The Anti Viral Properties Of Saliva Biology Essay

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Saliva is a potentially important barrier against viral infections; The aim of this essay is to understand the mechanisms surrounding antiviral properties. It is possible that they could be exploited for pharmaceutical purposes in the future.

The main body of my work concentrates on a few salivary proteins and their action for inhibiting HIV, but I will look at a herpes simplex virus and influenza viruses as well, because these viruses are common in the dental setting. Herpes can have long term latent symptoms, which may express themselves periodically in the oral cavity, and influenza is a common viral infection that can infect immunocompromised patients and is a yearly epidemic.

Inhibitory activity of saliva and the components involved and the mechanisms by which they work differ from virus to virus and from gland to gland, along with the variation in salivary protein concentrations, which has an effect on the ability of saliva to inhibit various viruses.

In this essay I am looking at the antiviral properties of saliva, I will concentrate on just a few of the various constituents in saliva, because the body of research available is too large for me to cover in depth every salivary component and its antiviral properties.

My main interest is to understand the mechanisms surrounding antiviral properties. It is possible that they could be exploited for pharmaceutical purposes in the future.

1.1 What is saliva?

Saliva is a glandular secretion that constantly bathes the teeth and oral mucosa. The presence is saliva is vital to the maintenance of healthy oral tissues. Salivary secretion is 'a unidirectional movement of fluid electrolytes and macromolecules into saliva in response to appropriate stimulation'{{59 Edgar,W.M. 2004; }}. Saliva is secreted from salivary glands, which are complex, tubular, exocrine glands that open into the mouth. There are 3 pairs of major salivary glands, the parotid, submandibular and sublingual glands, and 600 -1000 minor salivary glands the oral mucosa.

Salivary glands

The parotid lies between the mastoid process and the posterior border of the mandible and has serous acini. The submandibular gland extends from under the base of the tongue in the posterior part of the floor of the mouth around the posterior margin of the mylohyoid muscle into the

superior part of the neck. Submandibular saliva is produced by both serous and mucous acini, and so is thicker than that of the parotid. The sublingual gland lies under the tongue in the floor of the mouth. It produces very thick saliva because it is a mucous gland. The minor salivary glands scattered around the mouth beneath the mucosa are mucous glands of a similar histological structure to the sublingual gland and secrete spontaneously, apart from the glands of von Ebner, which are serous glands. See fig 1.1 for the structure of the salivary glands.{{59 Edgar,W.M. 2004; }}

The submandibular and sublingual glands make the greatest volume contribution to the resting flow rate, they contain large amounts of mucins. The parotid glands contribute a higher proportion of the stimulated flow, which is serous and watery. Figure 1.2 (below) shows the percentages each gland contributes to whole mouth saliva volume.

Salivary composition

The average salivary composition is shown in Fig. 1.3 [Appendix] as list of the constituents of saliva and their concentrations in unstimulated and stimulated saliva.

Many of the functions and properties of saliva are dependent upon proteins secreted by different glands. The composition of saliva varies according to many factors, varying from person to person and including from which gland it is secreted. Fig 1.4 is a list of factors affecting salivary composition.

Functions of saliva

Saliva has a myriad of functions, summarised in fig1.5 [appendix]. It acts as a lubricant, dissolves some constituents of food and softens the bolus in order to aid swallowing and reduce friction in the oesophagus. The ultimate role however is protection and most of the functions can be classified as such. The main protective factor of saliva is the flow rate, which flushes away food debris as well as oral and exogenous , often noxious microorganisms.{{59 Edgar,W.M. 2004; }}

The various components in saliva relate to different functions, as seen in fig. 1.6 [appendix] Salivary proteins contribute to the formation of the acquired pellicle, which is protective and influences the initial microbial colonisation on the teeth. Saliva contains a buffering bicarbonate ion, which helps prevent acid pH and demineralization, and saliva is supersaturated with calcium and phosphate to aid remineralisation. Mucins and statherins are two glycoproteins which help keep saliva supersaturated, they prevent the spontaneous precipitation of these salts by binding calcium. {{59 Edgar,W.M. 2004; }}

Many components of saliva contribute to its antibacterial, antifungal and antiviral properties.

