1. A composition of constructs of adenoviral product containing the modified DNA sequences coding specific peptides for use as a therapeutic against covid-19 for administration to a newly diagnosed patient of coronavirus to change the course of infection by developing rapid innate immune response and method of preparation of adenoviral therapeutic products thereof comprising of at least one or a combination of components as follows; a. S1 subunit of the S protein or RBD domain of the S protein, b. S1 subunit of the S protein or RBD domain of the S protein both tagged with an adjuvant peptide, c. S1 subunit of the S protein or RBD domain of the S protein or multiple proteins of SARS Co-2 for vaccination or all such proteins tagged with a FLAG peptide, d. specific DNA sequences in combination such that the size of an adenoviral vaccine genome will exceed the packaging capacity in case of homologous recombination event during expansion of virus in HEK-293 cells during manufacturing; where one sequence is naturally occurring variant of the RBD or S1 subunit, another sequence is codon-optimized version of the RBD or S1 subunit and third sequence is also a variant of the RBD or S1 subunit sequence such that none of the three sequences share a length over 20 to 30 bp which would increase the chance of homologous recombination within the added sequences, e. specific sequences of Chimpanzee Serotype 68 adenovirus such that they include minor sequences of serotype 5 adenovirus and include a variation of above mentioned one to three sequences of the coronavirus, f. RBD and/or S1 subunit sequences where the natural signal peptide is replaced by a synthetic signal peptide such that the antigen is secreted out of the cells and is made available for antibody generation and or a Foldon domain is attached at the 3’ terminal of the coding region such that that a trimer is formed after ribosomal synthesis of the peptide, g. And adenoviral vector which can be administered intranasally for development of immediate and rapid innate immune responses wherein, composition of DNA constructs of a tricistronic adenoviral vaccine candidate of serotype 5 as shown in SEQ ID NO: 6 and FIG. 9 comprises of three DNA sequences encoding; a.) the receptor binding domain (RBD) by using codon optimized RBD sequence (as shown in SEQ ID NO- 1 and FIG. 1), b.) natural sequence of the RBD SARS Cov-2 (as shown in SEQ ID NO- 17 and FIG. 24), and, c.) S1 subunit of Coronavirus fused with a Foldon tag (as shown in SEQ ID NO- 2 and FIG. 2) formulated for intranasal, oral, intramuscular or subcutaneous administration for prevention against covid-19, using plasmids shown in FIG. 13, FIG. 14, FIG. 15, FIG. 16 (as presented in sequences 8,9,10 and 11), such that the adenovirus generates innate immunity due to adenoviral envelope proteins and adaptive immunity to Coronavirus due to virtue of secreted synthetic secretory peptide used in combiation with the antigens such that the signal peptide is cleaved after translation and the RBD antigen of 193 aa length is secreted in to the extracellular space for the generation of neutralizing antibodies of IgA and IgG types mainly; and wherein composition of DNA constructs of a monocistronic adenoviral vaccine of serotype Chimpanzee 68 with minor sequences from Serotype 5 as shown in SEQ ID NO: 7 and FIG. 12 comprised of; a single DNA sequence encoding the receptor binding domain (RBD) by using codon optimized RBD sequence (as shown in Sequence 1 and FIG. 1 using plasmids shown in FIG. 18, FIG.19, FIG.20 and FIG. 21 (as presented in SEQ ID NO- 12, 13, 14 and 15). 2) The composition as claimed in claim 1, wherein DNA construct of SEQ ID NO: 2, comprises of, a DNA sequence encoding the codon optimized S1 subunit of the SARS Cov-2, formulated for adenovorial delivery via intranasal, oral, intramuscular or subcutaneous administration, such that the DNA constructs of SEQ ID NO: 2 leads to a biologically expressed S1 subunit, in the trimerized form due to its fusion part of foldon domain for the generation of neutralizing antibodies. 3) The composition as claimed in claim 1, wherein DNA construct of SEQ ID NO: 1 comprises of, a DNA sequence encoding the codon optimized RBD domain of the SARS Cov-2, formulated for adenovorial delivery via intranasal, oral, intramuscular or subcutaneous administration, such that the DNA constructs of SEQ ID NO: 1 leads to a biologically expressed RBD domain secreted by use of a synthetic secretory peptide used in combiation with the RBD domain such that the signal peptide is cleaved after translation and the S1 subunit is secreted in the extracellular space for the generation of neutralizing antibodies. 4) The composition as claimed in claim 1, wherein SEQ ID NO.- 16 as shown in FIG. 23 is map of plasmid pAD1129-ANS10-modified with DNA sequence created for vaccination against the South African strain B.1.351 which carries mutation from both UK and South African Strain (mutations shown in the black box). 5) The DNA constructs as claimed in claim 1, wherein the adenoviral vaccine candidates (SEQ ID NO.-6 and SEQ ID NO.-7) as produced are such that: i. RBD antigen produces neutralizing antibody only, and does not lead to disease enhancement, ii. Signal peptide attached to the RBD sequence helps extracellular secretion of the antigen once adenoviral particle produces the antigen in the cells, thus increasing the chances of formation of antibodies, iii. S1 subunit attached to foldon trimerization domain is expressed as well in the same viral particle increasing the range of antibodies produced and increasing chance to produce antibodies in the natural trimer form, iv. Adenoviral genome is made such that homologous recombination is not viable and this eliminates the risk of producing replication competent virus even in HEK 293 cell line, v. Intranasal or oral delivery of the candidates overcomes the drawbacks of thrombosis as the candidate vaccine does not enter the blood and provides high quality mucosal immunity, vi. Chimpanzee Adeno candidate using the RBD sequence is also able to be given intranasally or orally and thus provides mucosal immunity against the coronavirus without side effects of thrombosis as it never enters the bloodstream, vii. Using natural sequence of the Coronavirus RBD in the same viral genome where the codon-optimized sequence of the Coronavirus RBD is used, prevents any homologous recombination and yet produces the same protein the double amount (two sequences for the same peptide, one natural and one codon-optimized, increasing the overall protein production). viii. The adenoviral nature of the therapeutic drastically changes the outcome of the coronavirus infection and can be used any number of times to prevent development of complicated Covid19 disease by simple intranasal or oral administration of the candidate after the diagnosis of coronavirus infection. 6) The DNA constructs as claimed in claim 1, wherein the method of preparation of Adeno viral therapeutics product comprises the steps of:

1. Construction of the adenoviral candidates which includes the steps of: a. Creation of shuttle plasmids, b. Cosmid Construction, c. Rescue, d. Amplification, and,

