Patent Application
20200384095
The present invention relates to a novel consortium for a potent vaccine for enteric fever, comprised of specific strains Salmonella typhi and Salmonella paratyphi A in equal proportion.
The Present invention also relates to a methodology for preparing the said novel consortium based on outer membrane vesicles (OMV).
Enteric fever, a serious invasive bacterial infection, caused by Salmonella enterica serovers Typhi and Paratyphi A (hereafter, S. typhi and S. paratyphi A, respectively) is a major global burden in developed and developing countries like India. Although S. typhi is more prevalent, affects 21.7 million cases and 200,000 deaths per year worldwide, S. paratyphi (A, B and C) can cause significant enteric fever especially in Asia as well as in travelers returning from these endemic areas (1, 2, 3). Currently, there are only two licensed vaccines are available against S. typhi; a live attenuated galE mutant and a Vi-polysaccharide vaccine (4). Although somewhat effective, they have their limitations such as they do not provide long-term protection in children and they do not provide significant long-term immunity in adults too.
Presently, the focus of vaccine research is on acellular vaccine because of the certain drawbacks of conventional immunogens. In this changing state of vaccine research, Outer Membrane Vesicles (OMVs) has got significant importance. Neisseria meningitis OMV based licensed vaccine is now presently available in market (5). As OMVs has got both LPS and proteins, they do not need any artificial adjuvants (6).
Though OMVs are used in drug development against various microorganisms, such methods are often expensive and complicated as most of the techniques employ infusion of different proteins from outside.
As the enteric fever is most predominant in developing countries, the cost-effectivity should be the prime concern in field of vaccine development.
Further, the conventional technology often devoid of providing substantial protection against enteric fever caused by S. typhi and also results some hazardous side-effects in human.
Hence, there is always a need to provide an innovative formulation by using a process based on Outer Membrane Vesicles (OMV) which are overcoming the drawbacks of the conventional practice.
The present invention meets the above-mentioned long-felt need.
The principal object of the present invention is to provide a simple yet effective consortium comprised of isolated Outer Membrane Vesicles (OMV) of Salmonella typhi and Salmonella paratyphi A in equal proportion.
Another objective of the present invention is to provide a consortium which is effective in treating enteric fever.
Yet another objective of the present invention is to provide a simple consortium wherein any mutation or deletion of gene has not been adopted to provide the end product.
Further objective of the present invention is to provide a simple consortium wherein the mutation or deletion of gene is not incorporated to reduce the expression of immune-dominant non-protective antigens.
Another objective of the present invention is to provide a simple consortium wherein no antibiotics or any excipients have been used.
Yet another objective of the present invention is to provide a simple consortium wherein no protective proteins is incorporated from outside.
Further objective of the present invention is to provide a simple consortium wherein only isolated OMVs of Salmonella strain is used, hence, it is cost-effective and environment friendly.
It is to be noted, however, that the appended drawings illustrate only typical embodiments of the present subject matter and are therefore not to be considered for limiting of its scope, for the invention may admit to other equally effective embodiments. The detailed description is described with reference to the accompanying figures.
Some embodiments of system or methods in accordance with embodiments of the present subject matter are now described, by way of example, and with reference to the accompanying figures, in which:
a illustrate an electron micrograph of OMVs attached to bacteria and isolated OMVs and characterization of isolated OMVs.
The present subject matter relates to a novel formulation comprised of isolated outer membrane vesicles from two thyphoidal Salmonella strains such as Salmonella typhi C-6953 and Salmonella paratyphi A C-6915.
In the said formulations, two Salmonella strains are mixed in 1:1 ratio i.e. 50% of Salmonella typhi C-6953 and 50% of Salmonella paratyphi A-6915.
The immunogenicity and protective efficacy have been studied on adult mice after oral immunization with the said formulation.
The evaluation of the generation of humoral as well as cell mediated immune response after oral immunization by measuring different immunologic markers as well as anti-Vi polysaccharide specific serum immunoglobulin and Th1/Th17 specific cytokine response from splenic and DCs (Dendritic Cells) were performed.
This bivalent OMVs based vaccine could be an ideal human vaccine candidate against enteric fever.
The strains which are used in the said formulation are clinical isolates and thus do not have any modification or induced mutation in them.