1.2 What is a virus?

Viruses are very small microorganisms, ranging from about 20nm to 150nm in diameter. They are obligate intracellular parasites, and have a receptor-binding protein for 'docking' to cells to use for reproduction and they contain one species of nucleic acid; DNA or RNA. Viral genomes commonly have 10-15 genes and is protected by a capsid consisting of protein subunits (capsomeres) and sometimes a lipid envelope. The nucleic acid core is often complexed with a capsid, 'nucleocapsid' and the complete virus particle is known as a virion.

Classification of viruses

Viruses are classified according to the characteristics of their nucleic acid, the number of strands, and the polarity. There are some subdivisions of the families on the basis of gene structure, and further divided according to antigenic and biological properties. {{81 Collier,L.H. 2006; }}

Figure 1. 6 shows a list of both DNA and RNA viruses, and the characteristics of the genome. Evidently there are too many viruses for me to look at many in detail, so I will concentrate on a few viruses on which a lot of research has been done and I feel are relevant to the clinical setting; HIV-1, herpes simplex virus and influenza A virus.

2.1 Mucins

Structure

Mucins are salivary mucoglycoproteins with a protein core and a number of attached oligosaccharide chains. The two mucins found in saliva are MUC5B (MG1), a high molecular weight glycoprotein1, and the low molecular weight glycoprotein2 MUC7 (MG2). {{59 Edgar,W.M. 2004; }} MUC5B appears to be exclusive to the oral cavity, as no counterparts in other body fluids have been found.{{67 Amerongen,A.V. 1995; }} The have a multifunctional role and form an integral part of oral biofilms.

Mucins and HIV-1

Habte et al purified crude saliva into MUC5B and MUC7, to determine the possible anhti-HIV properties of salivary mucins. Their results showed 100% HIV-1 replication or infection of the CEM SS cells in their mucin-free control group (HIV-1 plus media). But when isolated with MUC5B or MUC7, the HIV inhibition assay revealed that both the crude saliva and salivary MUC5B and MUC7 mucins inhibited HIV-1 activity by 100%.. these results suggest that the mucins aggregated all the viruses, leaving no free viruses in the filtrates to cause viral infection. {{27 Habte,H.H. 2006; }}

Ergo the media, unlike the mucins did not aggregate the viruses, which resulted in free viruses in the filtrate, which were then infective to the CD4+ CEM SS cells.

The inhibition of HIV-1 by mucins is thought to be by aggregation of the virus into large, insoluble complexes prior to host cell entry {{23 Bergey,E.J. 1994; 82 Malamud,D. 1997;83 Malamud,D. 1993; ; 9 Shugars,D.C. 1999; }}the specificity of this aggregation is not at all clear, as previously Fox et al and Nagashunmugam et al showed little or no potency of saliva agsint a range of viruses. This indicates a possible specificity for salivary mucins to aggregate HIV-1. Another suggested theory is that the negative charges on mucins through sialic acid and sulphate groups could be responsible for specific interactions with receptors on the virus. {{27 Habte,H.H. 2006; }}

Mucins and Influenza

Whole, SM/SL and parotid saliva expressed anti-IAV activity to varying degrees. Large molecular weight mucins and thrombospondins were found to be more potent, but they are present in a higher concentration. However as serous parotid saliva also expressed anti IAV activity, the presence of other proteins in saliva are also playing a role. {{25 White,M.R. 2009;}}

White et al '09 reported that MUC5B had an inhibitory effect on PR-8 and Sendai virus strains of IAV at physiological concentrations (10'200 lg/ml). However MUC7 was not tested, and may have anti IAV activity. The mechanism by which MUC5B inhibits IAV is by presenting a sialic acid ligand for the viral hemagglutinin. -{{25 White,M.R. 2009; }}