2. Characterization of the adenoviral candidates which includes the steps of: i. Genome Restriction Analysis, ii. Physical titer, iii. Infectious titer, iv. VP/ IU ratio, v. Genome sequencing, vi. Transgene expression, vii. RCA assay. Dated this 28th day of April, 2021. For the applicant, ____________ Mrs. Allison Katariya Agent of the applicant -IN/PA-2190 , Description:FIELD OF THE INVENTION: The invention relates to the field of cell and molecular biology, immunology, adenoviral therapeutic and vaccine candidates for treatment of SARS-Cov-2, and more particularly relates to preparation of compositions of constructs of modified DNA sequences for treatment against Covid-19, and even further to the production of a large genome adenoviral product of serotype 5 and an optional booster of a different serotype. BACKGROUND: Pandemic of Covid-19 caused by the SARS Cov-2 has caused fatalities with life-long repercussions globally and demands urgent and critical address in the form of a treatment. This disease can cause fatalities amongst the elderly population especially above 70 years of age at a high rate, however it can also cause mass hospitalizations (up to 20% for all population) and a prolonged halt in the economy could lead much disastrous results for pediatric health as well as health of all those who depend on the daily wages for a surviving nutrition. The disease course ranges from mainly asymptomatic and mild to more severe and critical, in 10% to 20% of symptomatic patients who may be at considerable risk of fatality with many different organ systems. Every day there are hundreds of thousands of patients getting infected and there is no space in the hospitals or especially in the intensive care units. The present invention discloses life-saving solution to this deadly virus in the form of intranasal or oral mucosal route delivered therapeutic candidates that prevent the otherwise reported complications and deaths as well as the drawbacks like clotting which are associated with the other adenoviral products or mRNA or protein products which are administered by intramuscular route. An effective immune response against SARS-CoV-2 requires both arms of the immune system, the innate immune system and the adaptive immune system, however the innate immunity fails to control the Coronavirus infection in cases of serious and protracted illness. Current understanding suggests that the SARS-CoV-2 enters type II pneumocytes via ACE2 in the respiratory system leading to rapid virus replication as well as concomitantly to a proinflammatory state with elevated levels of cytokines such like interleukins IL-1, IL-6, and CXCL8, and TNF. Proinflammatory genes upregulated by the innate immune system belong to NH-kB pathway of proinflammatory mediators and they lead to accumulation of pathogenic inflammatory neutrophils and macrophages in the lung. The elevation of cytokines in severe disease is accompanied by elevated alanine aminotransferase, lactate dehydrogenase, C-reactive protein (CRP), ferritin, and D-dimers. The elevated interleukin IL-6 and TNF responses lead to inflammatory cell death with concomitant elevation of TNF and IFN-? to trigger pyroptosis, apoptosis and necrosis driving he tissue damage and mortality in the Covid-19 patients. If the immune response of mucosal infection can be shifted early on to bring in more natural killer cells, the serious disease would not develop. Adenovirus vectors induce the innate immunity of the immune system that leads to the inflammation of transduced tissues and efficient clearance of administered vectors. Unlike adaptive immunity, the innate response is mediated by the adenovirus particles and does not require viral transcription, thus the production of antigenic peptides is not required at this stage. Their presence rather has an enhancing effect for providing a repertoire of antigens to the T cells such that it comes without the replicating virus which may try to evade the immune system and thus leads to the development of antibodies. Severe acute respiratory syndrome (SARS) emerged as a new disease threat in late 2002 which spread to several countries with considerable loss of life. The etiological agent in the first outbreak was SARS Coronavirus- 1 (Cov-1). Coronaviruses can infect respiratory, gastrointestinal, hepatic and nervous systems of humans, birds, mice, livestock, bats and many other animals. The Severe Acute Respiratory Syndrome (SARS) outbreak of 2002 and Middle East Respiratory Syndrome (MERS) of 2012 outbreaks gave us warning of ‘animal to human’ and ‘human to human’ transmission of newly emerging coronaviruses. During late 2019, a severe respiratory disease was reported in the city of Wuhan, Hubei Province, China. This disease was named Covid-19 and was caused by another strain of Coronavirus; SARS Cov-2. The coronaviruses causing SARS of 2002 or Covid-19 are single-stranded positive sense RNA virus, whose genome encodes nonstructural replicase polyprotein (rep), and structural proteins such as Spike (S), envelope (E), membrane (M) and nucleocapsid (N). Phylogenetic analysis of the viral genome revealed that the SARS-Cov-2 was closely related to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus). These viruses were previously sampled from bats in China. The complete genome of SARS Cov-2 (Wuhan) has been analyzed after collecting broncho-alveolar lavage from one patient who exhibited the SARS from Cov-2. SARS Cov-2 (causing Covid-19) virus is not very different from SARS Cov-1 that caused the outbreak in year 2002/2003. What is significantly different with this virus is its high infectivity and relatively lower fatality rate. Our own comparisons of the two viruses for their sequences in the specific receptor binding domain as well as others have shown that the virus has significant amino acid sequence similarity (overall region in the RBD- similarity 83% and 50% similarity in the Receptor binding motif). Antibodies induced by anti-SARS vaccines can cross-neutralize bat SARS-like coronaviruses. The RBD domain of the Coronavirus binds the Angiotensin Convertase Enzyme receptor 2 (ACE2) which leads to the infection of cells in the respiratory as well as gastrointestinal tracts, hence the use of RBD as an antigen for therapeutic will provide neutralizing antibodies which are essential for the treatment of the infection to be successful. The inhaled virus SARS-CoV-2 likely binds to epithelial cells in the nasal cavity and starts replicating after gaining entry via attachment to the ACE2 receptors without much resistance by the innate immune response. For the mechanism of action for the therapeutic product it is essential to understand the body’s immune response against a pathogen like coronavirus. Body has two types of immune responses; innate and adaptive. Innate immunity consists of macrophages, dendritic cells, mast cells, granulocytes (WBCs including neutrophils, basophils and eosinophils) and complement protein. Adaptive immune response consists of B cells producing antibodies, memory B cells and T cells (CD4+ and CD8+). The natural killer T cells take part in both innate and adaptive immune responses. The innate immune response is immediate and rapid while the adaptive immune response is slow and takes up to 2 weeks to develop. During the local mucosal propagation of the Coronavirus (during which time the infected people are also infectious), there is limited local innate immune response. The adenoviral non-replicative vectors can generate an innate immune response rapidly which does not require viral transcription to occur and thus start the innate immune response immediately thus preventing the complicated Covid disease and by the time two weeks have passed the antigens transcribed and translated by the adenoviral vector also contribute to the adaptive immune response and development of antibodies against the coronavirus which brings the best of both worlds to the mechanism of action of this adenoviral therapeutic carrying a payload of antigens which can generate neutralizing antibodies. Thus, the advantage of the present invention over existing art is that the product are such that they recruit the innate system early on and ramps up the innate immune response which in turn prevents development of serious disease, the DNA constructs of the present invention are such that they provide antigens to the body which are transcribed by the adenovirus and are readily available for antibody generation (unlike the coronavirus which would cause infection and delay in the development of adaptive immune response). Another advantage of the present invention is that the product does not enter the blood stream when given intranasally or orally, thus it does not need to be produced in the serum free media. This can give a major advantage to a country like India which can feel shortages of serum free media supplies from Western Countries. Another advantage of the present invention is that it will also act as a vaccine in people who are not infected with the coronavirus and can be administered as many times desired with no known side effects. Adenoviral infections of upper respiratory tract are very common and pose no risk to humans and yet the vaccine mechanism bypasses any existing immunity against the adenoviruses. Currently marketed vaccines cause multiple side effects like blood clots and neurological disorders which are not expected with our invention. Another advantage of our invention is that it can be given without any aid from a doctor or a nurse, at outpatient venue, can remain stable at 2 to 8 degree Celsius up to a month without affecting its potency and thus can be delivered and used in developing countries in a much user-friendly manner. OBJECT OF THE INVENTION: The primary object of the invention is to provide compositions of DNA constructs either used alone or in combination to provide prevention, diagnosis and treatment of Covid-19. Yet another object of the present invention is to provide composition of DNA constructs specifically for treatment of coronavirus infection to avoid complications leading to severe covid-19 infection in the form of an adenoviral product that can be administered as a tablet given orally or drops for intranasal administration. SUMMARY OF THE INVNETION: Before the present invention is described, it is to be understood that the present invention is not limited to specific methodologies and materials described, as these may vary as per the person skilled in the art. It is also to be understood that the terminology used in the description is for the purpose of describing the particular embodiments only and is not intended to limit the scope of the present invention. The present invention discloses composition of constructs of modified DNA sequences and method of preparation of therapeutic candidates using adenoviral vectors and constructs of modified DNA sequences against covid-19. Two therapeutic candidates which are also useful as vaccine candidates are mainly described in the present patent application; one is a Serotype 5 adenoviral therapeutic and another is a Serotype Chimpanzee 68 adenoviral therapeutic. No serotype can be patented just as no species of animals or plants can be patented. Because an entire serotype of a Virus cannot be patented, in this invention, we apply to patent the use of Serotype 5 and Serotype 68 (not exclusively only these 2 serotypes) adenoviral vectors carrying DNA sequences generating neutralizing antibodies after intranasal or oral/ mucosal administration such that the DNA modifications and novel use of multiple coronavirus epitopes in a single viral particle for intranasal, oral as well as intramuscular use in the form of a Serotype 5 or Serotype 68 adenoviral therapeutic expressing such epitopes by virtue of modified sequences of RBD or S1 subunit alone or in combination with readily alternative use as a vaccine. The serotype 5 adenovirus is to be used as the primary therapeutic followed by administration of the serotype 68 virus candidate for the booster effect. The invention relates to the compositions of DNA constructs of SEQ ID NO: 1 to 5 for use in the treatment of Covid-19 in humans as well as animals. The invention relates to the compositions of DNA constructs of SEQ ID NO: 6 and 7 which are the complete genome sequences of two adenoviral therapeutic candidates successfully developed based on the sequences described in SEQ ID NO: 1 to 5. The invention relates to the compositions of DNA constructs of SEQ ID NO: 8,9,10,11,12,13,14 and 15 which are the plasmid sequences used for the creation of two adenoviral therapeutic candidates. These plasmids were used in creating the whole genome sequences of SEQ ID NO: 6 and SEQ ID NO: 7. The invention relates to the compositions of DNA constructs of SEQ ID NO: 16 which is the plasmid sequence used for the creation of adenoviral therapeutic candidate against the South African variant strain B.1.351. However, it is not essential to create a separate candidate for the South African variant or other variants unless the population spread of a specific strain is at large scale. The serotype 5 therapeutic candidate would sufficiently work for the purpose of treatment of all strains of coronavirus as generation of innate immune response is primary mode of mechanism of action. The said composition of therapeutic with constructs of modified DNA sequences comprises of at least 7 different important functional components as follows: a) Adenoviral vectors mainly of Serotype 5 with large genome capacity which incapacitates the packaging of adenovirus in case of a homologous recombination even during manufacturing b) S1 subunit of the S protein or RBD domain of the S protein. c) S1 subunit of the S protein or RBD domain of the S protein both tagged with an adjuvant peptide; for creating a better immune response. d) S1 subunit of the S protein or RBD domain of the S protein or multiple proteins of SARS Co-2 for generating innate as well as adaptive immune response; e) Use of specific DNA sequences in combination such that the size of an adenoviral genome will exceed the packaging capacity in case of homologous recombination event during expansion of virus in HEK-293 cells during manufacturing; where one sequence is naturally occurring variant of the RBD or S1 subunit, another sequence is codon-optimized version of the RBD or S1 subunit and third sequence is also a variant of the RBD or S1 subunit sequence such that none of the three sequences share a length over 20 to 30 bp which would increase the chance of homologous recombination within the added sequences. f) Specific sequences of Chimpanzee Serotype 68 adenovirus such that they include minor sequences of serotype 5 adenovirus and include a variation of above mentioned one to three sequences of the coronavirus. g) RBD and or S1 subunit sequences where the natural signal peptide is replaced by a synthetic signal peptide such that the antigen is secreted out of the cells and is made available for antibody generation and or a Foldon domain is attached at the 3’ terminal of the coding region so that a trimer is formed after ribosomal synthesis of the peptide. The primary mode of delivery of therapeutic targets would be intranasal, oral or parenteral (subcutaneous or intramuscular) delivery of a replication-incompetent adenoviral vehicle that can be produced by any of the available platforms. The present invention discloses DNA constructs that can be used for the prevention, diagnosis and treatment of Covid-19. For therapeutic purpose, the present invention describes composition of DNA construct of S1 subunit of the Spike Glycoprotein as well as DNA constructs of the Receptor Binding Domain (RBD) of the S protein (spike glycoprotein) tagged with a Foldon domain (FD) for trimerization and or antigens S1 or RBD attached with a synthetic signal peptide that will secrete the peptide out of the cell to make it available for antibody formation and methods to create adenoviral therapeutic product such that the adenoviral particles are replication non-competent and can be cultured in HEK-293 cells to remain replication non-competent by making candidates incapable to generate a viable replication competent adenovirus by homologous recombination. In summary, the DNA constructs of the present invention will be essential in controlling, treating and eliminating the Covid-19 disease, by virtue of diagnosis, prevention and treatment of the disease and the methods associated can be used in creation of therapeutic against many other infectious diseases like respiratory infections causing tuberculosis or infections of the gastrointestinal tract or genital tract like HPV or syphilis. This same technique can also be used in creating vaccine or therapeutic products against fungal infections, parasitic infections, viral as well bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS: The present invention, together with further objects and advantages thereof, is more particularly described in conjunction with the accompanying drawings in which: FIG. 1 is a diagrammatic representation of the DNA construct of SEQ ID NO: 1 carrying an optimized codon sequence of receptor binding domain (RBD) of SARS Cov-2 for expression via adenoviral delivery; FIG. 2 is a diagrammatic representation of the DNA construct of SEQ ID NO: 2 carrying an optimized codon sequence of predicted S1 subunit of S Protein with a Foldon trimerization domain attached at the 3’ end for expression via adenoviral delivery; FIG. 3 is a diagrammatic representation of the DNA construct of SEQ ID NO: 3, carrying an optimized codon sequence of receptor binding domain (RBD) of SARS Cov-2 and adjuvant peptide that would separate and get secreted to the extracellular space after translation for adenoviral delivery; FIG. 4 shows predicted 3-D structures for SARS Cov-1 epitope, SARS Cov-2 epitope and epitope RBD in our product; the three epitopes are seen having similar 3-D structure. FIG. 5 shows alignment of RBD domains from SARS Cov-2 and SARS Cov-1 (labeled as Cov-2 and Cov-1) with the rectangles showing specific sites that have been shown to generate neutralizing antibodies against the SARS Cov-1 virus; FIG. 6 shows the Western Blot assay expression results of the RBD protein expressed from the natural sequence detected in HEK-293 cell culture supernatant with help of a FLAG epitope under reducing conditions (only a monomer in Lane 1 and Lane 2) and under non-reducing conditions (monomer, and faint bands of dimer and trimer in Lane 3). FIG. 7 shows the Western Blot assay expression results of the RBD protein expressed from the codon-optimized sequence detected in HEK-293 cell culture supernatant with help of a FLAG epitope under reducing conditions (only a monomer in Lane 2 and Lane 3) and under non-reducing conditions (monomer, and faint bands of dimer and trimer in Lane 5). FIG. 8 shows the Western Blot assay expression results of the S1-FD protein expressed from the codon-optimized sequence detected in HEK-293 cell culture supernatant with help of a FLAG epitope under reducing conditions (only a monomer in Lane 1 and Lane 2) and under non-reducing conditions (monomer, and strong bands of dimer and trimer in Lanes 3 and 4). FIG. 9 shows the map of Ad-ANS06 Serotype 5 adenoviral candidate vaccine genome. FIG. 10 shows the map of Ad-ANS12 Serotype Chimpanzee 68 adenoviral candidate vaccine genome. FIG. 11 shows plasmid pAD1127-ANS05 with EF1a-RBD cassette in place of the E1 region; where the RBD sequence is tagged with a synthetic signal peptide for secretion of the antigen outside the cells. FIG. 12 shows the backbone pAD1128 plasmid for the Serotype 5 adenovirus with wild-type Ad5 E2 and late genes. FIG. 13 shows the plasmid pAD1129-ANS10 with CMV-S1FD cassette in place of the E3 region and RSV-OPT-RBD cassette between the L5 and E4 poly A signals used for the creation of Ad-ANS06 tricistronic serotype 5 adenoviral vaccine candidates; where the RBD sequence is codon-optimized and natural signal peptide is replaced by synthetic signal peptide and S1 subunit sequence still carries the natural signal peptide sequence but is attached to a Foldon trimerization domain at the 3’ end. FIG. 14 shows pAD1130 plasmid with E4 genes, this is one of the plasmids used in the creation of adenoviral vaccine candidate ANS06. FIG. 15 shows the restriction enzyme digest confirmation of genomic map of the cosmid created for the Ad-ANS06 genome. FIG. 16 shows Results of Western Blot Assay for created adenoviral candidate Ad-ANS06 in HEK-293 cells (confirmation of antigen expression of the actual therapeutic candidate created from the plasmids) where all lanes showed antigen in monomeric, dimeric and trimeric forms (marked within oval shapes). FIG 17 shows Results of RCA assay showing no replication competent viral particles for both Ad-ANS06 and Ad-ANS12. FIG. 18 shows plasmid pAD1144-ANS15 with CMV-codon optimized RBD cassette in place of the E1 region used for the creation of chimpanzee adenoviral candidate Ad-ANS12. FIG. 19 shows plasmid pAD1145 used for the creation of chimpanzee adenoviral candidate Ad-ANS12. FIG. 20 shows plasmid pAD1146 used for the creation of chimpanzee adenoviral candidate Ad-ANS12. FIG. 21 shows plasmid pAD1147 used for the creation of chimpanzee adenoviral candidate Ad-ANS12. FIG. 22 shows the restriction enzyme digest confirmation of genomic map of the cosmid created for the chimpanzee adenoviral candidate Ad-ANS12. FIG. 23 shows the map of the first plasmid pAD1129-ANS10-modified with DNA sequence created for vaccination against the South African strain B.1.351 as well as British Strain B.1.1.7 (common mutation N501Y) which carries mutations from both UK and South African Strain (mutations shown in the black box); FIG. 24 shows the map of the natural sequence of the RBD domain. FIG. 25 and FIG. 26 show the groups of rats used in the animal study where the two therapeutic candidates Ad-ANS06 and Ad-ANS12 were tested. FIG. 27, FIG. 28 and FIG. 29 show the results of detection of SARS-CoV-2 antibodies by bio layer interferometry in the rat sera vaccinated with the adenoviral products Ad-ANS06 and Ad-ANS12 in the animal studies. The curves show that high rate of antigen antibody binding was present in the sera of the tested rats. FIG. 30 and FIG. 31 show the groups of rabbits used in the animal study where the two therapeutic candidates Ad-ANS06 and Ad-ANS12 were tested. DETAILED DESCRIPTION OF THE INVENTION: The disclosure has been described with reference to the accompanying embodiments which do not limit the scope and ambit of the disclosure. The description provided is purely by way of example and illustration. The foregoing description of the specific embodiments so fully revealed the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the scope of the embodiments as described herein. Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. The present invention discloses novel compositions of constructs of modified DNA sequences (SEQ ID NO: 1-17) administered via adenoviral vector, for the treatment of Covid-19 disease. The compositions of the said constructs of the modified DNA sequences as described in the present invention for treatment of Covid-19 do not necessarily need a specific promoter or a specific plasmid for the delivery and expression. In one embodiment of the present invention, the first DNA cassette SEQ ID NO: 1 Receptor Binding Domain of the SARS Cov-2 is constructed and administered via adenoviral particles via intranasal, oral mucosal, routes for generation of innate immunity and to provide protection against the development of serious Covid-19 disease which is manifestation of a derailed and uncontrolled immune response. In another embodiment of this invention, the availability of the antigen for the generation of adaptive immunity via adenoviral viral vector delivery is enhanced because of presence of a synthetic signal peptide replacing the natural signal peptide associated with the RBD domain which gets released into extracellular space after translation due to the presence of synthetic signal peptide. The DNA cassettes described in various embodiments of the invention are delivered preferably via intranasal-mucosal route mucosal route intranasally or, orally or via other routes mainly subcutaneously for gene delivery by adenoviral systems. The different embodiments of the invention are mentioned below. However, different combinations of these constructs can be made and administered by replacing the epitopes for a specific virus or specific promoter for controlled or constitutive expression of described peptides. The present invention discloses compositions of constructs of modified DNA sequences of constructs of modified DNA sequences/ DNA cassettes (SEQ ID NO: 1 to SEQ ID NO: 17) encoding the Kozac sequence for increasing the expression of the epitope, codon-optimized gene sequence of the RBD epitope followed by a stop codon under expression control of any promoter, e.g. CMV promoter, EF1 promoter or RSV promoter. The RBD epitope is preserved among the SARS Cov-1 and SARS Cov-2, as shown in FIG. 4 and FIG. 5. Also as seen in FIG. 5, the rectangle marked epitopes are not only highly conserved but also generate neutralizing antibodies. The RBD protein 3-D structure also shows conservation across SARS Cov-1 and SARS Cov-2. The chosen therapeutic epitope also shows the same 3D structure which would be essential for generation of same neutralizing antibodies. The RBD protein 3-D structure also shows conservation across SARS Cov-1 and SARS Cov-2. The chosen epitope is essential for generation of same neutralizing antibodies. The adenoviral delivery of RBD epitope is a preferred way for our product because RBD can generate neutralizing epitopes, adenoviral particles can provide the mucosal immune response against the viruses like SARS Cov-2 along with the systemic immune response. Even when a strain variant may have a slightly different 3-D therapeutic the mechanism of action of the therapeutic remains unaffected because the first part of the mechanism of action consists of generation of innate immunity by presence of adenoviral particles. In another embodiment of the invention, S1 subunit of the S protein is used instead of RBD as the vaccine epitope or the therapeutic epitope. Hence the codon optimized sequence of S1 subunit is used for the generation of antibodies (FIG. 2) preferably tagged with Foldon Domain. The adenoviral viral vector efficacy is enhanced because of presence of a Foldon trimerization domain at the end of the S1 subunit such that the S1 subunit forms a trimer and generates high quality antibodies after translation due to the presence of Foldon domain (FIG. 2). In another embodiment of the invention (shown in FIG. 23), the DNA sequence is modified to reflect certain mutations in the virus such that a vaccine or a therapeutic can be created against the South African variant B.1.351 as well as a common mutation in the British Strain B.1.1.7 (N501Y). These Sequence variations are described below: Plasmid pAD1129-ANS10 sequence modifications: i. 6403 G to C ii. 1233 G to C iii. 2130-2133 CTG to CAC iv. 2142-2144 AGA to ATC v. 2655-2657 AAA to AAC vi. 2856-2858 GAA to AAG vii. 2907-2909 AAC to TAC In another embodiment, Ad-ANS06 the vector (shown in FIG. 9 as Ad-ANS06b) is designed such that the genome size (of 36.2 KB) becomes unpackageable to produce replication competent virus particles in case homologous recombination occurs in the HEK-293 cells (genome size would become over 38 KB if homologous recombination occurs and thus becomes unpackageable). Thus, this invention eliminates the need of specialized culture cells for production of adenoviral vaccines if the same method can be used for insertion of multiple antigens in the same viral genome. Another advantage this embodiment offers is to provide means of expressing multiple copies of genes or different genes at the same time by use of the same viral particle. In yet another embodiment, Ad-ANS12 candidate vaccine (shown in FIG. 10) was created by using a Chimpanzee adenoviral genome and few small sequences from serotype 5 adenovirus such that, it contains only one sequence of receptor binding domain to express the RBD epitope to generate immunogenicity in the vaccine. This patent for the invention does not patent the serotype itself as a species of organism cannot be patented, however the use of serotype 5 for expression of RBD 193 Covid 19 epitope is being described as a novel embodiment for intranasal use or oral use in the form of an adenoviral therapeutic agent. In addition, use of Serotype 68, is the second therapeutic to be used after the first is used for the generation of long lasting immunity whenever such use is appropriate. The present invention has many advantages as follows: 1) Immediate and quick generation of innate immune response to control the Coronavirus infection by virtue of adenoviral vectors administered to intranasal or oral mucosa. 2) Generation of mucosal immune response against the RBD domain of the SARS Cov-2 when used via an adenoviral vector. The immunity generating neutralizing antibodies against the RBD domain are extremely important because the SARS Cov-2 virus uses the RBD domain to bind ACE2 protein. Once the RBD is associated with ACE2, then the virus S protein transmembrane portions fuse with the cell membrane, giving entry into the cell. The route of entry of Coronavirus is mucosal (through respiratory as well as gastrointestinal tracts) hence the mucosal immunity provided by our vaccine candidates is of critical importance. 3) Antibodies generated against RBD and S1-subunit are mainly neutralizing type and are not expected to lead to antibody mediated disease enhancement. 4) Signal peptide used in the DNA constructs enhances the secretion of the antigen in the extracellular space making it available for the generation of antibodies. 5) The S1 subunit trimerizes due to Foldon domain and thus provides high quality antibodies for protective immunity against the disease. 6) The developed adenoviral therapeutic candidates can also be used for intranasal, oral as well as intramuscular delivery for use as a vaccine. Intranasal or oral delivery requires much smaller dose and generates the best quality of antibodies (IgA mucosal antibodies) that are required for blocking the entry of a respiratory pathogen. In another advantage, vaccines given by the intramuscular route may lead to thrombosis and intranasal or oral route eliminates or minimizes this risk completely. 7) Immunity provided against the South African variant and other variants is also of critical essence as described in one embodiment, the vaccine candidate contains the S1 subunit sequence with the mutations present in the variants and thus provides mucosal immunity against these variants. 8) Producing a tricistronic adenoviral vaccine candidate is advantageous such that because of use of natural and codon-optimized sequences in the same viral genome, the protein production (antigen production) is enhanced and yet chances of homologous recombination are minimized. 9) Another major advantage of the invention is that the adenoviral candidate Ad-ANS06 does not need a special cell line for its manufacturing even though it is of Serotype 5, where the DNA sequence is used to create an adenoviral vaccine of Serotype 5 type, in such a way that the genomic size of adenovirus prevents the viable homologous recombination and thus prevents inadvertent production of replication competent adenoviral particles, which is a major limiting factor in production of adenoviral vaccines in HEK cell line. United States Food and Drug Administration (USFDA) requires that there should be less than 1 replication competent adenoviral particles in 3x10e10 viral particles (vp) for human use. Although other health authorities including Indian FDA may require less than 1 replication competent particle per dose, in both instances the current embodiment of Serotype 5 adenoviral candidate is useful (Example-1). 10) Invention offers another advantage by creating an adenoviral candidate with Chimpanzee adenoviral serotype 68 in another embodiment, described below in example 2 (an adenoviral candidate vaccine Ad-ANS12), where the candidate vaccine was created using the said DNA sequence in such a way that the vector comprised of Chimp adenovirus type 68 carrying small type 5 adenoviral sequences. In this embodiment, an advantage is such that it prevents inadvertent production of replication competent adenoviral particles as homologous recombination is not possible between the genome of chimp adenovirus type 68 and adenoviral genes inserted in the Human Embryonic Kidney (HEK) cell line. The HEK cells are used for the manufacturing of adenoviral vaccines since a long time and homologous recombination has been a challenge for the production of vaccines, the approach of the present invention to produce both the vaccine candidates eliminates this challenge as described in example 1 and example 2 due to novel composition of DNA constructs. EXAMPLES To further elucidate the mechanism of the invention, two examples are described here for multiple candidate vaccine preparation. The methodology for using the said DNA sequences for the creation of vaccine candidates is described below in brief. In first example the three DNA sequences [first sequence with Coronavirus S1 subunit tagged with Foldon domain (S1-FD) (FIG. 2) is as in SEQ ID NO- 2)), second sequence with natural RBD sequence (FIG. 24 and as in SEQ ID NO- 17) and the third sequence codon-optimized RBD sequence (FIG. 1 and as in SEQ ID NO- 1)) were used by T4 ligation where a cosmid viral genome (as shown in FIG. 9 and SEQ ID NO- 6) was produced from four plasmid constructs to create a therapeutic candidate called as Ad-ANS06 (See FIG. 9, shown as Ad-ANS06b) which has a Serotype 5 Adenovirus genome and which would be expressing Receptor Binding Domain and S1 protein with trimerization Foldon domain. The Foldon domain sequence comprises of 27 amino acid (81 nucleotides) length C-terminal sequence from T4 bacteriophage. This Foldon sequence trimerizes any peptide it is attached with. The S glycoprotein of Coronavirus being a trimer, S1 subunit when in trimerized form produces not only antibodies to epitopes that are available on single chain but also those three-dimensional epitopes that are available only in the trimeric form. In second example, a codon-optimized single copy of RBD DNA sequence (FIG. 1 and SEQ ID NO- 1) was used to create a therapeutic candidate called as Ad-ANS12 (see FIG. 10 and SEQ ID NO- 7) which is a Serotype 68 Chimpanzee Adenovirus with a few Serotype 5 adenovirus sequences while carrying a Codon-Optimized sequence of the Receptor Binding Domain. The process again involves use of four construct plasmids which are combined by T4 ligation to create a cosmid viral genome sequence as shown in FIG. 10. This candidate can be used as a booster so that the immunity persists. Sequences for Receptor Binding Domain (193 aa) and S1 subunit of the Spike glycoprotein of the coronavirus bound to a Foldon domain were synthesized and tested for expression in HEK cells. FIG. 6 shows expression of RBD protein from a plasmid with natural sequence of RBD. FIG. 7 shows expression of RBD protein from a plasmid with codon-optimized sequence of RBD. FIG. 8 shows expression of S1 subunit protein bound to a Foldon tag by use of a plasmid with codon-optimized sequence of S1-FD. These three sequences were then inserted into plasmids that were used for the creation of cosmids (genomes for adenoviral candidate vaccines). In yet another embodiment of the present invention is described the method of preparation and characterization of the candidates Ad-ANS06 and Ad-ANS12 (DNA sequences SEQ ID NO-1 to SEQ ID NO-17). A.) Method to produce and test the Candidate Adenoviral therapeutic using the DNA sequences consists the steps of;

