Description:FIELD OF THE INVENTION
The present invention relates to custom outer membrane protein LipL32 mRNA (isoforms 1 - 15) sequences oligonucleotide primer sequences and amplicons for detection of the major etiological agent of the zoonotic disease Leptosporosis namely, Leptospira interrogans in human, veterinary animals and environmental samples. More particularly the present invention relates to designing of high-throughput oligonucleotide primer sequences for diagnosis and mRNA based human and veterinary vaccine against Leptospirosis.
BACKGROUND OF THE INVENTION
Leptospirosis (also known as Weil's disease, canicola fever, canefield fever, nanukayami fever, 7-day fever and many more) is a bacterial zoonotic disease caused by spirochaetes of the genus Leptospira that affects humans and a wide range of animals, including mammals, birds, amphibians, and reptiles. It was first described by Adolf Weil in 1886 when he reported an "acute infectious disease with enlargement of spleen, jaundice and nephritis". Leptospira was first observed in 1907 from a post mortem renal tissue slice. Leptospira is a genus of spirochaete bacteria, including a small number of pathogenic and saprophytic species. Members of Leptospira are also grouped into serovars according to their antigenic relatedness. There are about 20 species of Leptospira and currently over 200 recognized serovars.
The disease Leptospirosis is transmitted by the urine of an infected animal, and is contagious as long as it is prevailing in humid area. Earlier time there is a common misconception that the disease is associated only with rats. Rats, mice and voles are important primary hosts, but a wide range of other mammals including dogs, deer, rabbits, hedgehogs, cows, sheep, raccoons, possums, skunks, and even certain marine mammals are also able to carry and transmit the disease. Dogs may lick the urine of an infected animal off the grass or soil, or drink from an infected puddle. There have been reports of "house dogs" contracting leptospirosis apparently from licking the urine of infected mice that entered the house. Although this behavior is generally associated with some degree of infection risk. The disease therefore requires a population of mammalian carriers, normally together with wetted soil or polluted puddles, to keep the cycle going. The type of habitats most likely to carry infective bacteria are muddy riverbanks, ditches, gulleys and muddy livestock rearing areas where there is regular passage of either wild or farm mammals. The disease is endemic wherever open sewers or agricultural practices lead to contamination of water with animal urine. There is a direct correlation between the amount of rainfall and the incidence of leptospirosis, making it seasonal in temperate climates. In tropical regions of the world, leptospirosis is a wide spread and year-round public health problem.
Rats are one of the major reservoir hosts for serovars of Leptospira interrogans comprising many pathogenic serovars, and infected rats probably remain infected, and infectious, for life. Later the infection is commonly transmitted to humans by allowing water that has been contaminated by pathogen carrying animal urine to come in contact with unhealed breaks in the skin, the eyes, or with the mucous membranes. The bacterial spirochete colonizes the renal tubules of infected animals and human and is shed in the urine for varying periods of time. Although most human leptospirosis infections are self-limited, complications are common, involving hepato-renal failure, pulmonary hemorrhage, and cause death in 10%-50% of severe cases. W.H.O report states that the prevalence in tropical countries is nearly 100 per 1 million populations.
Highest prevalence of leptospirosis is found especially after the rainy season when heavy rainfall and inadequate civil engineering result in urban flooding. Since the laborious nature of Leptospira isolation and culture techniques, particularly from environmental samples, there was no systematic and gold standard method available to detect and identify of leptospires in various kinds of clinical and environmental samples. Problem with the detection of leptospirosis in clinical samples is that due to the analogous, ambiguous and wide range of indications which lead to wrong diagnose of infection every so often unfortunately. Other challenges in detecting Leptospirosis on clinical samples include the similarity of signs and symptoms with those of other bacterial, viral and parasitic infections and frequent occurrence of the disease in atypical forms. They have been reported worldwide and affect both wild as well as domestic animals and humans. The importance of the infections due to these agents has also increased over time due to several factors such as their fast spreading rate, difficulty in quick and prompt diagnosis, control and prevention, and the cost of treatment.
The MAT (microscopic agglutination test), is the biochemical test for diagnosing leptospirosis, which is both laborious and least specificity due to which this method is underused. The need for paired serum samples and delayed antibody response do not contribute much to the early diagnosis. Moreover, all the pathogenic Leptospira serovar are not included in the panel of typing organisms, hence it frequently results in false negative results. Several other alternatives of antibody detection assays namely test, slide assay, hemagglutination test, immunofluorescence test, indirect enzyme- linked immunosorbent assay for IgM antibodies, dot-ELISA for IgM, and LEPTO Stick have subsequently been developed for diagnosis of leptospirosis.