Mutating a specific gene for over-expression of protective antigen may reduce immune-dominant non-protective antigen by mutating or deleting it. Moreover, LPS mutants might have some adverse effects on the end product; i.e., secreted OMVs. Mutating a gene also changes the bacterial genetic make-up and might eventually produce a specific type of protein which is not needed. Many other useful proteins could be lost in the process.
But, the said formulation does not change the genetic make-up of the microorganism and hence no such unwanted protein is produced.
No stress has been induced on the cultured bacteria in the form of antibiotics or using minimal medium, which eventually increases the cost-effectiveness of the final product.
Further, the said formulation does not incorporate antibiotics in it. Thus, reducing the chance of spreading anti-microbial resistance.
Unlike conventional practice, the present invention does not add any protein from outside or any other excipients such as lactose, sucrose, gelatin, sorbitol, human serum albumin and hence it is free from post-isolation purification steps.
From the analysis of the consortium, it has found that the consortium also comprises substantially high number of outer, inner, periplasmic and cytoplasmic protein, which have not infused from outside but were found to be present naturally.
It also possesses high number of cytosolic proteins (even proteins like DNA polymerase III, helicase, primase).
Any mutant stains has not been used in the novel formulation, the instant invention only uses their native form to deliver their natural contents in the host's body.
Further, effective short duration of immunization schedule can be achieved by the novel formulation. The protection can stay for 3 to 6 months without any further booster doses than the regimen stated.
The process for preparing the novel consortium has used log-phase culture of bacteria to isolate OMVs thus increasing the amount of TTSS proteins which are more potent in nature as an immunogen.
Also, as per the present invention the OMVs contain Vi-polysaccharide of Salmonella typhi. The content of Vi-polysaccharide in the bivalent formulation has been measured. The presence of Vi-polysaccharide in vaccine constituents makes the vaccine more effective against Salmonella typhi infection because, Salmonella typhi is covered with Vi-polysaccharide, presence of anti-Vi antibiotics in the serum would certainly elevated the level of protection. Presence of Paratyphi A OMVs enlarges its protective nature further against Paratyphi A.
The detailed result has given below:
Hypothetical proteins found after MALDI-TOF/TOF of Salmonella typhi C-6953 OMV:
IITNVFLNAK
LTASLLLIYAK
YEKNWFLPIVTIGK
MLTASLLLIYAKNNGITLLVTK
DDLLSRINR
GFSVPTPIQR
GFSVPTPIQRK
FKPQETIFELGPKGK
IQEILVGITFLIAIAFIVK
IQEILVGITFLIAIAFIVKK
MIQEILVGITFLIAIAFIVK
MLLALARLK
PNILPTLPTLR
MPNILPTLPTLR
PNILPTLPTLRILPTLPILR
VLVIGDLR
VDKGIVSLDR
MYRLLLGDGK
LVIQGFVKGVMHWVVEGGK
ETPIQEEVKPLIEDILRTK
MVEIAAVRGR
VMELAKAALR
