Gold Nanoparticle based Lipoidic Oral Vaccine against Respiratory Viruses Including SARS-Cov 2
DrSharadendu Bali1, DrVipin Saini2, DrSuvarna Prasad3
1Professor General Surgery, MMIMSR, Ambala, India.
2 Professor of Pharmacology and Vice-Chancellor, MMDU, Solan,HP, India.
3 Professor and Head, Dept. of Biochemistry, MMIMSR, Mullana, Ambala, India
ABSTRACT
Defective functioning of the primary immune cells is now considered as the most important cause of the delayed response and hyper-inflammatory lung reactions seen in the patients that become seriously ill during infection with SARS CoV-2. These immune defects are seen either in the inefficient recognition of PAMPs by the Dendritic cells or manifested in the delayed and hyper-inflammatory cytokine release by the macrophages in the lung. The innate immune system response is now being postulated as the cri tical factor in the body‘s ability to survive the attack by the virus.
Gold Nanoparticles (GNP) have been studied over past few years because of their ability to act as carriers of vaccines and chemotherapeutic agents. Use of GNP in rats has been noticed to increase lymphocyte cell populations and cause enhanced activation of macrophages and dendritic cells. Gold ash (elemental gold) formulations have been used in India for ages, for medical conditions ranging from fevers and senility to memory loss . If employed with suitable modifications, such formulations can play the significant role of oral vaccine against the vast range of influenza and corona viruses..
Introduction
The SARS-CoV 2 pandemic has highlighted the fact that immunosenescence in the older age group is the underlying cause of the much greater mortality seen in the aged [1] . Ancient Indian Medicine has nano gold-containing remedies for rejuvenation of the aged, and interestingly, for oral vaccination of infants against microbial attack. In recent years, Gold nanoparticles (GNP) have been found to strengthen the innate and adaptive immunity, validating the efficacy claims of these Ayurvedic formulations [2,3]. If delivered orally along-with lipids, the potency of these formulations can be further enhanced by absorption into the intestinal lymphatics, and thence direct delivery to the cardio-pulmonary circulation through the thoracic duct. The portal circulation is bypassed by the lipoidic formulations and hence the first-pass metabolic degradation in the liver is avoided [4]. Since the Novel Coronavirus attacks the lungs, strengthening of the immune response by immune cells of the lung mucosa associated lymphatic tissue (MALT) will have a salutary role in combating this deadly disease.
The formulation described in ancient Indian Medicine Classics to immunize infants (5) is known as Gold Sup (SwarnaPrashan) and has three ingredients – GNP, honey and ghee (clarified butter). Since raw honey contains a large variety of amino acids, nucleotides, glycosylated peptides and oligosaccharides, conjugation of these with GNP occurs spontaneously. Ghee acts as a carrier and forms lipid microspheres containing the GNP- peptide conjugates due to its constituent phospholipids. The ancient Gold Sup formulation with suitable modifications can be utilized to function as vaccine against Covid 19.
2. Properties of Gold Nanoparticles and use in Vaccines
Gold NPs are chemically inert and relatively stable. Gold NPs are also customizable in their size (1nm to 100nm) and shape. In addition, the large surface energy and natural affinity of GNP allows them to easily form conjugates when they contact proteins, peptides, polysachharides or lipids. This conjugation (causing surface modification) is due to both covalent and non-covalent mechanisms (Figure 1).
Fig 1. Gold nano-particles conjugated to functional molecules through thiol (SH) groups.
Synthetic viral sized nanoparticles, including GNPs are taken up by immune cells including Antigen Presenting Cells (APCs) , as these cells are specialised to interact with foreign material.
Numerous studies suggest that GNPs conjugated with functional molecules (called Bio- decorated gold) induce a robust immunity. This is attributable to several viral mimicry features like similar size and multivalent antigen display, since GNP functionalized with hundreds of antigens can emulate the repetitive antigen display on viral particles (6,7). The antibody responses are enhanced by cross-linking of B cell receptors (BCR) and also complement is activated (Figure 2).