2.2 Lactoferrin (Lf)

Lactoferrin is an iron-binding glycoprotein, from the transferrin family. Lf is a single polypeptide chain that is folded into two symmetrical globular lobes (N- and C- lobe) [see Fig 2.1]. These lobes are connected by a 'hinge region', proving the molecule with flexibility. It is these lobes which can each bind one metal atom: Fe 2+ or Fe3+. {{18 van der Strate,B.W. 2001;}}

Lf 'exhibits bacteriostatic and bactericidal activity against diverse pathogenic microorganisms' {{4 Kazmi,S.H. 2006; }}

LF has antiviral properties in its iron-bound or iron-free state (apo-LF) and there is evidence for the two forms existing in human saliva simultaneously.

But evidence shows apo-LF to be more potently anti-viral than when wholly saturated (sat-LF) Iron saturation plays only a small role in the antiviral potency of LF, apo-LF remains more potent than sat-LF. {{18 van der Strate,B.W. 2001; }}

Lactoferrin and HIV

Lf seems to act against HIV-1 at a number of stages of infection. Studies using bovine Lf indicate that Lf can target HIV-1 reverse transcriptase and HIV-1 entry.

The mechanism by which LF acts on HIV-1 is during early infection, probably during absorbtion in to the target cell, because when administered at later times after infection, the antiviral effect diminishes. {{18 van der Strate,B.W. 2001; }}

LF is capable of binding to the GPGRAF domain in the v3 loop of HIV envelope glycoprotein gp120 but to a lesser extent than negatively charged albumins {{18 van der Strate,B.W. 2001; 4 Kazmi,S.H. 2006; }}. It is the negatively charged 'hinge' region on LF that is responsible for binding to gp120. Possibly it is gp120 which is responsible to LF's antiviral effect, because it plays an important role in the adsorption of HIV-1 and entry into target cells by binding to CD4

Lactoferrin and HSV

The progression of HSV-1 infection was found to be inhibited by Lf at various stages of the viral replication cycle, and the degree of saturation played no role.

Valimaa et al '02 found that the inhibition of HSV correlated with the levels of lactoferrin, again, both apo-LF and sat-LF were capable of inhibiting both [HSV-1 and HSV-2] viruses {{18 van der Strate,B.W. 2001; }}

Valimaa suggested that the Lf inhibition mechanism of HSV is by competitively binding to negatively charged heparin sulphate surface GAGs, preventing attachment to the cell. Lf also inhibits post-attachment events like the HSV replication and cell-to-cell spread. {{44 Valimaa,H. 2002; }} Valimaa et al's earlier article also suggested that Lf can affect the HSV reactivation phenotype in the oral region.

Lactoferrin and Influenza

White et al '09 failed to show Lactoferrin having any antiviral effects on Phil82 strain of IAV up to a concentration of 12.5 lg/ml (data not shown). {{25 White,M.R. 2009; }}

a.) Lactoferricin (Lfcin)

Lactoferricin is a peptide obtained from the N-terminal region of the N-lobe from cleavage of lactoferrin. Is has been proven to have anti HIV-1 proerties. Lfcin has anti HSV properties, but it has been shown to be less potent than its native protein, Lactoferrin. {{18 van der Strate,B.W. 2001; }}But van der Strate et al showed that it is ineffective against HCV

LFcin is at least partially responsible for the antimicrobial effect against bacteria and fungi, by the formation of pores in the cell wall of fungi and bacteria, this peptide apparently does not seem to be important for the antiviral effect.