ETIEAALAQR
DDIEARAIAK
QIEAAKPK
ILTVGKYPLMTL
LLDGNGLYLYVPVSGK
KFFVTDK
ESLTLETVLK
EIETLLTVQAPR
SEPLWRTLIGIR
DKIYGILGLLNEK
DGDTIAIIAGMGRAAILR
VPITYHGFLMHSRGTIHIR
GFAGVATPMIRDGDTIAIIAGMGR
AFYMHLPAAGK
KLLTILNAMLR
LLTILNAMLRK
MAALVVTWFNPVIKAFYMHLPAA
GK
IDWIASQIR
KIDWIASQIR
QLNDLLKIIFFNVIR
GIVDPDLR
MVELLDLIR
VASESRAVVLQVDSLLK
LLVTVALAFLLVLVMAIFSIRSVMR
LSASADLLRR
MLQSIFTALLGR
LQSIFTALLGRLSASADLLR
DDEILDLLR
ENGIKTVVNK
IITNVFLNAK
LTASLLLIYAK
YEKNWFLPIVTIGK
DGQDLVISVR
IVAPTQRIDSR
QLLRDVSHELR
LQALIGSQRQLLR
LPLAGPASRTSDDLASH
APGQTAAGHGLGLAIARR
DDGPGVADEHLPQLSEPFFRAPGQ
TAAGHGLGAI
YFDAARSYGR
VGLSLSGPQQAAVLR
GMAEAPQVYWTTR
PHTLGNSGPAGTSLGLGLAALGRP
GYITLGRAGDMGPDR
AALLIMLYSGK
NNMQQLAKPEK
MEIAESIEATRQSVIR
MFQVLERAALLIMLYSGK
NNMQQLAKPEKVYLDNMNLMYA
LSSSADIGNIR
TLLDGDLQHRIR
ATNNLAATTEAVAAGADR
DPLGGPGKPVWAEVVSVWAK
DPLGGPGKPVWAEVVSVWAKATN
NLAATTEAVAAGADR
LGVSVATIER
AVLIEAIEQIDR
LTYPEIALRLGVSVATIER
LPSRADAEDVTSETFAQVVENK
AYLQSLMLMPEASVLSPEERAVLI
EAIEQIDR
ETLEGVINAR
ETLEGVINARAK
LMVVVERYPELK
GIVDPDLR
MVELLDLIR
VSRGIVALSNGMNALAK
LLVTVALAFLLVLVMAIFSIRSVMR
ILSTTVPVYGR
QGVFKMSYHIR
RIAYDVHGQALYAISR
IVEFFEKNFPGITPDLIPTDNLQK
DNLSLSYAMQQKELPDTQAIVED
YLEQYTK
HTVTALSR
VAIVGAGGTVGSFLAAALLKTGK
AGVPYIMPNGYAGDIEHVKFGQD
VMLGPVAQANR
MFAETVIGAPHGILVSR
FAETVIGAPHGILVSRITVYLSNAK
MFAETVIGAPHGILVSRITVYLSNAK
IQEILVGITFLIAIAFIVK
RGQPALSR
GQPALSRR
CRLQQATLTD
LVITLRDYGR
Hypothetical proteins present after MALDI-TOF/TOF of Salmonella paratyphi A C-6915 OMV:
LDCTEGLDYCCICCPK
MIGHCKLDCTEGLDYCICCPK
WQHLINDLQNDRSVDDEPGTYR
MSEQLHGNMHYLLTSETYNGILVR
MDEWMDGRQSLAEETSM
ILALYCVTVMEAHEIVGVEWGMNR
LLLIAHK
HNSTSSTTPNQREGGPLSGIEFLSFGK
TAGQLINWGMPTLAAEMLNALDCQR
IAVSWAMPVVLTQYDSVMATVMGDS
IKEANEALIDAHNTQTGMLTEEAR
EANELSMQQTMMLIMNAGNAKAFAK
LWYCMMFGVTVATIYGAALILMV
GDPSAAVTIADIAMRAHDAGDLPEGIEGG
MDIMINASFLPHTDPEASLAFYR
VQASGAEVMQEPTDQPWGARDCAFR
YRSAPQAASAGSPTSPPASMQPTPSTSAS
ELRNSFIMDQDNQAAFINTHYK
SLHEDTIDPIENEADHLFIIRNSK
DNVQCAPSGKAAITVSFVLEMDFR
MNSRPPFPSTSSPPSQPSPYRYLGR
QRAGAGNGETGASGVGEQQANFLFNLK
THEEAYAAAVEEFEANPPQVQRGK
GKKPLKPYEGDMPFFDNGDGTTTFK
LICKASSAQGCSPSTTLNQNFMQK
ASSAQGCPSTTLNQNFMQKGILECR
ALLGKMER
FFRGSSQQSSGNPATDFFTVASPLPAAN
HICFEIESYMFRIAFHDFFLPS
MILLHKYSIPACSCFQNIYALNTK
MACVVSHQENQDCASLTPETFLPR
VEAARSER
MACSEQEGVGSPDEEALFASQEGVK
NLCTQPDGGYLTDEGIQMAER
VDGYTVVWDPETDMVVWAGGR
MRIPSTGR
LAVSAVVLLAALSVQGVR
VIAEQEGADSFVCQLAAWLHDLADDK
MGHMERSFVSEDWAGLASWR
SFVSEDWAGLASWRCTCTDVDLGLR
MAPWERK
NALFTPVR
QMTAGMADIMGTSGLAWHQWK
MITQRLR
MPLAEGVTGEGRDTQSRPVGDDLDLTR
KILTEDYVNLVK
SQLNFYDTSVYNFIKSLDYAEVER
NQCNILR
MLFFYQLPFIIPIPSMQGNTFSR
OMV antigens were prepared from S. typhi C-6953 and S. paratyphi A C-6915, and S. typhi C-6.946 and S. paratyphi A BCR 148 for challenge study were collected from National Institute of Cholera and Enteric Diseases (NICED) culture bank. All strains were kept in 20% glycerol in brain heart infusion broth (Difco, USA) at 80° C. Prior to experimentation, each strain was grown in Tryptic Soy Broth (TSB; Difco, USA) at 37° C. under shaking conditions (100 rpm) or on plates in Tryptic Soy Agar (TSA; Difco, USA).