Figure 2. Multivalent antigen display on GNPs leads to B cell receptor clustering and signalling, and facilitating internalization of antigen.
Sonia Carabineiro found that GNP in association with viral antigens and engineered virus-like particles elicited strong immunological responses (8, Sonia). It was further discovered that gold NPs conjugated with M2e, the ectodomain of M2 protein present on influenza A virus, induced high levels of antibody response, providing complete protection against influenza virus challenge (9, Tao). Antigen coated GNP can also enhance delivery to lymph nodes by as much as six times, as was demonstrated when coronavirus spike proteins were delivered with 100nm GNP (10).
3. Traditional use and manufacturing process of Calcined Gold ash (Swarnabhasma) : Gold, known as Swarna in Ayurveda, is used as ash in several preparations of Indian medicine for conditions like tuberculosis, anaemia, sterility and muscular dystrophy. It is also used for its anxiolytic and anti-depressant actions. Gold sup( SwarnPrashan ) is a mixture of gold ash, honey and ghee which has been recommended in Ayurveda forinfants and young children, to be started soon after birth (5). This is intended to act like a vaccine against common bacterial and viral infections, and also has miraculous effects on mental ability.
The Ayurvedic method of manufacture of Gold ash, though a time consuming process, is required to be able to impart its proper benefits [11 Thakur&Gudi) . Swarnabhasma (SB) preparation requires specialised incineration methods, and employs sulphur, castor leaf and aloe vera juice, using earthen pots. In a study carried out on mice to evaluate the effects of SB, it was found to increase the count of peritoneal macrophages and the phagocytic index [12 Bajaj, Ahmad].
4. Effects of Gold Nano Particles on Immune Cells
GNP are readily ingested by APCs (Dendritic cells and Macrophages) , and cause a variety of beneficial effects. GNPs functionalized ( by surface modification) with peptides or oligonucleotides have much greater effects as compared to bare particles (13 Bastusa). Surface
modification can be carried out with compounds carrying functional groups, such as cyano (- CN), thiol (-SH), carboxy (COOH) and amino (-NH2) groups, known for their high affinity for gold.
*Effects on Stem cells:
GNP are able to induce the hematopoietic system since their administration has been found to increase total WBC count and haemoglobin content. (3, Nirmala). Ingested GNP can provide mechanical stimuli that trigger off a chain of biological alterations and modulate cell behaviour.
There is increasing evidence to suggest that GNPs modulate the expression of differentiation- relevant genes that promote osteoblast differentiation from mesenchymal stem cells (MSCs). At the same time, GNPs inhibit the differentiation of the latter into adipocytes (14 Wang& Wang)
* Effects on Dendritic cells :
The entry of the virus into DCs can be either by phagocytosis of apoptotic cells infected by virus, or by primary infection with virus. After entry of virus, the DCs rush to the draining lymph nodes and present viral antigens to T cells (Figure 3).
Fig 3. Bio-decorated GNP taken up by Dendritic cells being presented to t-cells in lymph nodes and Peyer‘s patches.
GNPs increase the homing capabilities of DCs, resulting in faster activation of T cells (15 Zhou& Zhang). Gold nano-rods also favour the maturation process of DCs (16, 17 Xu& Liu, kieng&safina) and increase the expression of MHC-II molecules, which present antigens to T lymphocytes (18, Villiers). Thus, by enhancing DC maturation and antigen presentation which is crucial for induction of T cell proliferation, differentiation and activation, all of the latter are promoted by nanoparticles, including GNPs (19, Jia&zhang).
* Effects on NK cells :
12 nm gold nanoparticles have been found recently to induce cell mediated responses accompanied by inflammatory natural killer (NK) cell stimulation (20, Guevel et al)
*Effects on lymphocytes :
In a study carried out on rat mesenteric lymph nodes, oral administration of GNP was found to activate migration, proliferation and differentiation of lymphocytes (21, Zlobina). Lin et al
reported that a high peptide density on the GNP surface can stimulate cytotoxic T lymphocytes better than can free peptides (22). Similarly, GNP functionalized with antigens showed improved presentation of antigen by the MHC-1 complex to CD8+ cells which resulted in increased activation of an epitope-specific immune response by the cytotoxic T cells (23 Cheung et al).