However, these methods too have several limitations. Some of these methods are still and require maintenance of alive Leptospira cultures for preparing a broadly reactive antigen which itself is cumbersome and risky run-through. Further, most patients with leptospirosis develop antibodies after infection and it persists in serum for a long time, even after the pathogenic organisms have been eliminated. Similarly, it was found that IgM antibodies were detected by dot-ELISA test in patients up to the duration of six months to twelve months from the onset of disease. This indicates that the antibody detection assay is less sensitive during the early period of infection that is acute phase of illness and also it cannot be used for monitoring the efficacy of the treatment. Moreover, there are other (less common) infectious strains. It should however be noted that genetically different leptospira organisms may be identical serologically and vice versa. Hence, an argument exists on the basis of strain identification. The currently employed serological testing technique also suffers from limitations. The traditional serologic system is seemingly more useful from diagnostic and epidemiologic standpoint at the moment which may be changed with further development and spread of advanced molecular biology based detection technologies. Thus, there is a need of a diagnostic strategy with more reliable, rapid and simpler means to detect and identify Leptospiral DNA from clinical and environmental samples.
Leptospira interrogans is one of the responsible causative agent induce abortion in veterinary animals by infection. Also noted that simplex PCR-based approach for identification of the infection causing microbes in clinical sample and multiplex real-time PCR assay for the detection of Leptospira interrogans along with other pathogenic species Brucella interrogans and Campylobacter foetus by targeting conserved regions of LipL32, BCSP31 and gB genes, respectively.
Though, the technology utilized in the document is conventional multiplex PCR and is not able to provide rapid and reliable results. Further, the limit of detection of this assay is as low as 15pg and they have low sensitivity and specificity. Therefore, there is a need in the art for developing an advanced high throughput diagnostic technique capable of detecting and identifying various serovars and strains of Leptospira interrogans. With this backdrop information it is decided to design high-throughput primers oligonucleotides from the selected candidate gene of the pathogenic organism Leptospira interrogans which is a pre-requisite for conduction conventional polymerase chain reaction and even advance Real-Time PCR based diagnostic assays as a due course venture.
The efficacy of heat killed whole cell vaccine for preventing leptospirosis is shown soon after Leptospira is proven to be the causative agent of Weil’s disease in Japan. Current Licensed heat killed whole cell Vaccines for leptospirosis are 1. Weil’s disease and Akiyami combined vaccine (Denka Seiken, Japan); 2. SPIROLEPT (Thea Laboratories, France); 3. Leptospirosis vaccine (Polyvalent vaccine) (Shanghai Institute of Biological Products, China); 4. Vax-SPIRAL (Trivalent vaccine) (Finlay Institut, Cuba). Although killed whole cell vaccines are used for prevention of human leptospirosis, there still remain several problems. In addition to issues on persistence of immunity and adverse events, the effectiveness of the killed vaccine is generally serovar-specific. There are more than 230 serovars among the pathogenic leptospires. The local variability in serovars of endemic leptospiral strains complicates the development of a vaccine that can be used worldwide. Thus, new vaccine strategies are directed to the exploration for genome data directed antigens that can generate cross protection against various serovars. The mRNA conferring immunogenic proteins, especially the outer membrane surface proteins, of pathogenic Leptospira, may be effective as immunogens. The identification of mRNAs and subsequent peptides/proteins, which are conserved among pathogenic leptospires and can elicit cross-protection against various serovars, has become a major focus of leptospirosis research. mRNA based vaccine and subunit vaccines would also have fewer side effects than killed whole cell vaccine. Leptospira genome annotation data point to the potential use of leptospiral mRNAs as candidates for a new strategy vaccine that could induce good protection against diverse serovars of Leptospira.
Surface expressed outer membrane lipoprotein namely LipL32 is a conserved one, both genetically and immunologically, in the various pathogenic leptospires. Custom LipL32 is one of the most abundant antigenic proteins in Leptospira. Protein LipL32 is showing hemolytic activity alone or synergistically with another hemolysin in natural system. And so LipL32 based antigen induces the production of antibody in patients with leptospirosis. Vaccination using an adenovirus vector and plasmid vector encoding the Custom LipL32 gene induced cross-protection from leptospirosis. Perusal of literature and arts revealed that there is no mRNA based human and veterinary vaccine. With this backdrop information, it is decided to design various length of mRNA fragments of custom LipL32 gene for artificial synthesis of nucleic acid especially mRNA based human and veterinary vaccine against leptospirosis.