Conclusion

LF is capable of inhibiting replication of a wide range of viruses, and the consensus is that LF prevents infection of the host cell, rather than it inhibiting virus replication after the target cell has become infected

Secretory Leukocyte Protease Inhibitor (SLPI)

(also known as human mucus protease inhibitor / antileukoprotease)

SLPI is a 12-kDa non-glycosylated single polypeptide chain protein, {{48 Lin,A.L. 2004; }} secreted by the serous acinar cells of parotid, SMSL and von Ebner glands. {{57 Franken,C. 1989; 54 Ohlsson,M. 1984; }}

Structure

SLPI forms two domains , a C-terminal and an N-terminal. These are both structurally homologous. The C-terminal has the protease-inhibitory site while the N-terminal stabilizes protease-inhibitor interactions.The N-terminal also contains sites which are essential for heparin binding. The C-terminal is responsible for the killing of bacteria, e.g. Escherichia coli and Staphylococcus aureus, and fungi, e.g. Aspergillus fumigatus and Candida albicans. The acid stability of SLPI allows the protein to remain functional within an acidic environment such as the mouth.{{9 Shugars,D.C. 1999; }}

Functions

SLPI has been shown to have a broad 'antimicrobial activity and has been shown to inhibit candidal, bacterial and viral growth in vitro. SLPI also plays a role in wound healing '. {{48 Lin,A.L. 2004; }}

Other functions include the regulation of elastase found in gingival crevicular Fluid (GCF) in the modulation of periodontal disease process. (Cox, Rodriguez-Gonzalez et al. 2006)

SLPI and HIV

SLPI has been shown to inhibit HIV-1 activity in human adherent monocytes at physiological concentrations (1'10 lg/ml) {{64 Wahl,S.M. 1997; 52 McNeely,T.B. 1995, 1997, ;9 Shugars,D.C. 1999;74 Shugars,D.C. 1998; }}

Mechanism:

Independent experiments have indicated that SLPI acts early in the infection.{{64 Wahl,S.M. 1997; }}

In early infection SLPI does not block interactions between virus and CD4 (cellular receptor). McNeely,T.B. 1995) and in late infection SLPI has no effect on reverse transcription, virus assembly and budding. McNeely,T.B. 1995; {{74 Shugars,D.C. 1998; }}. This indicates that the inhibitor disrupts an early step in the entry process after CD4 binding, and prior to reverse transcription. {{9 Shugars,D.C. 1999;52 52 ; 74 Shugars,D.C. 1998; }}

'This step might include interactions with the chemokine coreceptor or another cellular molecule associated with viral entry, fusion between viral and cellular membranes, or viral-capsid uncoating.'{{ 9 Shugars,D.C. 1999; }}

SLPI appears to interact with a host cell-associated molecule rather than by inactivating the virus directly; In 95, mc Neely observed inhibition of HIV only when the target monocytes were pretreated with SLPI, but There was no inhibition observed when the virus was pretreated. The inhibitor does not bind to the viral envelope glycoprotein, gp120, or its precursor molecule, gp160 (McNeely et al., 1995).

Mc Neely 97 used radiolabelled SLPI to discover that it binds with high specificity to monocytes, in a dose-,pH-, temperature- and time-dependent manner. They also found a monocytic cell-surface protein of approx. 55 kDa SLPI binding protein, which has yet to be described.

SLPI and Influenza

White et al '09 reported that SLPI had no inhibitory effect against the Phil82 strain of IAV at concentrations up to 100 lg/ml)

Although earlier SLPI mediated inhibition has also been reported for influenza and Sendai viruses (Beppu et al., 1997).(Shugars et al 1999)

Conclusion

SLPI plays a major role in the inhibition of HIV-1 and HSV. However no antiviral activity has been demonstrated against IAV either other retroviruses such as murine leukaemia virus and human T-lymphotropic virus (D.C. Shugars, unpublished data) or unrelated viruses such as cytomegalovirus (Wahl et al., 1997a) and herpes simplex virus {{64 Wahl,S.M. 1997; }}'

Proline-rich proteins (PRPs)

PRPs bind to calcium, preventing the spontaneous precipitation of calcium phosphate salts, maintaining the supersaturation of saliva. PRPs are abundant salivary proteins, comprising as much as 25-30%.

