cold: current therapy and natural history. 95(5 Pt 2), 1133-1138.
Sperber S. J. Hayden F. G. 1989Protective effect of rhinovirus receptor blocking antibody in human fibroblast cells. 12(5-6), 231-238.
Sutton T. C. Scott M. D. 2010The effect of grafted methoxypoly(ethylene glycol) chain length on the inhibition of respiratory syncytial virus (RSV) infection and proliferation. 31(14), 4223-4230.
Szleifer I. 1997Protein adsorption on surfaces with grafted polymers: a theoretical approach. 72(2 Pt 1), 595-612.
Turner R. B. Wecker M. T. Pohl G. Witek T. J. Mc Nally E. St George. R. Winther B. Hayden F. G. 1999Efficacy of tremacamra, a soluble intercellular adhesion molecule 1, for experimental rhinovirus infection: a randomized clinical trial. 281(19), 1797-1804.
Van Damme L. Ramjee G. Alary M. Vuylsteke B. Chandeying V. Rees H. Sirivongrangson P. Mukenge-Tshibaka L. Ettiegne-Traore V. Uaheowitchai C. Karim S. S. Masse B. Perriens J. Laga M. 2002Effectiveness of COL-1492, a nonoxynol-9 vaginal gel, on HIV-1 transmission in female sex workers: a randomised controlled trial. 360(9338), 971-977.
Van Effelterre T. Soriano-Gabarro M. Debrus S. Claire Newbern. E. Gray J. 2010A mathematical model of the indirect effects of rotavirus vaccination. 138(6), 884-897.
Veronese F. M. Harris J. M. 2002Introduction and overview of peptide and protein pegylation. 54(4), 453-456.
Veronese F. M. Mero A. 2008The impact of PEGylation on biological therapies. 22(5), 315-329.
Veronese F. M. Pasut G. 2005PEGylation, successful approach to drug delivery. 10(21), 1451-1458.
Wang M. Guilbert L. J. Li J. Wu Y. Pang P. Basu T. K. Shan J. J. 2004A proprietary extract from North American ginseng (Panax quinquefolium) enhances IL-2 and IFN-gamma productions in murine spleen cells induced by Con-A. 4(2), 311-315.
Winther B. 2011Rhinovirus infections in the upper airway. 8(1), 79 89.
Yale S. H. Liu K. 2004Echinacea purpurea therapy for the treatment of the common cold: a randomized, double-blind, placebo-controlled clinical trial. 164(11), 1237-1241.