SUMMARY OF THE INVENTION
Thus, in one aspect the present invention provides custom outer membrane protein LipL32 mRNA (isoforms 1 - 15) sequences, oligonucleotide primer sequences for detection of the presence of Leptospira interrogans species in biological or environmental samples.
Accordingly, in one embodiment the present invention provides oligonucleotides sequences to be used as primer for detection of Leptospiral DNA in versatile samples using a molecular protocol for example polymerase chain reaction (PCR) techniques.
In one embodiment the present invention provides a method for detecting Leptospiral DNA in biological and environmental samples, wherein the strategy comprises of amplifying a segment of Leptospiral DNA by subjecting the designed primers of the present invention specific for leptospires, in combination with heat-stable DNA polymerase in the presence of sample DNA to temperature cycles and detecting the amplified DNA.
In one embodiment the present invention provides custom mRNA (isoforms 1 - 15) sequences and amplicons for the preparation of mRNA based human and veterinary leptospirosis vaccine.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Oligonucleotide sequences of newly designed Forward and Reverse Primers utilizable to amplify the Outer membrane protein custom LipL32 encoding mRNA, amplicon gene sequences and translated protein sequences.
Figure 2: Oligonucleotide sequences of newly designed Forward and Reverse Primers utilizable to amplify the Outer membrane protein custom LipL32 encoding mRNA, gene sequences and translated protein sequences of mRNA (isoforms 1 - 15).
DESCRIPTION OF THE INVENTION
The present disclosure relates to design and compositions of custom outer membrane protein LipL32 mRNA (isoforms 1 - 15) sequences, primers for molecular diagnosis of agents responsible for causing leptospirosis. More specifically, the present invention relies on a qualitative simplex and multiplex types polymerase chain reaction based method for rapid detection and identification of Leptospira interrogans. The application provides primer sequences for use in the PCR assay.
The term “biological sample” as used in the present application includes all members of the mammalians inclusive of human and veterinary animals. The assay provided by the present invention is specific to pathogenic Leptospira interrogans serovars and strains. The disclosed primer sequences allow for the detection of all serovars of the target pathogen present in a sample in high throughput manner. The PCR assay relies on the use of a specific combination of primers sequences either as conventional simplex or multiplex PCR and Real-Time PCR reactions based on SYBR green dye chemistry.
The Applicant notes that none of the processes or assays available in the art is able to provide for a rapid and reliable PCR assay result for the simultaneous detection of multiple locus of the candidate mRNA LipL32 of Leptospira interrogans with higher reliability rate.
The Applicant has specifically employed the technology of primer designing as semi manual and software depended for PCR reaction as it presents numerous advantages over simplex PCR assays which are generally employed in the prior technologies. Specifically, the multiplex priming assay of the present application has numerous advantages in terms of input time and turnaround time. Moreover, the multiplex priming assay allows for the detection of more than one locus of a target gene sequence in a single reaction as an outcome to generate higher quantity of amplified products. All the primers designed here show best quality range (rating of 75 and above out of 100 as highest scale) based on various primers parameters including salt adjusted melting temperature, GC contents and other secondary structures (Self dimer, Hair pin formation, Cross dimer, Repeats and Runs).
In an embodiment of the present invention, as opposed to complex and complicated primer designing methods which have been previously reported, the present assay involves a multiplex priming qualitative polymerase chain reaction. The assay can be easily performed even with a small amount of biological sample spiked with Leptospira interrogans.
In an embodiment of the present invention, technical improvements and advancements reported by the present designing procedure is attributable to the specific combination of newly designed primers sequences utilized in the invention. Designing of appropriate primers are essential to the successful outcome of any kind of PCR assays.
In an embodiment of the present invention, the primer sequences have been specifically designed in such a manner which allows them to work in a single reaction in a compatible manner. Further, the design and specific sequence of the primer sequences also does not produce any non-specific flanking when tested with genetically related pathogens based on NCBI BLAST report.
In an embodiment of the present invention, there is provided a primers composition for both conventional and Real-time polymerase chain reactions based detection and identification of Leptospira interrogans in biological and environmental samples, wherein the composition comprises:
In an embodiment of the present invention, the forward primer sequences comprises oligonuclotide sequence flanking the outer membrane protein coding custom mRNA (isoforms 1 - 15) sequences LipL32 at various locus are given in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, 15 SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, 5 SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, 5 SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO: 107.
In another embodiment of the present invention, the reverse primer sequences comprises oligonucleotide sequences flanking outer membrane protein coding custom mRNA (isoforms 1 - 15) sequences LipL32 at various locus are given in SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167 and SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213 and SEQ ID NO: 214.