1. Construction of the adenoviral candidate which includes the steps of: a. Creation of shuttle plasmids, b. Cosmid Construction, c. Rescue, d. Amplification, and

2. Characterization of the candidate which includes the steps of: i. Genome Restriction Analysis, ii. Physical titer, iii. Infectious titer, iv. VP/ IU ratio, v. Genome sequencing, vi. Transgene expression, vii. RCA assay. Example 1: Construction of candidate therapeutic Ad-ANS06; a serotype 5 Adenoviral candidate which can also be used as a vaccine before the infection with three gene inserts for more expression, where one gene sequence is of S1 subunit of the S glycoprotein tagged with FD domain, and two sequences are codon optimized sequence of the receptor binding domain (RBD) and natural sequence of the RBD. AD-ANS06:

1. Construction of the adenoviral therapeutic candidate which can also be used alternatively as a vaccine if used prior to infection: a. Creation of shuttle plasmids: Four plasmids were created for creation of ANS06. i. pAD1127-ANS05 (see FIG. 11) (which has EF1a promoter driving the expression of natural RBD sequence leading to expression of 193 amino acids). ii. pAD1128 (see FIG. 12) such that pAd1128 is a shuttle plasmid that contains WT Ad5 E2 and late genes. iii. pAD1129-ANS10 (see FIG. 13) (which has RSV promoter driving the expression of codon-optimized RBD sequence leading to expression of 193 amino acids). iv. pAD1130 (see FIG. 14) pAd1130 is a shuttle plasmid that contains the WT Ad5 E4 region. The importance of presenting natural as well as codon-optimized sequences of antigen provides multiple advantages: i. It increases the size of the final adenoviral genome such that it is incapable of producing replication competent adenoviral particles, because after homologous recombination the packaging capacity would be surpassed. ii. It increases the expression of the antigen without creating a self-repeating DNA sequence within the plasmid. iii. It eliminates any uncertainty whether natural or codon-optimized sequences should be used for antigen expression as both sequences are present. Another advantage of the present invention is that the S1 subunit and RBD subunit both are available for creation of antibodies against the antigenic epitope. b. Cosmid Construction Ad-ANS06: The entire genome of tricistronic virus Ad-ANS06 was reconstituted in a cosmid using the four shuttle plasmids mentioned above namely; pAD1127-ANS05 (E1/pIX shuttle), pAD1128 (E2 shuttle), pAD1129-ANS10 (E3/ fiber shuttle) and pAD1130 (E4 shuttle). These four plasmids were used to create a cosmid with restriction enzyme digestion and ligation with T4 DNA ligase. The ligation product was packaged into lambda phage heads which were subsequently used to infect competent E. Coli. E. coli were selected with the help of ampicillin and kanamycin selection and cosmid DNA was purified using the alkaline lysis method and confirmed with restriction enzyme digestion to be correct. Ad5-based tri-cistronic vector with a EF1a-RBD cassette in place of the E1 region (see FIG.- 11), a CMV-S1FD cassette in place of the E3 region (see FIG. 13), and an RSV-OPT-RBD cassette between the L5 and E4 polyA signals (see FIG.- 13). All 3 cassettes (placed in two plasmids overall during the creation of AD-ANS06; pAD1127-ANS05 carrying EF1a-RBD cassette and pAD1129-ANS10 plasmid carrying CMV-S1FD and RSV-OPT-RBD cassettes) feature ATG triplets upstream of the initiator codon of interest which reduces the efficiency of expression. A vector where there were no ATG triplets upstream of the initiator codon could not produce a viable candidate potentially due to interference of the expressed proteins with the candidate production, hence the extra ATG triplets were maintained for a little inefficiency in expression. c. Rescue: i. Transfection: Two 6-cm dishes of 293 cells were transfected with PacI-digested pAd5-ANS06 using the calcium phosphate-DNA co-precipitation method. Ad5.CMV-GFP DNA (a known GFP insert) was used as positive control. Cells were rinsed twice with DMEM about 12 hours after the transfection. ii. Harvest: Virus plaque harvest (day 11 post-transfection) was conducted when the first plaque was detected in one of the dishes of the HEK293 cells transfected with pAD5-ANS06; four plaques were harvested. d. Amplification: i. Pre-amplification (first round of expansion): A confluent 15-cm dish of 293 cells was split into 6-cm dishes at 30% confluence. The dishes were infected directly with Ad-ANS06 plaques #1-2-3-4 harvested earlier. On day 4 post-infection, the dish of 293 cells infected with Ad-ANS06 clone 3 showed complete cytopathic effect (CPE). Cells and medium were harvested, frozen/thawed three times (3x), clarified by centrifugation and aliquoted into sterile 1-mL vials. On day 6 post-infection, the dishes of 293 cells infected with Ad-ANS06 clones 1,2 and 4 showed complete CPE. Cells and medium were harvested, frozen/thawed 3x, clarified by centrifugation and aliquoted into sterile 1-mL vials. ii. Amplification (second round of expansion): Confluent 10-cm dishes of 293 cells were infected with the crude lysates of Ad-ANS06 clones harvested. On day 3 post-infection, all dishes showed strong CPE. Cells and medium were harvested in the following way. 1.5 mL infected cell suspension was centrifuged for 2 min at 3,000 rpm, RT. The cell pellets were washed twice with PBS, then frozen at -70°C. These cell pellets were used for Western Blot testing for expression studies (for both candidate vaccines). Viral DNA (vDNA) was extracted from 4 mL infected cells suspension following the Hirt method. The remaining infected cell suspension was frozen/thawed 3x, clarified by centrifugation and aliquoted into sterile 1-mL vials.