There is also growing evidence suggesting that GNPs increase the production of B lymphocytes, activate B cells and enhance IgG secretion (24 M sharma).
* Effects on Macrophages :
The most significant effect is macrophage polarization and differentiation into anti-inflammatory phenotype. Dendritic cells and macrophages residing in the alveolar epithelium are most important in the fight against viruses till adaptive immunity is established. Any invading pathogen is recognized by PRRs( pattern recognition receptors) present on cell membranes of these immune cells, which engage the Pathogen associated molecular patterns (PAMPs) located on the surfaces of the pathogens. This recognition by PRRs is hampered in Covid-19.
Broncho-alveolar fluid (BALF) from Covid 19 patients has revealed abundance of inflammatory monocyte-derived macrophages and a depletion of tissue-resident alveolar macrophages. The source of the large amounts of cytokines causing Acute Lung Injury ( ALI ) in Covid-19 patients is considered to be monocytes and macrophages ( 25 Diao& Wang). In the initial stages of lung infection (in patients who recover), M1 macrophages secrete large amounts of pro-inflammatory cytokines upon stimulation of PRRs, while inthe resolution phase, phenotype of macrophages shifts to the reparative M2 type. The M2 macrophages secrete anti-inflammatory cytokines which act to subdue the inflammation and promote tissue repair. Thus macrophage polarization and activation play a key role in both initiation and resolution of ALI ( 26 Wang & Zhang).
Mechanical forces have recently been found to affect the polarization of macrophages (27) . Tissue resident macrophages lining the rapidly filling and emptying alveoli are routinely subjected to huge amounts of stretch and mechanical loading, and these forces play a role in the differentiation of these macrophages. Macrophages by nature actively ingest nanoparticles including GNP utilizing macrophage scavenger receptor A, and these nanoparticles affect the shape and loading of the macrophages. In a recent study, it was found that GNP conjugated with IL-4 when injected into injured skeletal muscle of mice resulted in doubling of the percentage of M2a phenotype macrophages and a twofold decrease in M1 macrophages (28 Raimondo) . In another recent study in mice lung macrophages, it was found that peptide-coated Gold NP induced macrophage polarization from inflammatory M1 phenotype toward anti- inflammatory M2 phenotype (26 wang&zhang). Thus, use of GNP can effectively regulate lung inflammation, protecting lungs from injury and promoting inflammation resolution.
*Effects on neutrophils : The extracellular traps of neutrophils (NETs) entrap the GNPs soon after administration. The resultant cell-gold networks may contribute to alerting the immune system by activating DNA receptors like TLR9 ( 2, Luo and Chang. ).
5. Honey and its constituents as capping agents for Gold NPs
Honey is a complex mixture of oligosaccharides, amino acids, polypeptides and proteins. Some of these molecules can bind spontaneously to GNP, increasing their Immunogenicity. The amino acid Proline, which is part of the protein antigen in a large number of viruses, has a significant presence in honey (29 yamada) . Polyreactive natural antibodies normally present in the body, can recognize these proline-containing structures. Repeated exposure to the vast array of conjugated GNP-antigen molecules including proline would result in ―trained immunity‖ of hemopoitic stem cells, dendritic cells and NK cells and also help enlarge the repertoire of T cell receptors.
Raw honey also contains glycoprotein peptides, which is significant because glycosylation increases immunogenicity of the peptides (30 rudd) . An example is Peptidoglycan, which is a well-known PRR agonist that stimulates innate responses. Several studies have shown that glycosylation has a significant influence on uptake of antigenic proteins. (31 Baum, cobb) ).Thus the glycosylated peptides in honey can act as potent stimulants of the immune system. Since the innate immune system is antigen non-specific, activation of this system can lead to priming against a large variety of new and unknown antigens by the mechanism of trained immunity, discussed later.