OMVs were prepared from two Salmonella enterica strains with slight modifications where cells were grown at 37° C. under shaking condition followed by centrifugation at 8000 rpm for 40 minutes at 4° C. Following filtration by 0.22 pm bacterial filters (Millipore, USA), OMVs were subsequently purified by ultracentrifugation (4 h, 140,000×g, 4° C.) using a Sorvall T-865 rotor, and re-suspended in Phosphate-Buffered Saline (PBS, pH 7.4). The protein concentration was determined by the modified Lowry protein assay kit (Pierce, USA). LPS O—Ag concentration was determined by a method used by Dubois et al.
A 5 μlaliquot of secreted OMVs were placed on a carbon coated grid and left for 1 minute for proper absorption. The grid was then washed with two drops of Tris-HCl buffer. After blotting excess fluid, the sample was stained with 2% aqueous solution of uranyl acetate. In case of negative staining of bacteria-secreting OMVs, the same procedure was followed with log-phase live bacterial cells. Both the negatively stained OMVs and bacteria-secreting OMVs were observed under Tecnai 12 (as given in
From
The experiments which have performed are given below:
Seven weeks old, BALB/c mice of either sex were taken from the animal resource division of NICED, Kolkata. Male and female mice were caged separately groups of 10 and maintained at a temperature of 25° C. with humidity at 75%. Mice were fed sterile food and water. All the animal experiments were conducted following the standard operating procedure as outlined by Committee for the Purpose of Control and Supervision of Experiments on Animal (CPCSEA), Ministry of environment and forest, Government of India. The animal experimental protocol was approved by the Institutional Animal Ethical Committee of NICED with the project approval no. PRO/108 May, 2014-July, 2017.
Oral Immunization 7 weeks old female BALB/c mice were kept empty stomach 24 hours before the immunization date, water adlibitum. Mice were immunized orally on days 0th, 14th and 28th (
Blood was collected from the lateral tail vein at different time intervals on the 0th, 14th, 21st, 28th, 35th, 78th, 90th day of first oral immunization. The collected blood was taken in BD Microtainer (BD, NJ, USA) followed by centrifugation (1000 rpm, 10 min and 4° C.). Stools from immunized and non-immunized mice were collected in an aseptic Eppendorf by pressing the abdominal region. Stools were then homogenized by a plastic homogenizer and centrifuged at 10000×g for 10 min to remove the debris. The supernatant was collected and stored.
The results of representative immunoblot analysis against OMVs, from two typhoidal strains are given in
The protein content of the OMVs recovered from Salmonella strains were determined as described earlier in this paper. 80 μg of proteins were boiled in 5× SDS-PAGE buffer and loaded onto a 12% SDS-PAGE gel. The gel was then stained by either Coomassie or silver stain. For immunoblot assay, gel was transferred onto nitrocellulose membrane (Bio-Rad, USA) by using the ATTO AE-6687 (Japan) blot apparatus. The polyclonal antibody rose in mice and HRP-conjugated rabbit anti-mouse secondary IgG were used to detect the proteins which were immunogenic.
Here, 1, 2, and 3 denotes three different concentrations of LPSs against which the dot blot analysis was performed.