2. Characterization of the candidate: i. Genome Restriction Analysis: vDNA was extracted from Ad-ANS06-infected 293 cells according to the Hirt method. The DNAs were resuspended into 50 µL TE pH 7.5. The identity of the virus was confirmed by restriction digestion with HindIII, Acc65I and SpeI (5 µL DNA per lane) (see FIG.- 15). All 3 clones show identical restriction patterns, similar to the cosmid DNA restriction pattern. This indicate that the virus is stable. The presence of the 4155 bp HindIII fragment, 1313 bp Acc65I fragment and the 1024 bp SpeI fragment demonstrates the presence of the EF1a-RBD expression cassette in the vector. The presence of the 3982 bp and 2849 bp HindIII fragments, the 3321 bp and 2250 bp Acc65I fragments and the 3747 bp SpeI fragment demonstrates the presence of the CMV-S1FD expression cassette in the vector. The presence of the 4149 bp HindIII fragment, the 4353 bp Acc65I fragment and the 1193 bp SpeI fragment demonstrates the presence of the RSV-OPT-RBD expression cassette in the vector. ii. Physical titer: The OD260-SDS method was used to determine the concentration of virus particles in the purified stocks of Ad-ANS06. The concentration of VP in the preparations was calculated given the extinction coefficient of 1.1 x 10e12 virus particles (VP) per Abs260 unit in presence of SDS. Ad-ANS06 physical quantity from one batch was calculated as 1.4 x 10e13 at a concentration of 1.6 x 10e12 vp/ml. iii. Infectious Titer: Titration of the Ad-ANS06 adenovirus was conducted by using the hexon detection immune-histochemistry using an Adeno-X Rapid Titer kit. The CsCl-purified virus stock of Ad-ANS06 was diluted 10-fold serially in DMEM/FBS (10e1 to 10e6). The 293 cells from one 15-cm dish (passage 44) were trypsinized, washed once in DMEM/FBS and resuspended in 30 mL DMEM/FBS. 300 µL of cell suspension were added to tubes containing 50 µL of virus dilutions 10e3 to 10e6. The infected cells were then transferred to a well of a 24-well plate. After 3 days culture at 37 °C, the infected cell monolayers were fixed with ice-cold methanol, stained with an anti-Ad5 hexon primary antibody, a HRP-conjugated secondary antibody and DAB as substrate. Positive cells were counted under the microscope (10x10 magnification). The infectious titer (IU/mL) was calculated using the following formula: (average # of infected cells/field) x 47.4 x dilution factor / (volume of virus used for the infection in mL) = infectious titer. iv. VP/IU Ratio: VP/IU (viral particle/ infectious units) ratio for AD-ANS06 was calculated to be 20. v. Genome sequencing: The entire genomes of Ad-ANS06 was sequenced by NGS on an Illumina iSEQ100 instrument. a. vDNA purification: vDNA was extracted from purified VP of Ad-ANS06 using proteinase K. The DNA was resuspended in 30 µl TE pH 7.5. vDNA concentration of 0.48 µg/ µl from 3.9 x 10e12 VP/ml of stock concentration of virus. b. The sequencing and analysis confirmed the presence of the 3 intact expression cassettes: the EF1a-RBD-ßglobin pA cassette in the E1 region, the CMV-S1FD-SV40pA cassette in the E3 region and the RSV-OPT-RBD-bGHpA cassette between the L5 and E4 polyA signals. vi. Transgene expression: Expression of vaccine antigen coronavirus spike glycoprotein subunit S1 was confirmed by western blot assay conducted under non-reducing and reducing conditions. Expression of the antigen was confirmed where monomeric, dimeric and trimeric forms of the S1 subunit as well as receptor binding domain were seen on the western blot (see FIG 16). Primary antibodies against the Receptor Binding Domain and S1 subunit were used to detect the protein. Results showed detectable RBD protein in the Ad-ANS06 infected cells from present from 1.17 mg/ml to 1.46 mg/ml). vii. RCA assay: The presence of replication-competent adenovirus (RCA) in the purified preparations of Ad-ANS06 was assessed using a modified infectivity/PCR method, which combines the amplification of infectious RCA by cell culture with the sensitivity of detection of E1-specific sequences by PCR. For each construct, 10 x 15-cm dishes of A549 cells (~ 2.5x 10e8 cells) were infected with 3 x 10e10 VP. Five days after the infection, the infected cells were harvested, pooled, and frozen/thawed 3 times in order to release the virus particles. New monolayers of A549 cells seeded in 10-cm dishes were infected with these crude virus lysates and cultured for another 5 days. Cells were harvested and viral DNA was purified according to the Hirt method. It was used as template for the detection of a 220 bp-long E1-specific sequence by PCR. As controls, two dishes of A549 cells were infected with a RCA-free control virus spiked with 10 and 100 VP of WT Ad5. The results showed the absence of detectable E1-specific sequence in the DNA extracted from A549 cells infected with Ad-ANS04, Ad-ANS06 and Ad-ANS12. Thus, no RCA was detected in 3 x 10e10 VP of the purified preparations of Ad-ANS06 (see FIG. 17). Animal Study: The candidate Ad-ANS06 was tested in two animal studies, dosing outline and groups of the rat repeat dose toxicity and immunogenicity study are shown in FIG.- 25 and FIG.- 26 respectively. The candidate was given intranasally at a dose of 2x10e10 vp per dose given at day 0, day 14 and day 28. Also, the repeated dose toxicity and immunogenicity study was conducted in New Zealand white rabbits; dosing outline and groups are shown in Figures 30 and 31 respectively. The candidate Ad-ANS06 was safe with no safety concerns revealed in rats or rabbits. Day 28 (after 2 doses) immunogenicity results showed presence of neutralizing antibodies against the Coronavirus as shown in FIG. 28. The figures show results of detection of SARS-CoV-2 antibodies by bio layer interferometry. The curves show that high rate of antigen antibody binding was present in the sera of the tested rats. Example 2: Construction of candidate Ad-ANS12; an Adenoviral candidate comprised of chimpanzee serotype 68 with small serotype 5 sequences, with single gene insert with codon-optimized sequence of the receptor binding domain (RBD) for more expression, is prepared. AD-ANS12:

1. Construction of the adenoviral candidate: a. Creation of shuttle plasmids: Four plasmids were created for creation of ANS12. i. pAD1144-ANS15 (E1/pIX) (see FIG 18) which has CMV promoter driving the expression of codon-optimized RBD leading to expression of 193 amino acids ii. pAD1145 (see FIG 19) (E2/ late genes) iii. pAD1146-01 (see FIG 20) (E3/ fiber genes) with 4.4 kb deletion in E3 gene iv. pAD1147 (see FIG 21) The importance of codon-optimized sequence of antigen provides an advantage because of creation of only neutralizing antibodies against the receptor binding domain of the coronavirus. b. Cosmid Construction Ad-ANS12: The entire genome of Ad-ANS12 was reconstituted in a cosmid using the four shuttle plasmids mentioned above namely; pAD1144-ANS15 (E1/pIX shuttle), pAD1145 (E2/late shuttle), pAD1146-01 (E3/ fiber shuttle) and pAD1147 (E4 shuttle). These four plasmids were used to create a cosmid with restriction enzyme digestion and ligation with T4 DNA ligase. The ligation product was packaged into lambda phage heads which were subsequently used to infect competent E. Coli. E. coli were selected with the help of ampicillin and kanamycin selection and cosmid DNA was purified using the alkaline lysis method and confirmed with restriction enzyme digestion to be correct. c. Rescue: i. Transfection: Two 6-cm dishes of 293 cells were transfected with PacI-digested pAd5-ANS12 using the calcium phosphate-DNA co-precipitation method. Ad5.CMV-GFP DNA (a known GFP insert) was used as positive control. Cells were rinsed twice with DMEM about 12 hours after the transfection. ii. Harvest: First plaque was detected on day 5 after the transfection. Virus plaque harvest (day 10 post-transfection) was conducted from the dishes of the HEK293 cells transfected with pAD-ANS12; four plaques were harvested. d. Amplification: i. Pre-amplification (first round of expansion): A confluent 15-cm dish of 293 cells was split into 6-cm dishes at 30% confluence. The dishes were infected directly with Ad-ANS12 plaques #1-2-3-4 harvested earlier. On day 6 post-infection, the dish of 293 cells infected with Ad-ANS12 showed complete cytopathic effect (CPE). Cells and medium were harvested, frozen/thawed three times (3x), clarified by centrifugation and aliquoted into sterile 1-mL vials. ii. Amplification (second round of expansion): Confluent 10-cm dishes of 293 cells were infected with the crude lysates of Ad-ANS12 clones harvested. On day 2 post-infection, all dishes showed strong CPE. Cells and medium were harvested in the following way. 1.5 mL infected cell suspension was centrifuged for 2 min at 3,000 rpm, RT. The cell pellets were washed twice with PBS, then frozen at -70°C. These cell pellets were used for Western Blot testing for expression studies (for both candidate vaccines). Viral DNA (vDNA) was extracted from 4 mL infected cells suspension following the Hirt method. The remaining infected cell suspension was frozen/thawed 3x, clarified by centrifugation and aliquoted into sterile 1-mL vials.

2. Characterization of the candidate vaccine: i. Genome restriction analysis: vDNA was extracted from Ad-ANS12-infected 293 cells according to the Hirt method. The identity of the virus was assessed by restriction digestion with MfeI, AflIII and XcmI (see FIG. 22). The insertion of the 5’ UTR-optimized CMV-OPT-RBD cassette in the virus is demonstrated by the presence of the 1249- and 2634-bp MfeI fragments, the 1353- and 3020-bp AflIII fragments and the 1224- and 1675-bp XcmI fragments (bars in the gel pictures) (see FIG. 22). All 3 clones show identical restriction patterns, which indicates that the virus is stable. ii. Physical titer: The OD260-SDS method was used to determine the concentration of virus particles in the purified stocks of Ad-ANS12. The concentration of VP in the preparations was calculated given the extinction coefficient of 1.1 x 10e12 virus particles (VP) per Abs260 unit in presence of SDS. Ad-ANS12 physical titer from one batch was calculated as 1.1 x 10e13 at a concentration of 2.7 10e12 vp/ml. iii. Infectious Titer: The concentration of infectious particles in the purified stocks of Ad-ANS12 was determined by end-point dilution assay (TCID50), using virus growth as indicator. The infectious units per ml ratio was determined to be 1 x 10e11/ml. iv. VP/IU Ratio: VP/IU (viral particle/ infectious units) ratio for AD-ANS12 was calculated to be 27. v. Genome sequencing: The entire genomes of Ad-ANS12 was sequenced by NGS on an Illumina iSEQ100 instrument. a. vDNA purification: vDNA was extracted from purified VP of Ad-ANS12 using proteinase K. The DNA was resuspended in 30 µl TE pH 7.5. vDNA concentration of 0.35 µg/ µl from 2.7 x 10e12 VP/ml of stock concentration of virus. b. The sequencing and analysis confirmed the presence of the intact CMV-OPT-RBD-SV40pA expression cassette in the E1 region. vi. Transgene expression: Expression of vaccine antigen coronavirus spike glycoprotein subunit S1 was confirmed by western blot assay conducted under non-reducing and reducing conditions. Expression of the antigen was confirmed where monomeric, dimeric and trimeric forms of the RBD were seen on the western blot (see FIG. 16). Primary antibodies against the Receptor Binding Domain were used to detect the protein. Results showed detectable RBD protein in the Ad-ANS12 infected cells from 3.7 mg/ml to 4.48 mg/ml). vii. RCA assay: The presence of replication-competent adenovirus (RCA) in the purified preparations of Ad-ANS12 was assessed using a modified infectivity/PCR method, which combines the amplification of infectious RCA by cell culture with the sensitivity of detection of E1-specific sequences by PCR. For each construct, 10 x 15-cm dishes of A549 cells (~ 2.5 x10e8 cells) were infected with 3 x 10e10 VP. Five days after the infection, the infected cells were harvested, pooled, and frozen/thawed 3 times in order to release the virus particles. New monolayers of A549 cells seeded in 10-cm dishes were infected with these crude virus lysates and cultured for another 5 days. Cells were harvested and viral DNA was purified according to the Hirt method. It was used as template for the detection of a 220 bp-long E1-specific sequence by PCR. As controls, two dishes of A549 cells were infected with a RCA-free control virus spiked with 10 and 100 VP of WT Ad5. The results showed the absence of detectable E1-specific sequence in the DNA extracted from A549 cells infected with Ad-ANS04, Ad-ANS06 and Ad-ANS12. Thus, no RCA was detected in 3 x 10e10 VP of the purified preparations of Ad-ANS12 (see FIG. 17). Animal Studies: The candidate vaccine Ad-ANS12 was tested in two animal studies, dosing outline and groups of the rat repeated dose toxicity and immunogenicity study are shown in FIG. 25 and FIG. 26 respectively. Also, the repeated dose toxicity was conducted in New Zealand rabbits; dosing outline and groups are shown in FIG.- 30 and FIG.- 31 respectively. The candidate was given intranasally at a dose of 2x10e10 vp per dose given at day 0, day 14 and day 28 and intramuscularly in a different group of animals at a dose of 5x10e10 vp per dose given at day 0 and day 28. The vaccine was safe with no safety concerns revealed in rats and rabbits. Day 28 immunogenicity (after 2 doses) results showed presence of neutralizing antibodies against the Coronavirus as shown in FIG. 28 (for intranasal dose) and FIG. 29 respectively (for intramuscular dose). The figures show results of detection of SARS-CoV-2 antibodies by bio layer interferometry. The curves show that high rate of antigen antibody binding was present in the sera of the tested rats. The embodiments herein above and the various features and advantageous details thereof are explained with reference to the non-limiting embodiments in the following description. Descriptions of well-known components and processing techniques are omitted to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skilled in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein. While considerable emphasis has been placed herein on the components and component parts of the preferred embodiments, it will be appreciated that many embodiments can be made and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other changes in the preferred embodiment as well as other embodiments of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.