In another embodiment, the amplicon sequences of outer membrane protein coding custom mRNA (isoforms 1 - 15) sequences LipL32 as variable length are given in SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO:, 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO:, 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 315, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320 and SEQ ID NO: 321.
In an embodiment of the present invention, the protein sequences as translate of amplicon sequences of outer membrane protein coding custom mRNA (isoforms 1 - 15) sequences LipL32 as variable length are given in SEQ ID NO: 322, SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, SEQ ID NO: 328, SEQ ID NO: 329, SEQ ID NO: 330, SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 334, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 345, SEQ ID NO: 346, SEQ ID NO: 347, SEQ ID NO: 348, SEQ ID NO: 349, SEQ ID NO: 350, SEQ ID NO: 351, SEQ ID NO: 352, SEQ ID NO: 353, SEQ ID NO: 354, SEQ ID NO: 355, SEQ ID NO: 356, SEQ ID NO: 357, SEQ ID NO: 358, SEQ ID NO: 359, SEQ ID NO: 360, SEQ ID NO: 361, SEQ ID NO: 362, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 366, SEQ ID NO: 367, SEQ ID NO: 368, SEQ ID NO: 369, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 372, SEQ ID NO: 373, SEQ ID NO: 374, SEQ ID NO: 375, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380, SEQ ID NO: 381, SEQ ID NO: 382, SEQ ID NO: 383, SEQ ID NO: 384, SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 387, SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO: 401, SEQ ID NO: 402, SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 405, SEQ ID NO: 406, SEQ ID NO: 407, SEQ ID NO: 408, SEQ ID NO: 409, SEQ ID NO: 410, SEQ ID NO: 411, SEQ ID NO: 412, SEQ ID NO: 413, SEQ ID NO: 414, SEQ ID NO: 415, SEQ ID NO: 416, SEQ ID NO: 417, SEQ ID NO: 418, SEQ ID NO: 419, SEQ ID NO: 420, SEQ ID NO: 421, SEQ ID NO: 422, SEQ ID NO: 423, SEQ ID NO: 424, SEQ ID NO: 425, SEQ ID NO: 426, SEQ ID NO: 427 and SEQ ID NO: 428.
As used herein, the term “vaccine” refers to a production of inactivated or dead or weakened pathogens or components of pathogens or derived antigenic determinants used to induce antibodies or immunity against the pathogenic microorganism. In addition, the term “vaccine” is administered into subjects in the form of suspension or solution of the pathogen to produce immunity against the disease caused by that pathogen. The present invention provides selected messenger ribonucleic acid (mRNA) comprising at least one open reading frame coding for at least one protein wherein the mRNA comprises at least one modification, which increases the expression of its encoded protein. In the meaning of the present invention, the term “Tailored mRNA” comprises that any mRNA that is modified in a way that leads to increases the amount of protein translation in comparison with other mRNA lacking the modification.
The tailored mRNA according to the present invention may encode a protein which comprises a pathogenic antigen or a fragment, variant or derivative thereof. Such pathogenic antigens are derived from outer membrane protein LipL32 of pathogen Leptospira interrogans which evoke an immunological reaction in human and veterinary animal subjects.
In another embodiment, the tailored mRNA relates to ribonucleotide sequence in particular an mRNA sequence modified for enhanced expression of mRNA encoded protein LipL32 of Leptospira interrogans.
In a preferred embodiment, the modified custom mRNA encodes an outer membrane protein LipL32 of Leptospira interrogans or an antigenic fragment thereof, is a component of the vaccine injected into the dermis or intramuscular of a subject may stimulate immune reactions.
In one embodiment, all inventive custom mRNA sequences comprise a coding region, encoding antigenic protein derived from outer membrane protein LipL32 of Leptospira interrogans or a fragment, variant or derivative thereof or from any engineered LipL32 protein.
In a preferred embodiment, the forward primer sequences comprise oligonuclotide sequence flanking complete custom mRNA (isoforms 1 - 15) sequences of the outer membrane protein coding LipL32 at various locus are given in SEQ ID NO: 429, SEQ ID NO: 430, SEQ ID NO: 431, SEQ ID NO: 432, SEQ ID NO: 433, SEQ ID NO: 434, SEQ ID NO: 435, SEQ ID NO: 436, SEQ ID NO: 437, SEQ ID NO: 438, SEQ ID NO: 439, SEQ ID NO: 440, SEQ ID NO: 441, SEQ ID NO: 442 and SEQ ID NO: 443.