Dot blot analysis was done as described previously. Briefly, LPS of the two strains were taken and blotted onto a nitrocellulose membrane. The membrane was then washed with Tris-Buffered Saline (TBS) contains 0.1% Tween-20. The membrane was then incubated with primary and secondary antibody successively, where OMV-immunized mice serum was serving the purpose of a primary antibody and the blot was then finally developed by chemiluminescence.
Different immunoglobulins; e.g. IgG and its sub-types (IgG1, IgG2a, IgG3), and IgA, sIgA and IgM were measured by ELISA as stated by Keren (23). Briefly, disposable polystyrene micro-titer wells (Nunc, Denmark) were separately coated with OMVs (5 μg/well) from either strains of the immunogens (Table 1) and incubated for 18 h at 4 C. Wells were washed and blocked with Bovine Serum Albumin (BSA; Sigma Chemical, USA). After washing the wells with PBS-T (PBS with 0.5% Tween-20, Sigma Chemicals, USA) and incubated with serially diluted serum samples, 100 μL HRP conjugated goat anti-mouse immunoglobulin was added and incubated. After washing with PBS, the substrate o-phenyl-Di-amine (OPD) was added to each well followed by stopping the reaction after 10 min by adding 100 μL of 2 N sulphuric acid. OD492 was taken. The experiments were repeated three times for each immunoglobulin, with the immunized and non-immunized serum, collected from individual mice, before, during and after immunization. The same procedure was carried out when ELISA were done against Vi-polysaccharide of S. typhi.
A serum immunoglobulin titer in immunized sera were separately measured against each component OMVs of bivalent OMV and heat-killed (HK) formulations. A. Serum IgG (i), IgG1 (ii), IgG2a (iii), IgG3 (iv); B. Serum IgA; C. Serum IgM response against each of the two OMVs and heat-killed immunogens at pre-immunization, immunization and postimmunization. The horizontal axis indicates the days of blood collection. Data represented here are the mean values +/− Standard Deviation (SD) of three independent experiments. The differences in post-immunization day wise response of each of the studied antibodies against each of the two OMVs were highly significant (P value<0.005) (shown in
Dendritic cells from bone marrow of non-immunized BALB/c mice were cultured for 7 days in complete RPMI containing 10% FBS in the presence of 20 ng/ml GM-CSF (Tonbo). Cells were then treated with 100 ng/ml bivalent OMV and incubated in 37° C. for 24 hours in presence of 5% CO2. Different cytokines, namely IFN-γ, IL-4, IL-12p70, IL-1β and IL-23 were then measured (refer
After 2 weeks from the end of last immunization, splenic cells from immunized BALB/c mice were cultured for 2 hours in complete RPMI containing 10% FBS. Cells were then treated with 100 ng/ml bivalent OMV and incubated in 37 C for 24 hours in presence of 5% CO2. Different cytokines, namely IFN-γ, IL-6 and IL-17 were then measured (
Motility assay was done as previously described, with modifications. Briefly, the immunized and non-immunized serum samples were mixed with PBS at a concentration of 1:400 and poured on soft agar (0.3%) plates. The plates were kept for an hour to get the serum mixed PBS soak in the plate. After the plates became dry, log-phase bacteria (OD600=0.8) were pricked in the middle of the plate. The plates were then incubated at 37° C. for 24 hours. After 24 hours, the results were seen as in
Bivalent S. typhi and S. paratyphi A OMV Protect Adult Mice From S. typhi and S. paratyphi A Challenge
After four successive oral immunizations with bivalent OMVs formulation, protective efficacy was observed in an adult mice intra-peritoneal model (
Tissue homogenates from liver reveals only 10-100 organisms/gm in case of immunized mice, whereas, in non-immunized mice, 5-log fold higher colonization ability was observed.
Both immunized and non-immunized mice were challenged with 2×106 CFU/ml intra-peritoneally and kept them for 12 days for survival assay. In case of non-immunized mice, all the mice died within 1 4 days. But, 80% and 100% immunized mice were still alive. This result suggests that, our bivalent formulation is inhibiting the systemic infection of typhoidal salmonellae in mice and it indeed protecting the mice from lethal infection.
In the majority of cases, the data presented are not normally distributed due to biological variation. Therefore, non-parametric tests were used for all data analysis. Comparison between two categorical variables was made using the two-tailed student's test.