Claims:We Claim,
1. A composition of constructs of adenoviral product containing the modified DNA sequences coding specific peptides for use as a therapeutic against covid-19 for administration to a newly diagnosed patient of coronavirus to change the course of infection by developing rapid innate immune response and method of preparation of adenoviral therapeutic products thereof comprising of at least one or a combination of components as follows;
a. S1 subunit of the S protein or RBD domain of the S protein,
b. S1 subunit of the S protein or RBD domain of the S protein both tagged with an adjuvant peptide,
c. S1 subunit of the S protein or RBD domain of the S protein or multiple proteins of SARS Co-2 for vaccination or all such proteins tagged with a FLAG peptide,
d. specific DNA sequences in combination such that the size of an adenoviral vaccine genome will exceed the packaging capacity in case of homologous recombination event during expansion of virus in HEK-293 cells during manufacturing; where one sequence is naturally occurring variant of the RBD or S1 subunit, another sequence is codon-optimized version of the RBD or S1 subunit and third sequence is also a variant of the RBD or S1 subunit sequence such that none of the three sequences share a length over 20 to 30 bp which would increase the chance of homologous recombination within the added sequences,
e. specific sequences of Chimpanzee Serotype 68 adenovirus such that they include minor sequences of serotype 5 adenovirus and include a variation of above mentioned one to three sequences of the coronavirus,
f. RBD and/or S1 subunit sequences where the natural signal peptide is replaced by a synthetic signal peptide such that the antigen is secreted out of the cells and is made available for antibody generation and or a Foldon domain is attached at the 3’ terminal of the coding region such that that a trimer is formed after ribosomal synthesis of the peptide,
g. And adenoviral vector which can be administered intranasally for development of immediate and rapid innate immune responses
wherein, composition of DNA constructs of a tricistronic adenoviral vaccine candidate of serotype 5 as shown in SEQ ID NO: 6 and FIG. 9 comprises of three DNA sequences encoding;
a.) the receptor binding domain (RBD) by using codon optimized RBD sequence (as shown in SEQ ID NO- 1 and FIG. 1),
b.) natural sequence of the RBD SARS Cov-2 (as shown in SEQ ID NO- 17 and FIG. 24), and,
c.) S1 subunit of Coronavirus fused with a Foldon tag (as shown in SEQ ID NO- 2 and FIG. 2) formulated for intranasal, oral, intramuscular or subcutaneous administration for prevention against covid-19, using plasmids shown in FIG. 13, FIG. 14, FIG. 15, FIG. 16 (as presented in sequences 8,9,10 and 11),
such that the adenovirus generates innate immunity due to adenoviral envelope proteins and adaptive immunity to Coronavirus due to virtue of secreted synthetic secretory peptide used in combiation with the antigens such that the signal peptide is cleaved after translation and the RBD antigen of 193 aa length is secreted in to the extracellular space for the generation of neutralizing antibodies of IgA and IgG types mainly; and
wherein composition of DNA constructs of a monocistronic adenoviral vaccine of serotype Chimpanzee 68 with minor sequences from Serotype 5 as shown in SEQ ID NO: 7 and FIG. 12 comprised of; a single DNA sequence encoding the receptor binding domain (RBD) by using codon optimized RBD sequence (as shown in Sequence 1 and FIG. 1 using plasmids shown in FIG. 18, FIG.19, FIG.20 and FIG. 21 (as presented in SEQ ID NO- 12, 13, 14 and 15).

2) The composition as claimed in claim 1, wherein DNA construct of SEQ ID NO: 2, comprises of, a DNA sequence encoding the codon optimized S1 subunit of the SARS Cov-2, formulated for adenovorial delivery via intranasal, oral, intramuscular or subcutaneous administration, such that the DNA constructs of SEQ ID NO: 2 leads to a biologically expressed S1 subunit, in the trimerized form due to its fusion part of foldon domain for the generation of neutralizing antibodies.

3) The composition as claimed in claim 1, wherein DNA construct of SEQ ID NO: 1 comprises of, a DNA sequence encoding the codon optimized RBD domain of the SARS Cov-2, formulated for adenovorial delivery via intranasal, oral, intramuscular or subcutaneous administration, such that the DNA constructs of SEQ ID NO: 1 leads to a biologically expressed RBD domain secreted by use of a synthetic secretory peptide used in combiation with the RBD domain such that the signal peptide is cleaved after translation and the S1 subunit is secreted in the extracellular space for the generation of neutralizing antibodies.

4) The composition as claimed in claim 1, wherein SEQ ID NO.- 16 as shown in FIG. 23 is map of plasmid pAD1129-ANS10-modified with DNA sequence created for vaccination against the South African strain B.1.351 which carries mutation from both UK and South African Strain (mutations shown in the black box).

5) The DNA constructs as claimed in claim 1, wherein the adenoviral vaccine candidates (SEQ ID NO.-6 and SEQ ID NO.-7) as produced are such that:
i. RBD antigen produces neutralizing antibody only, and does not lead to disease enhancement,
ii. Signal peptide attached to the RBD sequence helps extracellular secretion of the antigen once adenoviral particle produces the antigen in the cells, thus increasing the chances of formation of antibodies,
iii. S1 subunit attached to foldon trimerization domain is expressed as well in the same viral particle increasing the range of antibodies produced and increasing chance to produce antibodies in the natural trimer form,
iv. Adenoviral genome is made such that homologous recombination is not viable and this eliminates the risk of producing replication competent virus even in HEK 293 cell line,
v. Intranasal or oral delivery of the candidates overcomes the drawbacks of thrombosis as the candidate vaccine does not enter the blood and provides high quality mucosal immunity,
vi. Chimpanzee Adeno candidate using the RBD sequence is also able to be given intranasally or orally and thus provides mucosal immunity against the coronavirus without side effects of thrombosis as it never enters the bloodstream,
vii. Using natural sequence of the Coronavirus RBD in the same viral genome where the codon-optimized sequence of the Coronavirus RBD is used, prevents any homologous recombination and yet produces the same protein the double amount (two sequences for the same peptide, one natural and one codon-optimized, increasing the overall protein production).
viii. The adenoviral nature of the therapeutic drastically changes the outcome of the coronavirus infection and can be used any number of times to prevent development of complicated Covid19 disease by simple intranasal or oral administration of the candidate after the diagnosis of coronavirus infection.

6) The DNA constructs as claimed in claim 1, wherein the method of preparation of Adeno viral therapeutics product comprises the steps of:
1. Construction of the adenoviral candidates which includes the steps of:
a. Creation of shuttle plasmids,
b. Cosmid Construction,
c. Rescue,
d. Amplification, and,
2. Characterization of the adenoviral candidates which includes the steps of:
i. Genome Restriction Analysis,
ii. Physical titer,
iii. Infectious titer,
iv. VP/ IU ratio,
v. Genome sequencing,
vi. Transgene expression,
vii. RCA assay.

Dated this 28th day of April, 2021.
For the applicant,
____________
Mrs. Allison Katariya Agent of the applicant -IN/PA-2190
, Description:FIELD OF THE INVENTION:
The invention relates to the field of cell and molecular biology, immunology, adenoviral therapeutic and vaccine candidates for treatment of SARS-Cov-2, and more particularly relates to preparation of compositions of constructs of modified DNA sequences for treatment against Covid-19, and even further to the production of a large genome adenoviral product of serotype 5 and an optional booster of a different serotype.

BACKGROUND:
Pandemic of Covid-19 caused by the SARS Cov-2 has caused fatalities with life-long repercussions globally and demands urgent and critical address in the form of a treatment. This disease can cause fatalities amongst the elderly population especially above 70 years of age at a high rate, however it can also cause mass hospitalizations (up to 20% for all population) and a prolonged halt in the economy could lead much disastrous results for pediatric health as well as health of all those who depend on the daily wages for a surviving nutrition. The disease course ranges from mainly asymptomatic and mild to more severe and critical, in 10% to 20% of symptomatic patients who may be at considerable risk of fatality with many different organ systems. Every day there are hundreds of thousands of patients getting infected and there is no space in the hospitals or especially in the intensive care units. The present invention discloses life-saving solution to this deadly virus in the form of intranasal or oral mucosal route delivered therapeutic candidates that prevent the otherwise reported complications and deaths as well as the drawbacks like clotting which are associated with the other adenoviral products or mRNA or protein products which are administered by intramuscular route. An effective immune response against SARS-CoV-2 requires both arms of the immune system, the innate immune system and the adaptive immune system, however the innate immunity fails to control the Coronavirus infection in cases of serious and protracted illness. Current understanding suggests that the SARS-CoV-2 enters type II pneumocytes via ACE2 in the respiratory system leading to rapid virus replication as well as concomitantly to a proinflammatory state with elevated levels of cytokines such like interleukins IL-1, IL-6, and CXCL8, and TNF. Proinflammatory genes upregulated by the innate immune system belong to NH-kB pathway of proinflammatory mediators and they lead to accumulation of pathogenic inflammatory neutrophils and macrophages in the lung. The elevation of cytokines in severe disease is accompanied by elevated alanine aminotransferase, lactate dehydrogenase, C-reactive protein (CRP), ferritin, and D-dimers. The elevated interleukin IL-6 and TNF responses lead to inflammatory cell death with concomitant elevation of TNF and IFN-? to trigger pyroptosis, apoptosis and necrosis driving he tissue damage and mortality in the Covid-19 patients.
If the immune response of mucosal infection can be shifted early on to bring in more natural killer cells, the serious disease would not develop. Adenovirus vectors induce the innate immunity of the immune system that leads to the inflammation of transduced tissues and efficient clearance of administered vectors. Unlike adaptive immunity, the innate response is mediated by the adenovirus particles and does not require viral transcription, thus the production of antigenic peptides is not required at this stage. Their presence rather has an enhancing effect for providing a repertoire of antigens to the T cells such that it comes without the replicating virus which may try to evade the immune system and thus leads to the development of antibodies.
Severe acute respiratory syndrome (SARS) emerged as a new disease threat in late 2002 which spread to several countries with considerable loss of life. The etiological agent in the first outbreak was SARS Coronavirus- 1 (Cov-1). Coronaviruses can infect respiratory, gastrointestinal, hepatic and nervous systems of humans, birds, mice, livestock, bats and many other animals. The Severe Acute Respiratory Syndrome (SARS) outbreak of 2002 and Middle East Respiratory Syndrome (MERS) of 2012 outbreaks gave us warning of ‘animal to human’ and ‘human to human’ transmission of newly emerging coronaviruses. During late 2019, a severe respiratory disease was reported in the city of Wuhan, Hubei Province, China. This disease was named Covid-19 and was caused by another strain of Coronavirus; SARS Cov-2.
The coronaviruses causing SARS of 2002 or Covid-19 are single-stranded positive sense RNA virus, whose genome encodes nonstructural replicase polyprotein (rep), and structural proteins such as Spike (S), envelope (E), membrane (M) and nucleocapsid (N).
Phylogenetic analysis of the viral genome revealed that the SARS-Cov-2 was closely related to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus). These viruses were previously sampled from bats in China.
The complete genome of SARS Cov-2 (Wuhan) has been analyzed after collecting broncho-alveolar lavage from one patient who exhibited the SARS from Cov-2. SARS Cov-2 (causing Covid-19) virus is not very different from SARS Cov-1 that caused the outbreak in year 2002/2003. What is significantly different with this virus is its high infectivity and relatively lower fatality rate. Our own comparisons of the two viruses for their sequences in the specific receptor binding domain as well as others have shown that the virus has significant amino acid sequence similarity (overall region in the RBD- similarity 83% and 50% similarity in the Receptor binding motif). Antibodies induced by anti-SARS vaccines can cross-neutralize bat SARS-like coronaviruses. The RBD domain of the Coronavirus binds the Angiotensin Convertase Enzyme receptor 2 (ACE2) which leads to the infection of cells in the respiratory as well as gastrointestinal tracts, hence the use of RBD as an antigen for therapeutic will provide neutralizing antibodies which are essential for the treatment of the infection to be successful. The inhaled virus SARS-CoV-2 likely binds to epithelial cells in the nasal cavity and starts replicating after gaining entry via attachment to the ACE2 receptors without much resistance by the innate immune response.
For the mechanism of action for the therapeutic product it is essential to understand the body’s immune response against a pathogen like coronavirus. Body has two types of immune responses; innate and adaptive. Innate immunity consists of macrophages, dendritic cells, mast cells, granulocytes (WBCs including neutrophils, basophils and eosinophils) and complement protein. Adaptive immune response consists of B cells producing antibodies, memory B cells and T cells (CD4+ and CD8+). The natural killer T cells take part in both innate and adaptive immune responses. The innate immune response is immediate and rapid while the adaptive immune response is slow and takes up to 2 weeks to develop.
During the local mucosal propagation of the Coronavirus (during which time the infected people are also infectious), there is limited local innate immune response. The adenoviral non-replicative vectors can generate an innate immune response rapidly which does not require viral transcription to occur and thus start the innate immune response immediately thus preventing the complicated Covid disease and by the time two weeks have passed the antigens transcribed and translated by the adenoviral vector also contribute to the adaptive immune response and development of antibodies against the coronavirus which brings the best of both worlds to the mechanism of action of this adenoviral therapeutic carrying a payload of antigens which can generate neutralizing antibodies.
Thus, the advantage of the present invention over existing art is that the product are such that they recruit the innate system early on and ramps up the innate immune response which in turn prevents development of serious disease, the DNA constructs of the present invention are such that they provide antigens to the body which are transcribed by the adenovirus and are readily available for antibody generation (unlike the coronavirus which would cause infection and delay in the development of adaptive immune response).
Another advantage of the present invention is that the product does not enter the blood stream when given intranasally or orally, thus it does not need to be produced in the serum free media. This can give a major advantage to a country like India which can feel shortages of serum free media supplies from Western Countries.
Another advantage of the present invention is that it will also act as a vaccine in people who are not infected with the coronavirus and can be administered as many times desired with no known side effects. Adenoviral infections of upper respiratory tract are very common and pose no risk to humans and yet the vaccine mechanism bypasses any existing immunity against the adenoviruses. Currently marketed vaccines cause multiple side effects like blood clots and neurological disorders which are not expected with our invention.
Another advantage of our invention is that it can be given without any aid from a doctor or a nurse, at outpatient venue, can remain stable at 2 to 8 degree Celsius up to a month without affecting its potency and thus can be delivered and used in developing countries in a much user-friendly manner.

OBJECT OF THE INVENTION:
The primary object of the invention is to provide compositions of DNA constructs either used alone or in combination to provide prevention, diagnosis and treatment of Covid-19.