6. Ghee (Clarified butter)
The milk fat globule membrane of ghee contains phospholipids like glycerophospholipids and sphingolipids, alongwith glycerides, free fatty acids, glycoproteins, cerebrosides, gangliosides, and cholesterol (32 Bezelgues). The phospholipids in ghee act as emulsifying agents and can create liposome-like particles when used in emulsions. These liposomal particles containing the bio-decorated GNP can be taken up by lymphatics, which then directly exposes the immune cells to the conjugated antigens, since a large number of immune cells inhabit the lymphatics.
7. Tinosporacordifolia.
T. cordifolia (TC) is a versatile herb having a wide range of biologically active compounds, and several medicinal properties like, hepatoprotective, immunomodulatory and anti-neoplastic (33 Saha, Ghosh). It has also been found to have significant anti-oxidant, anti-diabetic, anti- inflammatory, anti-microbial activities. A large number of studies also support the immunomodulatory effects of TC. More and Pai found the extract of TC increased the production of nitric oxide in macrophages, and of reactive oxygen species in human neutrophils, thus boosting the phagocytic activity of these cells (34 ). It was also found to increase cytokine production. TC extracts have been documented to enhance B cell differentiation and activate cytotoxic T cells (35 ). Significantly, an immunomodulatory protein from the stem of TC plant was found to be a strong immunogen by itself and also worked as a powerful adjuvant (36).
8. The Oral GNP vaccine - formulation and dosage
Gold sup (see sections 1 & 3) is made simply by mixing gold ash with honey and ghee. The important rule to be followed is to use honey and ghee in unequal proportions by weight. For the purpose of using gold sup as a vaccine against flu viruses, extract of Tinosporacordifolia (TC) is added. TC extracts are available as traditional generic ―Giloy sat‖, which is in powder form, or the distilled essential oil may be used, in amount of 500mg. The final vaccine formulation may then be called as GST (Gold Sup Tinospora).
The formulation of the oral GST vaccine is as below : Ingredients : Gold ash, raw honey, ghee, T cordifolia extract.
Amounts used per dose for adults : 25mg gold ash, 2 gm honey, 8 gm ghee, 500 mg TC extract.
Total weight – 10.525 gm.
Conversely, it can be : 25mg gold ash, 2 gm ghee, 8 gm honey, 500 mg TC extract.
One dose of 10.525 gm of this gold sup would measure 2tsf ( two tea-spoons full).
The important point to note is to thoroughly stir or whip the mixture before use, so as to create a uniform, homogeneous suspension. Vigorous mixing will not only ensure separation of agglomerated gold particles, but also their thorough conjugation with the various amino acids, peptides and saccharides in honey.
Dosing schedule : One dose of GST oral vaccine ( 10.525 gm) on empty stomach, twice a day for 3 days, then twice a week for 4 wks. This schedule may be repeated every three months if further protection is required.
9. Immunological derangements in SARS-CoV-2 and the role of Gold Sup Tinospora (GST) formulation in conferring protection
9a. Pathogenesis of severe SARS-CoV 2 infection
In the severe cases of Covid-19 infection, uncontrolled inflammation in the lungs occurs, as a result of cytokine storm. Some other immunological features that have been clearly identified are
Lymphopenia , particularly reduction in T cells.
The SARS- CoV 2 virus antagonizes interferon responses (37 Tay and Poh) at various stages of the interferon signalling pathway, including by preventing PRR recognition. ( 38, 39Ling&Lu, Yuki) .
Depletion and exhaustion of NK cells. (40 Zheng&Gao 2020).
Impediments in the differentiation and function of DCs, hindering subsequent adaptive immune responses (41 Li& Fan 2020).
Decrement of alveolar macrophages and proliferation of inflammatory monocyte- derived macrophages, leading to so-called hyperinflammation and cytokine storm ( 42 meradmiriam).
T cell exhaustion (39 Yuki )
Increased apoptosis of neutrophils in the lung parenchyma.