PRPs are found in parotid saliva, but has not been reported in SM/SL salivary secretions so the research done by MR Robinovitch et al 2001 is presumed to be specific to parotid saliva at this point. {{70 Robinovitch,M.R. 2001; }}

Studies on the structure

PRPs and HIV

Robinovitch et al have reported certain specific basic PRPs to significantly inhibit both T-tropic and M-tropic lab adapted strains of HIV-1, but the extent to which it does this is yet unknown. The mechanism is also unestablished but involves virus-host cell interaction prior to and the knowledge that PRPs bind specifically to gp120 of HIV-1, indicates that this may be a way for the basic PRPs to block virus interaction with the CD4 receptor or the coreceptors. {{70 Robinovitch,M.R. 2001; }}

Mechanism:

Since the inhibition is detectable with the MAGI assay, its mechanism of action prior to the introduction of the tat gene product into the host cell and may be through the binding of the basic proline-rich proteins to the HIV-1 gp120 coat of the virus.

PRPs and HSV

Heineman & Greenberg established that whole human saliva can prevent the infection of epithelial cells, in vitro, of HSV-1, and that this was a result of the direct action of saliva on the cells rather than the inactivation of virus. {{73 Heineman,H.S. 1980; }} This was confirmed by Bergey et al, who demonstrated the direct inactivation of HSV-1 by human saliva, {{23 Bergey,E.J. 1994; }}.

Gu et al have identified that it is likely PRPs play a large part in the inhibition of HSV and concluded that PRP activity involved direct inactivation of the virus. This was decudced using a solid-phase assay, which demonstrated HSV particle adsorption and that pre-treatment of HSV-1 with PRPs reduced the virus titer. They suggest that this activity arises from the ability of PRPs to bind to selective glycoprotein(s) at the viral cell surface, possibly preventing attachment of the virus and/or penetration mediated by specific receptors at the host cell surface. {{78 Gu,M. 1995; }}

PRPs and Influenza

White et al failed to show PRP antiviral activity on IAV at concentrations up to 100 lg/ml.{{25 White,M.R. 2009; }}

Hypotonicity

As well as these non-specific endogenous proteins inhibiting diverse viruses as various stages,

Several studies have suggested that the hypotonicity of saliva could possibly have an antiviral effect. {{68 Baron,S. 1999; 4 Kazmi,S.H. 2006; }}

within the ductal region of the salivary glands there is significant electrolyte reabsorption, rendering the saliva hypotonic. The final saliva that enters the mouth, on average, contains -25 mEq/L of NaC1 and --2-5 mg/ml protein{{30 Kaplan,M.D. 1993; }}.

Baron et al suggested that hypotonicity plays a large role in the inhibition of HIV-1 by lysis of infected mononuclear leukocytes, and preventing the attachment to mucosal epithelial cells, preventing the replication and transmission of HIV-1. this conclusion has been drawn due to the limited inhibition of HIV by the reported salivary inhibitors (2- to 5-fold) compared with the 10 000-fold or higher inhibition by salivary lysis of infected leukocytes.

Also indicated in the results is the reduction of oral protection in conditions hypotonicity is overcome by ingested isotonic solutions, such as milk and seminal fluid, or severe oral bleeding. Additionally, when acute HIV infection where cell-free HIV is high; or advanced AIDS cell-associated HIV is high; or when saline irrigation of the mouth overcomes hypotonicity of saliva and creates aerosols oral transmission to contacts and health care workers may be increased under these particular circumstances. {{68 Baron,S. 1999; }}

Conclusion

3.1 Differences in the composition of glandular secretions

There was no apparent correlation between individual anti-HIV-1 activity in the three types of saliva; the majority of parotid saliva possessed low antiviral activity, the majority of sm/sl saliva possessed moderate antiviral activity, and the majority of whole saliva possessed high antiviral activity. In addition, in four subjects anti-HIV-1 activity was higher in parotid than in sm/sl saliva and in six subjects the reverse. This indicates that the antiviral activity of whole saliva is unlikely to be derived from parotid saliva and only partially derived from sm/sl saliva, leaving the possibility that minor salivary glands also contribute to the anti-HIV-1 activity.