Yet another object of the present invention is to provide composition of DNA constructs specifically for treatment of coronavirus infection to avoid complications leading to severe covid-19 infection in the form of an adenoviral product that can be administered as a tablet given orally or drops for intranasal administration.

SUMMARY OF THE INVNETION:
Before the present invention is described, it is to be understood that the present invention is not limited to specific methodologies and materials described, as these may vary as per the person skilled in the art. It is also to be understood that the terminology used in the description is for the purpose of describing the particular embodiments only and is not intended to limit the scope of the present invention.
The present invention discloses composition of constructs of modified DNA sequences and method of preparation of therapeutic candidates using adenoviral vectors and constructs of modified DNA sequences against covid-19. Two therapeutic candidates which are also useful as vaccine candidates are mainly described in the present patent application; one is a Serotype 5 adenoviral therapeutic and another is a Serotype Chimpanzee 68 adenoviral therapeutic. No serotype can be patented just as no species of animals or plants can be patented. Because an entire serotype of a Virus cannot be patented, in this invention, we apply to patent the use of Serotype 5 and Serotype 68 (not exclusively only these 2 serotypes) adenoviral vectors carrying DNA sequences generating neutralizing antibodies after intranasal or oral/ mucosal administration such that the DNA modifications and novel use of multiple coronavirus epitopes in a single viral particle for intranasal, oral as well as intramuscular use in the form of a Serotype 5 or Serotype 68 adenoviral therapeutic expressing such epitopes by virtue of modified sequences of RBD or S1 subunit alone or in combination with readily alternative use as a vaccine. The serotype 5 adenovirus is to be used as the primary therapeutic followed by administration of the serotype 68 virus candidate for the booster effect.
The invention relates to the compositions of DNA constructs of SEQ ID NO: 1 to 5 for use in the treatment of Covid-19 in humans as well as animals.
The invention relates to the compositions of DNA constructs of SEQ ID NO: 6 and 7 which are the complete genome sequences of two adenoviral therapeutic candidates successfully developed based on the sequences described in SEQ ID NO: 1 to 5.
The invention relates to the compositions of DNA constructs of SEQ ID NO: 8,9,10,11,12,13,14 and 15 which are the plasmid sequences used for the creation of two adenoviral therapeutic candidates. These plasmids were used in creating the whole genome sequences of SEQ ID NO: 6 and SEQ ID NO: 7.
The invention relates to the compositions of DNA constructs of SEQ ID NO: 16 which is the plasmid sequence used for the creation of adenoviral therapeutic candidate against the South African variant strain B.1.351. However, it is not essential to create a separate candidate for the South African variant or other variants unless the population spread of a specific strain is at large scale. The serotype 5 therapeutic candidate would sufficiently work for the purpose of treatment of all strains of coronavirus as generation of innate immune response is primary mode of mechanism of action.
The said composition of therapeutic with constructs of modified DNA sequences comprises of at least 7 different important functional components as follows:
a) Adenoviral vectors mainly of Serotype 5 with large genome capacity which incapacitates the packaging of adenovirus in case of a homologous recombination even during manufacturing
b) S1 subunit of the S protein or RBD domain of the S protein.
c) S1 subunit of the S protein or RBD domain of the S protein both tagged with an adjuvant peptide; for creating a better immune response.
d) S1 subunit of the S protein or RBD domain of the S protein or multiple proteins of SARS Co-2 for generating innate as well as adaptive immune response;
e) Use of specific DNA sequences in combination such that the size of an adenoviral genome will exceed the packaging capacity in case of homologous recombination event during expansion of virus in HEK-293 cells during manufacturing; where one sequence is naturally occurring variant of the RBD or S1 subunit, another sequence is codon-optimized version of the RBD or S1 subunit and third sequence is also a variant of the RBD or S1 subunit sequence such that none of the three sequences share a length over 20 to 30 bp which would increase the chance of homologous recombination within the added sequences.
f) Specific sequences of Chimpanzee Serotype 68 adenovirus such that they include minor sequences of serotype 5 adenovirus and include a variation of above mentioned one to three sequences of the coronavirus.
g) RBD and or S1 subunit sequences where the natural signal peptide is replaced by a synthetic signal peptide such that the antigen is secreted out of the cells and is made available for antibody generation and or a Foldon domain is attached at the 3’ terminal of the coding region so that a trimer is formed after ribosomal synthesis of the peptide.
The primary mode of delivery of therapeutic targets would be intranasal, oral or parenteral (subcutaneous or intramuscular) delivery of a replication-incompetent adenoviral vehicle that can be produced by any of the available platforms.
The present invention discloses DNA constructs that can be used for the prevention, diagnosis and treatment of Covid-19. For therapeutic purpose, the present invention describes composition of DNA construct of S1 subunit of the Spike Glycoprotein as well as DNA constructs of the Receptor Binding Domain (RBD) of the S protein (spike glycoprotein) tagged with a Foldon domain (FD) for trimerization and or antigens S1 or RBD attached with a synthetic signal peptide that will secrete the peptide out of the cell to make it available for antibody formation and methods to create adenoviral therapeutic product such that the adenoviral particles are replication non-competent and can be cultured in HEK-293 cells to remain replication non-competent by making candidates incapable to generate a viable replication competent adenovirus by homologous recombination.
In summary, the DNA constructs of the present invention will be essential in controlling, treating and eliminating the Covid-19 disease, by virtue of diagnosis, prevention and treatment of the disease and the methods associated can be used in creation of therapeutic against many other infectious diseases like respiratory infections causing tuberculosis or infections of the gastrointestinal tract or genital tract like HPV or syphilis. This same technique can also be used in creating vaccine or therapeutic products against fungal infections, parasitic infections, viral as well bacterial infections.

BRIEF DESCRIPTION OF THE DRAWINGS:
The present invention, together with further objects and advantages thereof, is more particularly described in conjunction with the accompanying drawings in which:

FIG. 1 is a diagrammatic representation of the DNA construct of SEQ ID NO: 1 carrying an optimized codon sequence of receptor binding domain (RBD) of SARS Cov-2 for expression via adenoviral delivery;

FIG. 2 is a diagrammatic representation of the DNA construct of SEQ ID NO: 2 carrying an optimized codon sequence of predicted S1 subunit of S Protein with a Foldon trimerization domain attached at the 3’ end for expression via adenoviral delivery;

FIG. 3 is a diagrammatic representation of the DNA construct of SEQ ID NO: 3, carrying an optimized codon sequence of receptor binding domain (RBD) of SARS Cov-2 and adjuvant peptide that would separate and get secreted to the extracellular space after translation for adenoviral delivery;

FIG. 4 shows predicted 3-D structures for SARS Cov-1 epitope, SARS Cov-2 epitope and epitope RBD in our product; the three epitopes are seen having similar 3-D structure.

FIG. 5 shows alignment of RBD domains from SARS Cov-2 and SARS Cov-1 (labeled as Cov-2 and Cov-1) with the rectangles showing specific sites that have been shown to generate neutralizing antibodies against the SARS Cov-1 virus;

FIG. 6 shows the Western Blot assay expression results of the RBD protein expressed from the natural sequence detected in HEK-293 cell culture supernatant with help of a FLAG epitope under reducing conditions (only a monomer in Lane 1 and Lane 2) and under non-reducing conditions (monomer, and faint bands of dimer and trimer in Lane 3).

FIG. 7 shows the Western Blot assay expression results of the RBD protein expressed from the codon-optimized sequence detected in HEK-293 cell culture supernatant with help of a FLAG epitope under reducing conditions (only a monomer in Lane 2 and Lane 3) and under non-reducing conditions (monomer, and faint bands of dimer and trimer in Lane 5).

FIG. 8 shows the Western Blot assay expression results of the S1-FD protein expressed from the codon-optimized sequence detected in HEK-293 cell culture supernatant with help of a FLAG epitope under reducing conditions (only a monomer in Lane 1 and Lane 2) and under non-reducing conditions (monomer, and strong bands of dimer and trimer in Lanes 3 and 4).

FIG. 9 shows the map of Ad-ANS06 Serotype 5 adenoviral candidate vaccine genome.

FIG. 10 shows the map of Ad-ANS12 Serotype Chimpanzee 68 adenoviral candidate vaccine genome.

FIG. 11 shows plasmid pAD1127-ANS05 with EF1a-RBD cassette in place of the E1 region; where the RBD sequence is tagged with a synthetic signal peptide for secretion of the antigen outside the cells.

FIG. 12 shows the backbone pAD1128 plasmid for the Serotype 5 adenovirus with wild-type Ad5 E2 and late genes.

FIG. 13 shows the plasmid pAD1129-ANS10 with CMV-S1FD cassette in place of the E3 region and RSV-OPT-RBD cassette between the L5 and E4 poly A signals used for the creation of Ad-ANS06 tricistronic serotype 5 adenoviral vaccine candidates; where the RBD sequence is codon-optimized and natural signal peptide is replaced by synthetic signal peptide and S1 subunit sequence still carries the natural signal peptide sequence but is attached to a Foldon trimerization domain at the 3’ end.

FIG. 14 shows pAD1130 plasmid with E4 genes, this is one of the plasmids used in the creation of adenoviral vaccine candidate ANS06.

FIG. 15 shows the restriction enzyme digest confirmation of genomic map of the cosmid created for the Ad-ANS06 genome.

FIG. 16 shows Results of Western Blot Assay for created adenoviral candidate Ad-ANS06 in HEK-293 cells (confirmation of antigen expression of the actual therapeutic candidate created from the plasmids) where all lanes showed antigen in monomeric, dimeric and trimeric forms (marked within oval shapes).

FIG 17 shows Results of RCA assay showing no replication competent viral particles for both Ad-ANS06 and Ad-ANS12.

FIG. 18 shows plasmid pAD1144-ANS15 with CMV-codon optimized RBD cassette in place of the E1 region used for the creation of chimpanzee adenoviral candidate Ad-ANS12.

FIG. 19 shows plasmid pAD1145 used for the creation of chimpanzee adenoviral candidate Ad-ANS12.

FIG. 20 shows plasmid pAD1146 used for the creation of chimpanzee adenoviral candidate Ad-ANS12.

FIG. 21 shows plasmid pAD1147 used for the creation of chimpanzee adenoviral candidate Ad-ANS12.

FIG. 22 shows the restriction enzyme digest confirmation of genomic map of the cosmid created for the chimpanzee adenoviral candidate Ad-ANS12.

FIG. 23 shows the map of the first plasmid pAD1129-ANS10-modified with DNA sequence created for vaccination against the South African strain B.1.351 as well as British Strain B.1.1.7 (common mutation N501Y) which carries mutations from both UK and South African Strain (mutations shown in the black box);

FIG. 24 shows the map of the natural sequence of the RBD domain.

FIG. 25 and FIG. 26 show the groups of rats used in the animal study where the two therapeutic candidates Ad-ANS06 and Ad-ANS12 were tested.

FIG. 27, FIG. 28 and FIG. 29 show the results of detection of SARS-CoV-2 antibodies by bio layer interferometry in the rat sera vaccinated with the adenoviral products Ad-ANS06 and Ad-ANS12 in the animal studies. The curves show that high rate of antigen antibody binding was present in the sera of the tested rats.

FIG. 30 and FIG. 31 show the groups of rabbits used in the animal study where the two therapeutic candidates Ad-ANS06 and Ad-ANS12 were tested.

DETAILED DESCRIPTION OF THE INVENTION:
The disclosure has been described with reference to the accompanying embodiments which do not limit the scope and ambit of the disclosure. The description provided is purely by way of example and illustration.
The foregoing description of the specific embodiments so fully revealed the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the scope of the embodiments as described herein.
Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results.
The present invention discloses novel compositions of constructs of modified DNA sequences (SEQ ID NO: 1-17) administered via adenoviral vector, for the treatment of Covid-19 disease. The compositions of the said constructs of the modified DNA sequences as described in the present invention for treatment of Covid-19 do not necessarily need a specific promoter or a specific plasmid for the delivery and expression.
In one embodiment of the present invention, the first DNA cassette SEQ ID NO: 1 Receptor Binding Domain of the SARS Cov-2 is constructed and administered via adenoviral particles via intranasal, oral mucosal, routes for generation of innate immunity and to provide protection against the development of serious Covid-19 disease which is manifestation of a derailed and uncontrolled immune response.
In another embodiment of this invention, the availability of the antigen for the generation of adaptive immunity via adenoviral viral vector delivery is enhanced because of presence of a synthetic signal peptide replacing the natural signal peptide associated with the RBD domain which gets released into extracellular space after translation due to the presence of synthetic signal peptide.
The DNA cassettes described in various embodiments of the invention are delivered preferably via intranasal-mucosal route mucosal route intranasally or, orally or via other routes mainly subcutaneously for gene delivery by adenoviral systems.
The different embodiments of the invention are mentioned below. However, different combinations of these constructs can be made and administered by replacing the epitopes for a specific virus or specific promoter for controlled or constitutive expression of described peptides.
The present invention discloses compositions of constructs of modified DNA sequences of constructs of modified DNA sequences/ DNA cassettes (SEQ ID NO: 1 to SEQ ID NO: 17) encoding the Kozac sequence for increasing the expression of the epitope, codon-optimized gene sequence of the RBD epitope followed by a stop codon under expression control of any promoter, e.g. CMV promoter, EF1 promoter or RSV promoter. The RBD epitope is preserved among the SARS Cov-1 and SARS Cov-2, as shown in FIG. 4 and FIG. 5. Also as seen in FIG. 5, the rectangle marked epitopes are not only highly conserved but also generate neutralizing antibodies. The RBD protein 3-D structure also shows conservation across SARS Cov-1 and SARS Cov-2. The chosen therapeutic epitope also shows the same 3D structure which would be essential for generation of same neutralizing antibodies. The RBD protein 3-D structure also shows conservation across SARS Cov-1 and SARS Cov-2. The chosen epitope is essential for generation of same neutralizing antibodies. The adenoviral delivery of RBD epitope is a preferred way for our product because RBD can generate neutralizing epitopes, adenoviral particles can provide the mucosal immune response against the viruses like SARS Cov-2 along with the systemic immune response. Even when a strain variant may have a slightly different 3-D therapeutic the mechanism of action of the therapeutic remains unaffected because the first part of the mechanism of action consists of generation of innate immunity by presence of adenoviral particles.
In another embodiment of the invention, S1 subunit of the S protein is used instead of RBD as the vaccine epitope or the therapeutic epitope. Hence the codon optimized sequence of S1 subunit is used for the generation of antibodies (FIG. 2) preferably tagged with Foldon Domain. The adenoviral viral vector efficacy is enhanced because of presence of a Foldon trimerization domain at the end of the S1 subunit such that the S1 subunit forms a trimer and generates high quality antibodies after translation due to the presence of Foldon domain (FIG. 2).
In another embodiment of the invention (shown in FIG. 23), the DNA sequence is modified to reflect certain mutations in the virus such that a vaccine or a therapeutic can be created against the South African variant B.1.351 as well as a common mutation in the British Strain B.1.1.7 (N501Y). These Sequence variations are described below:
Plasmid pAD1129-ANS10 sequence modifications:

i. 6403 G to C
ii. 1233 G to C
iii. 2130-2133 CTG to CAC
iv. 2142-2144 AGA to ATC
v. 2655-2657 AAA to AAC
vi. 2856-2858 GAA to AAG
vii. 2907-2909 AAC to TAC
In another embodiment, Ad-ANS06 the vector (shown in FIG. 9 as Ad-ANS06b) is designed such that the genome size (of 36.2 KB) becomes unpackageable to produce replication competent virus particles in case homologous recombination occurs in the HEK-293 cells (genome size would become over 38 KB if homologous recombination occurs and thus becomes unpackageable). Thus, this invention eliminates the need of specialized culture cells for production of adenoviral vaccines if the same method can be used for insertion of multiple antigens in the same viral genome. Another advantage this embodiment offers is to provide means of expressing multiple copies of genes or different genes at the same time by use of the same viral particle.
In yet another embodiment, Ad-ANS12 candidate vaccine (shown in FIG. 10) was created by using a Chimpanzee adenoviral genome and few small sequences from serotype 5 adenovirus such that, it contains only one sequence of receptor binding domain to express the RBD epitope to generate immunogenicity in the vaccine.
This patent for the invention does not patent the serotype itself as a species of organism cannot be patented, however the use of serotype 5 for expression of RBD 193 Covid 19 epitope is being described as a novel embodiment for intranasal use or oral use in the form of an adenoviral therapeutic agent. In addition, use of Serotype 68, is the second therapeutic to be used after the first is used for the generation of long lasting immunity whenever such use is appropriate.
The present invention has many advantages as follows:
1) Immediate and quick generation of innate immune response to control the Coronavirus infection by virtue of adenoviral vectors administered to intranasal or oral mucosa.
2) Generation of mucosal immune response against the RBD domain of the SARS Cov-2 when used via an adenoviral vector. The immunity generating neutralizing antibodies against the RBD domain are extremely important because the SARS Cov-2 virus uses the RBD domain to bind ACE2 protein. Once the RBD is associated with ACE2, then the virus S protein transmembrane portions fuse with the cell membrane, giving entry into the cell. The route of entry of Coronavirus is mucosal (through respiratory as well as gastrointestinal tracts) hence the mucosal immunity provided by our vaccine candidates is of critical importance.
3) Antibodies generated against RBD and S1-subunit are mainly neutralizing type and are not expected to lead to antibody mediated disease enhancement.
4) Signal peptide used in the DNA constructs enhances the secretion of the antigen in the extracellular space making it available for the generation of antibodies.
5) The S1 subunit trimerizes due to Foldon domain and thus provides high quality antibodies for protective immunity against the disease.
6) The developed adenoviral therapeutic candidates can also be used for intranasal, oral as well as intramuscular delivery for use as a vaccine. Intranasal or oral delivery requires much smaller dose and generates the best quality of antibodies (IgA mucosal antibodies) that are required for blocking the entry of a respiratory pathogen. In another advantage, vaccines given by the intramuscular route may lead to thrombosis and intranasal or oral route eliminates or minimizes this risk completely.
7) Immunity provided against the South African variant and other variants is also of critical essence as described in one embodiment, the vaccine candidate contains the S1 subunit sequence with the mutations present in the variants and thus provides mucosal immunity against these variants.
8) Producing a tricistronic adenoviral vaccine candidate is advantageous such that because of use of natural and codon-optimized sequences in the same viral genome, the protein production (antigen production) is enhanced and yet chances of homologous recombination are minimized.
9) Another major advantage of the invention is that the adenoviral candidate Ad-ANS06 does not need a special cell line for its manufacturing even though it is of Serotype 5, where the DNA sequence is used to create an adenoviral vaccine of Serotype 5 type, in such a way that the genomic size of adenovirus prevents the viable homologous recombination and thus prevents inadvertent production of replication competent adenoviral particles, which is a major limiting factor in production of adenoviral vaccines in HEK cell line. United States Food and Drug Administration (USFDA) requires that there should be less than 1 replication competent adenoviral particles in 3x10e10 viral particles (vp) for human use. Although other health authorities including Indian FDA may require less than 1 replication competent particle per dose, in both instances the current embodiment of Serotype 5 adenoviral candidate is useful (Example-1).
10) Invention offers another advantage by creating an adenoviral candidate with Chimpanzee adenoviral serotype 68 in another embodiment, described below in example 2 (an adenoviral candidate vaccine Ad-ANS12), where the candidate vaccine was created using the said DNA sequence in such a way that the vector comprised of Chimp adenovirus type 68 carrying small type 5 adenoviral sequences. In this embodiment, an advantage is such that it prevents inadvertent production of replication competent adenoviral particles as homologous recombination is not possible between the genome of chimp adenovirus type 68 and adenoviral genes inserted in the Human Embryonic Kidney (HEK) cell line.

The HEK cells are used for the manufacturing of adenoviral vaccines since a long time and homologous recombination has been a challenge for the production of vaccines, the approach of the present invention to produce both the vaccine candidates eliminates this challenge as described in example 1 and example 2 due to novel composition of DNA constructs.
EXAMPLES
To further elucidate the mechanism of the invention, two examples are described here for multiple candidate vaccine preparation. The methodology for using the said DNA sequences for the creation of vaccine candidates is described below in brief.
In first example the three DNA sequences [first sequence with Coronavirus S1 subunit tagged with Foldon domain (S1-FD) (FIG. 2) is as in SEQ ID NO- 2)), second sequence with natural RBD sequence (FIG. 24 and as in SEQ ID NO- 17) and the third sequence codon-optimized RBD sequence (FIG. 1 and as in SEQ ID NO- 1)) were used by T4 ligation where a cosmid viral genome (as shown in FIG. 9 and SEQ ID NO- 6) was produced from four plasmid constructs to create a therapeutic candidate called as Ad-ANS06 (See FIG. 9, shown as Ad-ANS06b) which has a Serotype 5 Adenovirus genome and which would be expressing Receptor Binding Domain and S1 protein with trimerization Foldon domain. The Foldon domain sequence comprises of 27 amino acid (81 nucleotides) length C-terminal sequence from T4 bacteriophage. This Foldon sequence trimerizes any peptide it is attached with. The S glycoprotein of Coronavirus being a trimer, S1 subunit when in trimerized form produces not only antibodies to epitopes that are available on single chain but also those three-dimensional epitopes that are available only in the trimeric form.
In second example, a codon-optimized single copy of RBD DNA sequence (FIG. 1 and SEQ ID NO- 1) was used to create a therapeutic candidate called as Ad-ANS12 (see FIG. 10 and SEQ ID NO- 7) which is a Serotype 68 Chimpanzee Adenovirus with a few Serotype 5 adenovirus sequences while carrying a Codon-Optimized sequence of the Receptor Binding Domain. The process again involves use of four construct plasmids which are combined by T4 ligation to create a cosmid viral genome sequence as shown in FIG. 10. This candidate can be used as a booster so that the immunity persists.
Sequences for Receptor Binding Domain (193 aa) and S1 subunit of the Spike glycoprotein of the coronavirus bound to a Foldon domain were synthesized and tested for expression in HEK cells. FIG. 6 shows expression of RBD protein from a plasmid with natural sequence of RBD. FIG. 7 shows expression of RBD protein from a plasmid with codon-optimized sequence of RBD. FIG. 8 shows expression of S1 subunit protein bound to a Foldon tag by use of a plasmid with codon-optimized sequence of S1-FD. These three sequences were then inserted into plasmids that were used for the creation of cosmids (genomes for adenoviral candidate vaccines).
In yet another embodiment of the present invention is described the method of preparation and characterization of the candidates Ad-ANS06 and Ad-ANS12 (DNA sequences SEQ ID NO-1 to SEQ ID NO-17).
A.) Method to produce and test the Candidate Adenoviral therapeutic using the DNA sequences consists the steps of;
1. Construction of the adenoviral candidate which includes the steps of:
a. Creation of shuttle plasmids,
b. Cosmid Construction,
c. Rescue,
d. Amplification, and
2. Characterization of the candidate which includes the steps of:
i. Genome Restriction Analysis,
ii. Physical titer,
iii. Infectious titer,
iv. VP/ IU ratio,
v. Genome sequencing,
vi. Transgene expression,
vii. RCA assay.
Example 1:
Construction of candidate therapeutic Ad-ANS06; a serotype 5 Adenoviral candidate which can also be used as a vaccine before the infection with three gene inserts for more expression, where one gene sequence is of S1 subunit of the S glycoprotein tagged with FD domain, and two sequences are codon optimized sequence of the receptor binding domain (RBD) and natural sequence of the RBD.
AD-ANS06:
1. Construction of the adenoviral therapeutic candidate which can also be used alternatively as a vaccine if used prior to infection:
a. Creation of shuttle plasmids: Four plasmids were created for creation of ANS06.
i. pAD1127-ANS05 (see FIG. 11) (which has EF1a promoter driving the expression of natural RBD sequence leading to expression of 193 amino acids).
ii. pAD1128 (see FIG. 12) such that pAd1128 is a shuttle plasmid that contains WT Ad5 E2 and late genes.
iii. pAD1129-ANS10 (see FIG. 13) (which has RSV promoter driving the expression of codon-optimized RBD sequence leading to expression of 193 amino acids).
iv. pAD1130 (see FIG. 14) pAd1130 is a shuttle plasmid that contains the WT Ad5 E4 region.