In a preferred embodiment, the reverse primer sequences comprise oligonucleotide sequence flanking complete custom mRNA (isoforms 1 - 15) sequences of the outer membrane protein coding LipL32 at various locus are given in SEQ ID NO: 444, SEQ ID NO: 445, SEQ ID NO: 446, SEQ ID NO: 447, SEQ ID NO: 448, SEQ ID NO: 449, SEQ ID NO: 450, SEQ ID NO: 451, SEQ ID NO: 452, SEQ ID NO: 453, SEQ ID NO: 454, SEQ ID NO: 455, SEQ ID NO: 456, SEQ ID NO: 457 and SEQ ID NO: 458.
In a preferred embodiment, the tailored mRNA (isoforms 1 - 15) sequences of outer membrane protein coding LipL32 as variable length are given in SEQ ID NO: 459, SEQ ID NO: 460, SEQ ID NO: 461, SEQ ID NO: 462, SEQ ID NO: 463, SEQ ID NO: 464, SEQ ID NO: 465, SEQ ID NO: 466, SEQ ID NO: 467, SEQ ID NO: 468, SEQ ID NO: 469, SEQ ID NO: 470, SEQ ID NO: 471, SEQ ID NO: 472 and SEQ ID NO: 473.
In a preferred embodiment, the protein sequences as translate of tailored mRNA (isoforms 1 - 15) sequences of outer membrane protein coding LipL32 as variable length are given in SEQ ID NO: 474, SEQ ID NO: 475, SEQ ID NO: 476, SEQ ID NO: 477, SEQ ID NO: 478, SEQ ID NO: 479, SEQ ID NO: 480, SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO: 483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO: 487 and SEQ ID NO: 488. , C , Claims:We claim,
1. A custom outer membrane protein LipL32 mRNA (isoforms 1 - 15) sequences comprising a coding and non-coding regions of forward and reverse primers encode frame 1, frame 2 and frame 3, amplicon gene fragments, mRNA, translated protein sequences and mRNA coding regions of forward and reverse primers encoding frame 1 of mRNA.
2. The forward primer sequences as claimed in claim 1, comprises oligonucleotide sequences flanking the outer membrane protein coding custom LipL32 mRNA of Leptospira interrogans at various locus selected from sequences with SEQ ID NO: 1 to SEQ ID NO: 107.
3. The reverse primer sequences as claimed in claim 1, comprises oligonucleotide sequences flanking the outer membrane protein coding custom LipL32 mRNA of Leptospira interrogans at various locus selected from sequences with SEQ ID NO: 108 to SEQ ID NO: 214.
4. The amplicon gene fragments, DNA sequences and the translated protein sequences as claimed in claim 1, comprises DNA nucleic acid sequences flanking the outer membrane protein coding custom LipL32 mRNA or amplicon gene fragments DNA of Leptospira interrogans at various locus selected from sequences with SEQ ID NO: 215 to SEQ ID NO: 321.
5. The translated protein sequences as claimed in claim 1, comprises nucleic acid sequences flanking the outer membrane protein coding custom LipL32 mRNA or amplicon gene fragments of Leptospira interrogans at various locus selected from sequences with SEQ ID NO: 322 to SEQ ID NO: 428.
6. The forward primer sequences as claimed in claim 1, comprises oligonucleotide sequences flanking complete mRNA (isoforms 1 - 15) of the outer membrane protein coding custom LipL32 mRNA of Leptospira interrogans at defined locus selected from sequences with SEQ ID NO: 429 to SEQ ID NO: 443.
7. The reverse primer sequences as claimed in claim 1, comprises oligonucleotide sequences flanking complete mRNA (isoforms 1 - 15) of the outer membrane protein coding custom LipL32 mRNA of Leptospira interrogans at defined locus selected from sequences with SEQ ID NO: 444 to SEQ ID NO: 458.
8. The mRNA (isoforms 1 - 15) sequences as claimed in claim 1, comprises nucleotide sequences of outer membrane protein coding custom LipL32 mRNA of Leptospira interrogans selected from sequences with SEQ ID NO: 459 to SEQ ID NO: 473.
9. The protein sequences as translation of mRNA (isoforms 1 - 15) sequences as claimed in claim 1, comprises protein sequences of the outer membrane protein custom LipL32 mRNA of Leptospira interrogans selected from sequences with SEQ ID NO: 474 to SEQ ID NO: 488.
10. A custom outer membrane protein LipL32 mRNA isoforms (1 - 15) sequences as claimed in claims 1 to 9, are used for diagnosis of Leptospira interrogans from clinical, veterinary and environmental samples and mRNA based human and veterinary vaccine against Leptospirosis.