The minor salivary glands may also play a role, because Kazmi et al found that whole saliva was very highly anti-HIV (2nd most potent fluid in body, colostrum being the most potent). But that franctioned saliva was less inhibitory. SM/SL saliva is the most potent but less so than whole saliva, and parotid saliva, although low in mucins and had low inhibitory effects on HIV still had some effect. {{4 Kazmi,S.H. 2006; }}

Different glands secrete saliva with different components and concentrations. Parotid saliva, which is serous and does not contain mucins also has anti viral properties, indicating that mucins are not the sole inhibitor of HIV, but serous saliva contains proteins which have inhibitory effects.

Submandibular and sublingual saliva are often classed and tested together. However they have significantly different visco-elastic properties and different (glyco) protein concentrations. {{7 Bolscher,J.G. 2002; }} [See results figs 3.1 & 3.2 Appendix]

Bolscher et al acknowledged that cleared whole human saliva contains exudates from the blood and tissues and these may contribute to the ability to inhibit viruses, and therefore collection directly from the glandular orifices is best.

3.2 Variation in individual salivary composition

Not only do the different glands play roles of differing importance in viral inhibitory but salivary composition varies from person to person. This is mediated by gene polymorphism or post translational mechanisations {{7 Bolscher,J.G. 2002; }}

Of course there is a large difference between individuals so studying the genetics could be worthwhile, to discern if there are any detectable genes which predispose an individual to viral susceptibility or natural immune resistance.

The compositions of proteins in saliva vary widely from person to person but are also affected by many other factors excluding the type of gland from which it is secreted. [see appendix fig 1.4]

Each of these factors has a potential effect on the antiviral activity of the saliva.

3.3 Synergy amongst salivary proteins

It is essential to understand that the proteins do not act in isolation, but form many complexes in the mouth, with cofactors and other proteins as was identified by Bolscher et al. So the isolation of specific protein from crude saliva also filters out possibly important heterotypic complexes, which may be potential inhibitory components. For example, t =he presence of such complexes of mucins with amylase, proline-rich protein, staterin and histatins (33), as well as with SLPI (34) has been described.

3.1 Clinical applications

The antiviral properties of saliva are important to investigate as the mechanisms by which they work could ultimately be exploited pharmaceutically in active microbicidal formulations in order to prevent viral transmission or in the generation of new anti-viral drugs or vaccines.

X-ray crystallography is used to determine the tertiary structure of the capsid proteins of viruses, and this knowledge can be used with nucleotide sequencing of the corresponding gene to help chemotherapists design inhibitors that will be to these proteins and use them clinically.

The antiviral properties of saliva are varied and there has been a plethora of research dedicated to the salivary inhibition HIV-1 in vitro, and many salivary factors with potential anhi-HIV-1 properties have been identified, as seen in figure [see fig balh appendix]. However strictly the lab adapted strain, which may lack clinical relevance when looking at epidemiologic studies, As previously the findings by Fox et al. and Nagashunmugam et al. demonstrated little or no potency of saliva against (HSV), HIV-2, simian immunodeficiency virus, Epstein-Barr virus, cytomegalovirus, hepatitis B virus and adenovirus. {{85 Fox,P.C. 1988;84 Nagashunmugam,T. 1997; }}

More emphasis needs to be placed on common diseases. Research needs to be done on viruses like influenza; important to investigate as flu can affect the elderly and immunocomprimised and cause morbidity and mortality, and because of the new strains of avian and porcine flu.

APPENDIX 1

APPENDIX 2

APPENDIX 3

APPENDIX 4

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