The importance of presenting natural as well as codon-optimized sequences of antigen provides multiple advantages:
i. It increases the size of the final adenoviral genome such that it is incapable of producing replication competent adenoviral particles, because after homologous recombination the packaging capacity would be surpassed.
ii. It increases the expression of the antigen without creating a self-repeating DNA sequence within the plasmid.
iii. It eliminates any uncertainty whether natural or codon-optimized sequences should be used for antigen expression as both sequences are present.
Another advantage of the present invention is that the S1 subunit and RBD subunit both are available for creation of antibodies against the antigenic epitope.
b. Cosmid Construction Ad-ANS06:
The entire genome of tricistronic virus Ad-ANS06 was reconstituted in a cosmid using the four shuttle plasmids mentioned above namely; pAD1127-ANS05 (E1/pIX shuttle), pAD1128 (E2 shuttle), pAD1129-ANS10 (E3/ fiber shuttle) and pAD1130 (E4 shuttle). These four plasmids were used to create a cosmid with restriction enzyme digestion and ligation with T4 DNA ligase. The ligation product was packaged into lambda phage heads which were subsequently used to infect competent E. Coli. E. coli were selected with the help of ampicillin and kanamycin selection and cosmid DNA was purified using the alkaline lysis method and confirmed with restriction enzyme digestion to be correct. Ad5-based tri-cistronic vector with a EF1a-RBD cassette in place of the E1 region (see FIG.- 11), a CMV-S1FD cassette in place of the E3 region (see FIG. 13), and an RSV-OPT-RBD cassette between the L5 and E4 polyA signals (see FIG.- 13). All 3 cassettes (placed in two plasmids overall during the creation of AD-ANS06; pAD1127-ANS05 carrying EF1a-RBD cassette and pAD1129-ANS10 plasmid carrying CMV-S1FD and RSV-OPT-RBD cassettes) feature ATG triplets upstream of the initiator codon of interest which reduces the efficiency of expression. A vector where there were no ATG triplets upstream of the initiator codon could not produce a viable candidate potentially due to interference of the expressed proteins with the candidate production, hence the extra ATG triplets were maintained for a little inefficiency in expression.
c. Rescue:
i. Transfection: Two 6-cm dishes of 293 cells were transfected with PacI-digested pAd5-ANS06 using the calcium phosphate-DNA co-precipitation method. Ad5.CMV-GFP DNA (a known GFP insert) was used as positive control. Cells were rinsed twice with DMEM about 12 hours after the transfection.
ii. Harvest: Virus plaque harvest (day 11 post-transfection) was conducted when the first plaque was detected in one of the dishes of the HEK293 cells transfected with pAD5-ANS06; four plaques were harvested.
d. Amplification:
i. Pre-amplification (first round of expansion): A confluent 15-cm dish of 293 cells was split into 6-cm dishes at 30% confluence. The dishes were infected directly with Ad-ANS06 plaques #1-2-3-4 harvested earlier. On day 4 post-infection, the dish of 293 cells infected with Ad-ANS06 clone 3 showed complete cytopathic effect (CPE). Cells and medium were harvested, frozen/thawed three times (3x), clarified by centrifugation and aliquoted into sterile 1-mL vials. On day 6 post-infection, the dishes of 293 cells infected with Ad-ANS06 clones 1,2 and 4 showed complete CPE. Cells and medium were harvested, frozen/thawed 3x, clarified by centrifugation and aliquoted into sterile 1-mL vials.
ii. Amplification (second round of expansion): Confluent 10-cm dishes of 293 cells were infected with the crude lysates of Ad-ANS06 clones harvested. On day 3 post-infection, all dishes showed strong CPE. Cells and medium were harvested in the following way. 1.5 mL infected cell suspension was centrifuged for 2 min at 3,000 rpm, RT. The cell pellets were washed twice with PBS, then frozen at -70°C. These cell pellets were used for Western Blot testing for expression studies (for both candidate vaccines). Viral DNA (vDNA) was extracted from 4 mL infected cells suspension following the Hirt method. The remaining infected cell suspension was frozen/thawed 3x, clarified by centrifugation and aliquoted into sterile 1-mL vials.
2. Characterization of the candidate:
i. Genome Restriction Analysis: vDNA was extracted from Ad-ANS06-infected 293 cells according to the Hirt method. The DNAs were resuspended into 50 µL TE pH 7.5. The identity of the virus was confirmed by restriction digestion with HindIII, Acc65I and SpeI (5 µL DNA per lane) (see FIG.- 15). All 3 clones show identical restriction patterns, similar to the cosmid DNA restriction pattern. This indicate that the virus is stable. The presence of the 4155 bp HindIII fragment, 1313 bp Acc65I fragment and the 1024 bp SpeI fragment demonstrates the presence of the EF1a-RBD expression cassette in the vector. The presence of the 3982 bp and 2849 bp HindIII fragments, the 3321 bp and 2250 bp Acc65I fragments and the 3747 bp SpeI fragment demonstrates the presence of the CMV-S1FD expression cassette in the vector. The presence of the 4149 bp HindIII fragment, the 4353 bp Acc65I fragment and the 1193 bp SpeI fragment demonstrates the presence of the RSV-OPT-RBD expression cassette in the vector.
ii. Physical titer: The OD260-SDS method was used to determine the concentration of virus particles in the purified stocks of Ad-ANS06. The concentration of VP in the preparations was calculated given the extinction coefficient of 1.1 x 10e12 virus particles (VP) per Abs260 unit in presence of SDS. Ad-ANS06 physical quantity from one batch was calculated as 1.4 x 10e13 at a concentration of 1.6 x 10e12 vp/ml.
iii. Infectious Titer: Titration of the Ad-ANS06 adenovirus was conducted by using the hexon detection immune-histochemistry using an Adeno-X Rapid Titer kit. The CsCl-purified virus stock of Ad-ANS06 was diluted 10-fold serially in DMEM/FBS (10e1 to 10e6). The 293 cells from one 15-cm dish (passage 44) were trypsinized, washed once in DMEM/FBS and resuspended in 30 mL DMEM/FBS. 300 µL of cell suspension were added to tubes containing 50 µL of virus dilutions 10e3 to 10e6. The infected cells were then transferred to a well of a 24-well plate. After 3 days culture at 37 °C, the infected cell monolayers were fixed with ice-cold methanol, stained with an anti-Ad5 hexon primary antibody, a HRP-conjugated secondary antibody and DAB as substrate. Positive cells were counted under the microscope (10x10 magnification). The infectious titer (IU/mL) was calculated using the following formula: (average # of infected cells/field) x 47.4 x dilution factor / (volume of virus used for the infection in mL) = infectious titer.
iv. VP/IU Ratio: VP/IU (viral particle/ infectious units) ratio for AD-ANS06 was calculated to be 20.
v. Genome sequencing: The entire genomes of Ad-ANS06 was sequenced by NGS on an Illumina iSEQ100 instrument.
a. vDNA purification: vDNA was extracted from purified VP of Ad-ANS06 using proteinase K. The DNA was resuspended in 30 µl TE pH 7.5. vDNA concentration of 0.48 µg/ µl from 3.9 x 10e12 VP/ml of stock concentration of virus.
b. The sequencing and analysis confirmed the presence of the 3 intact expression cassettes: the EF1a-RBD-ßglobin pA cassette in the E1 region, the CMV-S1FD-SV40pA cassette in the E3 region and the RSV-OPT-RBD-bGHpA cassette between the L5 and E4 polyA signals.
vi. Transgene expression:
Expression of vaccine antigen coronavirus spike glycoprotein subunit S1 was confirmed by western blot assay conducted under non-reducing and reducing conditions. Expression of the antigen was confirmed where monomeric, dimeric and trimeric forms of the S1 subunit as well as receptor binding domain were seen on the western blot (see FIG 16). Primary antibodies against the Receptor Binding Domain and S1 subunit were used to detect the protein. Results showed detectable RBD protein in the Ad-ANS06 infected cells from present from 1.17 mg/ml to 1.46 mg/ml).
vii. RCA assay: The presence of replication-competent adenovirus (RCA) in the purified preparations of Ad-ANS06 was assessed using a modified infectivity/PCR method, which combines the amplification of infectious RCA by cell culture with the sensitivity of detection of E1-specific sequences by PCR. For each construct, 10 x 15-cm dishes of A549 cells (~ 2.5x 10e8 cells) were infected with 3 x 10e10 VP. Five days after the infection, the infected cells were harvested, pooled, and frozen/thawed 3 times in order to release the virus particles. New monolayers of A549 cells seeded in 10-cm dishes were infected with these crude virus lysates and cultured for another 5 days. Cells were harvested and viral DNA was purified according to the Hirt method. It was used as template for the detection of a 220 bp-long E1-specific sequence by PCR. As controls, two dishes of A549 cells were infected with a RCA-free control virus spiked with 10 and 100 VP of WT Ad5. The results showed the absence of detectable E1-specific sequence in the DNA extracted from A549 cells infected with Ad-ANS04, Ad-ANS06 and Ad-ANS12. Thus, no RCA was detected in 3 x 10e10 VP of the purified preparations of Ad-ANS06 (see FIG. 17).
Animal Study: The candidate Ad-ANS06 was tested in two animal studies, dosing outline and groups of the rat repeat dose toxicity and immunogenicity study are shown in FIG.- 25 and FIG.- 26 respectively. The candidate was given intranasally at a dose of 2x10e10 vp per dose given at day 0, day 14 and day 28. Also, the repeated dose toxicity and immunogenicity study was conducted in New Zealand white rabbits; dosing outline and groups are shown in Figures 30 and 31 respectively. The candidate Ad-ANS06 was safe with no safety concerns revealed in rats or rabbits. Day 28 (after 2 doses) immunogenicity results showed presence of neutralizing antibodies against the Coronavirus as shown in FIG. 28. The figures show results of detection of SARS-CoV-2 antibodies by bio layer interferometry. The curves show that high rate of antigen antibody binding was present in the sera of the tested rats.
Example 2:
Construction of candidate Ad-ANS12; an Adenoviral candidate comprised of chimpanzee serotype 68 with small serotype 5 sequences, with single gene insert with codon-optimized sequence of the receptor binding domain (RBD) for more expression, is prepared.
AD-ANS12:
1. Construction of the adenoviral candidate:
a. Creation of shuttle plasmids: Four plasmids were created for creation of ANS12.
i. pAD1144-ANS15 (E1/pIX) (see FIG 18) which has CMV promoter driving the expression of codon-optimized RBD leading to expression of 193 amino acids
ii. pAD1145 (see FIG 19) (E2/ late genes)
iii. pAD1146-01 (see FIG 20) (E3/ fiber genes) with 4.4 kb deletion in E3 gene
iv. pAD1147 (see FIG 21)
The importance of codon-optimized sequence of antigen provides an advantage because of creation of only neutralizing antibodies against the receptor binding domain of the coronavirus.
b. Cosmid Construction Ad-ANS12: The entire genome of Ad-ANS12 was reconstituted in a cosmid using the four shuttle plasmids mentioned above namely; pAD1144-ANS15 (E1/pIX shuttle), pAD1145 (E2/late shuttle), pAD1146-01 (E3/ fiber shuttle) and pAD1147 (E4 shuttle). These four plasmids were used to create a cosmid with restriction enzyme digestion and ligation with T4 DNA ligase. The ligation product was packaged into lambda phage heads which were subsequently used to infect competent E. Coli. E. coli were selected with the help of ampicillin and kanamycin selection and cosmid DNA was purified using the alkaline lysis method and confirmed with restriction enzyme digestion to be correct.
c. Rescue:
i. Transfection: Two 6-cm dishes of 293 cells were transfected with PacI-digested pAd5-ANS12 using the calcium phosphate-DNA co-precipitation method. Ad5.CMV-GFP DNA (a known GFP insert) was used as positive control. Cells were rinsed twice with DMEM about 12 hours after the transfection.
ii. Harvest: First plaque was detected on day 5 after the transfection. Virus plaque harvest (day 10 post-transfection) was conducted from the dishes of the HEK293 cells transfected with pAD-ANS12; four plaques were harvested.
d. Amplification:
i. Pre-amplification (first round of expansion): A confluent 15-cm dish of 293 cells was split into 6-cm dishes at 30% confluence. The dishes were infected directly with Ad-ANS12 plaques #1-2-3-4 harvested earlier. On day 6 post-infection, the dish of 293 cells infected with Ad-ANS12 showed complete cytopathic effect (CPE). Cells and medium were harvested, frozen/thawed three times (3x), clarified by centrifugation and aliquoted into sterile 1-mL vials.
ii. Amplification (second round of expansion): Confluent 10-cm dishes of 293 cells were infected with the crude lysates of Ad-ANS12 clones harvested. On day 2 post-infection, all dishes showed strong CPE. Cells and medium were harvested in the following way. 1.5 mL infected cell suspension was centrifuged for 2 min at 3,000 rpm, RT. The cell pellets were washed twice with PBS, then frozen at -70°C. These cell pellets were used for Western Blot testing for expression studies (for both candidate vaccines). Viral DNA (vDNA) was extracted from 4 mL infected cells suspension following the Hirt method. The remaining infected cell suspension was frozen/thawed 3x, clarified by centrifugation and aliquoted into sterile 1-mL vials.
2. Characterization of the candidate vaccine:
i. Genome restriction analysis: vDNA was extracted from Ad-ANS12-infected 293 cells according to the Hirt method. The identity of the virus was assessed by restriction digestion with MfeI, AflIII and XcmI (see FIG. 22). The insertion of the 5’ UTR-optimized CMV-OPT-RBD cassette in the virus is demonstrated by the presence of the 1249- and 2634-bp MfeI fragments, the 1353- and 3020-bp AflIII fragments and the 1224- and 1675-bp XcmI fragments (bars in the gel pictures) (see FIG. 22). All 3 clones show identical restriction patterns, which indicates that the virus is stable.
ii. Physical titer: The OD260-SDS method was used to determine the concentration of virus particles in the purified stocks of Ad-ANS12. The concentration of VP in the preparations was calculated given the extinction coefficient of 1.1 x 10e12 virus particles (VP) per Abs260 unit in presence of SDS. Ad-ANS12 physical titer from one batch was calculated as 1.1 x 10e13 at a concentration of 2.7 10e12 vp/ml.
iii. Infectious Titer: The concentration of infectious particles in the purified stocks of Ad-ANS12 was determined by end-point dilution assay (TCID50), using virus growth as indicator. The infectious units per ml ratio was determined to be 1 x 10e11/ml.
iv. VP/IU Ratio: VP/IU (viral particle/ infectious units) ratio for AD-ANS12 was calculated to be 27.
v. Genome sequencing: The entire genomes of Ad-ANS12 was sequenced by NGS on an Illumina iSEQ100 instrument.
a. vDNA purification: vDNA was extracted from purified VP of Ad-ANS12 using proteinase K. The DNA was resuspended in 30 µl TE pH 7.5. vDNA concentration of 0.35 µg/ µl from 2.7 x 10e12 VP/ml of stock concentration of virus.
b. The sequencing and analysis confirmed the presence of the intact CMV-OPT-RBD-SV40pA expression cassette in the E1 region.
vi. Transgene expression: Expression of vaccine antigen coronavirus spike glycoprotein subunit S1 was confirmed by western blot assay conducted under non-reducing and reducing conditions. Expression of the antigen was confirmed where monomeric, dimeric and trimeric forms of the RBD were seen on the western blot (see FIG. 16). Primary antibodies against the Receptor Binding Domain were used to detect the protein. Results showed detectable RBD protein in the Ad-ANS12 infected cells from 3.7 mg/ml to 4.48 mg/ml).
vii. RCA assay: The presence of replication-competent adenovirus (RCA) in the purified preparations of Ad-ANS12 was assessed using a modified infectivity/PCR method, which combines the amplification of infectious RCA by cell culture with the sensitivity of detection of E1-specific sequences by PCR. For each construct, 10 x 15-cm dishes of A549 cells (~ 2.5 x10e8 cells) were infected with 3 x 10e10 VP. Five days after the infection, the infected cells were harvested, pooled, and frozen/thawed 3 times in order to release the virus particles. New monolayers of A549 cells seeded in 10-cm dishes were infected with these crude virus lysates and cultured for another 5 days. Cells were harvested and viral DNA was purified according to the Hirt method. It was used as template for the detection of a 220 bp-long E1-specific sequence by PCR. As controls, two dishes of A549 cells were infected with a RCA-free control virus spiked with 10 and 100 VP of WT Ad5. The results showed the absence of detectable E1-specific sequence in the DNA extracted from A549 cells infected with Ad-ANS04, Ad-ANS06 and Ad-ANS12. Thus, no RCA was detected in 3 x 10e10 VP of the purified preparations of Ad-ANS12 (see FIG. 17).
Animal Studies: The candidate vaccine Ad-ANS12 was tested in two animal studies, dosing outline and groups of the rat repeated dose toxicity and immunogenicity study are shown in FIG. 25 and FIG. 26 respectively. Also, the repeated dose toxicity was conducted in New Zealand rabbits; dosing outline and groups are shown in FIG.- 30 and FIG.- 31 respectively. The candidate was given intranasally at a dose of 2x10e10 vp per dose given at day 0, day 14 and day 28 and intramuscularly in a different group of animals at a dose of 5x10e10 vp per dose given at day 0 and day 28. The vaccine was safe with no safety concerns revealed in rats and rabbits. Day 28 immunogenicity (after 2 doses) results showed presence of neutralizing antibodies against the Coronavirus as shown in FIG. 28 (for intranasal dose) and FIG. 29 respectively (for intramuscular dose). The figures show results of detection of SARS-CoV-2 antibodies by bio layer interferometry. The curves show that high rate of antigen antibody binding was present in the sera of the tested rats.
The embodiments herein above and the various features and advantageous details thereof are explained with reference to the non-limiting embodiments in the following description. Descriptions of well-known components and processing techniques are omitted to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skilled in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
While considerable emphasis has been placed herein on the components and component parts of the preferred embodiments, it will be appreciated that many embodiments can be made and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other changes in the preferred embodiment as well as other embodiments of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.