9b. Mechanism of Action of GST oral vaccine in priming immunity by “Trained Immunity”
and other processes
The ways in which Gold nano-particles enhance the activity and functioning of most immune cells has already been discussed in section 4 above. Some other significant effects of GNP based oral GST vaccine are described below.
Trained Immunity (TI), considered by many workers to be the reason for the less severe Covid- 19 disease seen in children (43 Cristiani), is a recently coined term that denotes the adaptive characteristics of the innate immune system, signifying its ability to build immunological memory and respond in a generalized manner to reinfection (44 Heijden). Trained innate immunity has been found to reside in monocytes, macrophages and NK cells.
The establishment of innate immune memory is due to epigenetic modifications, which persist, permitting cells to remain in a ‗‗trained‘‘ functional state, facilitating faster and increased responsiveness after re-challenge . This epigenetic modification also takes place at the level of myeloid bone marrow progenitors, resulting in a myelopoiesis bias and formation of monocytes having an increased preparedness to respond to pathogens (45 Mitroulis ). Such trained monocytes, after release from bone marrow, will circulate all through the body including the lungs.
Trained immunity is also induced in the local environments of the tissues. Experimental studies have shown that alveolar macrophages can also be programmed for long-term after infection ( 46netea). The non-specific cross-protection offered by TI enhances immune responses during subsequent infections by same or different microorganisms and this discovery has led to development of Trained-Immunity based Vaccines(47 sanchez).
The effect of GNP on alveolar macrophage polarization towards anti-inflammatory phenotype has already been discussed in section 4. The effect of glycosylated peptides and other antigenic molecules present in honey on stimulating APCs has also been discussed. The activated macrophages and DCs after phagocytosis rush to the lymph nodes(Peyer‘s patches) stimulating the T cells, including NK cells (Figure 3).
Since stimulation of PRRs by peptide-associated-GNP is much more than by peptide alone (48 Chatopadhyay), repeated exposure to the large assortment of immunogenic stimuli (present in GST), leads to better recognition of PAMPs by the PRRs on mononuclear phagocytes (trained immunity), with more efficient IF-g responses and downstream signalling (49 Netea). This primed functional state of trained cells appears to last long, and it can provide protection from causative agents that comes into contact with the host during this period of time (47 sanchez). In Covid-19 patients, MHC class I and II upregulation in Dendritic Cells was found lacking (50 spiegel) suggesting that DC function was impaired. Since NK cells attack the virus, inadequate expression and presentation of viral antigens by DCs to NK cells leads to diminished activation of the latter. There is also evidence of decreased numbers and exhaustion of NK and cytotoxic CD8+ cells in patients exhibiting severe disease ( 40 Zheng and Gao). Since Gold NPs improve expression of MHC on DCs, activate CD 8+ cells and cause NK cell stimulation, there is more efficient viral elimination.
Discussion
To overcome the poor immune response to vaccination by Influenza vaccines in the elderly, the various strategies employed include mucosal route of delivery, use of adjuvants and utilizing increased amounts of antigen (51, Oh & Lee). All three of these strategies are employed in the oral preparation of gold sup vaccine. The existence of a vast array of antigenic molecules in raw honey, including nucleotides, saccharides, proteins and lipids, introduces these to the immune cells, keeping the entire immune system in prime condition to respond to any new micro- organisms that it may encounter. Gold NP acts as a powerful adjuvant , and absorption through the mucosa of the gut ensures the introduction of the vaccine components to the vastly spread out mass of lymphatic tissue in the gut (GALT). The GNP vaccine is avidly taken up by the gut macrophages, mucosal M cells and DCs, and presented by these APCs to the lymphocytes in the Peyer‘s patches (Figure 3), stimulating both the innate and adaptive immunity.
Figure 4. Natural IgM antibodies show enhanced binding to bio-decorated GNP through multi-valent, high avidity interactions.