Comparison between multiple categorical variables was made using the one-tailed student's test. Each experiment was repeated at least three times. A P value of <0.05 or <0.01 were considered significant GraphPad Prism 5 for Windows OS was used for all statistical analyses.
The effectiveness of both Salmonella typhi and paratyphi A OMVs have been studied through various experiments.
When the mice were immunized with Salmonella typhi OMVs, they were protected from Salmonella typhi infection (evaluated from the bacterial count from spleen and liver 3 days' post infection). But when the same Salmonella typhi OMVs-immunized mice were challenged with Salmonella paratyphi A, very less amount of protection was found. The same trend was seen when monovalent Salmonella paratyphi A OMVs is used to immunize mice. They were protected from Salmonella paratyphi A infection, but not protected from Salmonella typhi infection. The results are following:
The effect of OMVs isolated from 24 hours' culture was assessed. Although this result shows that the immunized mice are significantly protected from the challenge, but the level of protection is much higher in case of OMVs isolated from 5 hours' culture (refer to Patent application
The OMVs from log-phase culture bacteria caused much less colonization in spleen and liver and substantially much more effective them any other formulations. It is rich in many proteins which were reported to be secreting via OMVs until recently (
The OMVs of two strains of bacteria as claimed contains proteins of different sizes as well as LPS in them. Western blot analysis indicates the presence of strong immune response against the immunogens present in BOMVs. Dot blot analysis serves the purpose of proving this immunogen to be effective and immunogenic against LPS of these two strains. Three doses of oral immunization of these BOMVs formulation in mice induces a significant rise in the level of immunoglobulins specific for isolated OMVs. Presence of sIgA against OMVs formulation and IgG specific for Vi-polysaccharide of S. typhi shows the potency of our bivalent formulation against S. typhi infection.
As Vi-polysaccharide is the outer covering of S. typhi, immunoglobulins present against this component indicates the presence of Vi-polysaccharide in the BOMVs. Because of their intra-cellular nature, both S. typhi and S. paratyphi A can only be eradicated from the host in the presence of significant Th1 cell-mediated immune response along with humoral immune response. A Th1 biased immune response was seen in the ELISA data.
Also, a significant up-regulation in the level of IFN-γ, IL-6, IL-12p70, IL-1β and IL-23 from the isolated BMDCs and IFN-γ, IL-6 and IL-17 from splenocytes shows that the induced response was a result of mainly a Th1 and Th17 cell mediated immune response. Moreover, as verified by sera and splenocytes adoptive transfer experiments, the protective effect of BOMVs vaccination was dependent on both humoral and cellular immunity. So, both humoral as well as cellular arms of the host's immune system are being activated upon the exposure of BOMVs in mice. Our BOMVs immunized mice sera can also inhibit the motility of the wild type strains of typhoidal salmonellae. Inhibition of motility means the bacteria will no longer be able to find their receptors for binding on the human epithelium thus, rendering their inability to cause infection. MTT assay was done to check the reactogenicity of BOMVs. It was found BOMVs were less reactogenic than the conventional heat-killed and whole cell lysate immunogens.
Inhibition to cause infection in mice was further confirmed by anti-colonization and survival assays. In our anti-colonization assay, immunized mice were challenged with circulating strains of typhoidal salmonellae via the intra-peritoneal route. Significant increase in the level of survival in the BOMVs immunized group was seen. Also, the presence of typhoidal salmonellae in spleen and liver were found to be significantly less in immunized mice. Taken together, these findings suggested us that BOMVs could be used as a novel non-living human vaccine candidate against S. typhi and S. paratyphi A infections in future.
In the majority of cases, the data presented are not normally distributed due to biological variation. Therefore, non-parametric tests were used for all data analysis. Comparison between two categorical variables was made using the two-tailed student's t test. Comparison between multiple categorical variables was made using the one-tailed student's t test. Each experiment was repeated at least three times. A P value of <0.05 or <0.01 were considered significant. GraphPad Prism 5 for Windows OS was used for all statistical analyses.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201711011707 | Mar 2017 | IN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IN2018/050158 | 3/21/2018 | WO | 00 |