Further, highly repetitive surfaces are known to bind strongly to natural IgM antibodies through multivalent, high-avidity interactions (52 Zinkernagel). Uptake of particles by macrophages and
DCs is also facilitated by IgM antibody binding (Figure 4), and this in turn can enhance immune processing through increased antigen presentation (48 Chattopadhyay 2017).
As discussed in section 4, the thiol and amine groups on peptides result in bio-conjugation with GNP. Raw honey contains large numbers of plant and animal peptides and amino acids, and when GNP come into contact with the thiol group on a cysteine amino acid, they can spontaneously form strong covalent linkage (Fig 1). This covalent bond forms because of the strong interaction between sulfur (of thiol) and gold. Amine groups can also form bonds using amides. Since GNPs have large surface area and consequently high surface energy, physical adsorption will also take place spontaneously (10 Chen) resulting in the formation of protein corona over GNP (53, Rong&Peng). This protein corona acts like a repetitive antigenic surface, as described above, to enhance immune processing.
Ghee in the formulation of the vaccine acts as a carrier and alongwithhoney, forms a thick suspension which coats the bio- conjugated gold particles. The phospholipid content and other emulsifying lipids in ghee form liposome like particle encapsulating the biodecoratedgoldnano- particles (see section 2). These hydrophobic GST nano- particles will be preferentially taken up by gut macrophages and DCs in comparison to their hydrophilic counterparts (54 Tabata). Also, being lipoidic, the conjugated GST particles will be absorbed by the lacteals, and travelling through the mesenteric lymphatics into the thoracic duct, will reach the Subclavian vein and then the cardio-pulmonary circulation (4). By this route, the liver is bypassed, preventing most of the GST from being taken up by the liver macrophages( Kuppfer cells). The GST particles can then directly reach the lungs, be taken up by alveolar cells and MALT, and exert their action on the lung macrophages, activating them and changing their phenotype. Also, the gut and lung APCs (which have ingested the GSTnano-particle), being highly mobile, can also travel within the lymphatics, to reach other body organs as also the bone marrow. Increasing evidence points towards the ―training‖ of the HSCs in the bone marrow, leading to global immune activation and protection from microbial invasion of all sorts, including viruses. In fact, MihaiNetea has proposed using Trained Immunity as a tool for reducing susceptibility to and the severity of SARS-CoV-2 infection (49).
Conclusion
It is now clear that children escape the severe form of SARS-CoV-2 disease presumably through trained immunity, having been freshly immunized against viral illnesses like polio, measles and influenza. Also, the more severe disease seen in the aged population is plausibly due to immuno-senescence . Gold NPs have already been discovered to significantly reverse several of the disorders related to IS. GNP, administered along with raw honey and ghee ( gold sup) further primes the innate immunity through the mechanism of trained immunity. The lipids contained in ghee would direct the Gold Sup to the lymphatic system for targeted delivery. Powerful immunomodulatory herbal extracts added to gold sup suspension would enhance the immuno- protective effects, offering a simple and potent modality to combat SARS Coronavirus 2, and also others that may crop up in the future. Also importantly, the GST formulation can instantly be made at home by procuring the pure gold ash, TC extract and honey from the market or online.
Abbreviations : SARS-Severe Acute Respiratory Syndrome, CoV – Corona Virus, GNP—Gold Nano Particles, PAMP- Pathogen Associated Molecular Patterns, IS —Immunosenescence, APC – Antigen presenting cells, ICU – Intensive Care Unit, , NK cells- Natural Killer cells, IL- Interleukin, , IFN-Interferon, MALT- Mucosa Associated Lymphatic Tissue, PRR- Pathogen Recognition Receptors, TLR- Toll Like Receptors, HSC- Haematopoietic stem cells, CMP-
common myeloid progenitor, ALI- acute lung injury, MHC-major histocompatibility complex, CD-Cluster DIFFERENTIATION, SB- swarnabhasma, BALF – Broncho-alveolar lavage fluid, DC- Dendritic Cells, SGNP- Solid Gold Nano Particle, HGG- Honey-ghee-gold suspension, SP- SwarnPrashan, GIT- gastro-intestinal tract
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