MXPA06015159A - Adjuvant-free vaccine composition containing salmonella enterica serovar typha porins. - Google Patents

Abstract

The present invention is related to the use of OmpC and OmpF porins from Salmonella enterica serovar Typhi for preparing adjuvant-free vaccine compositions, which are useful for conferring a long-lasting immunologic response mediated by bactericide antibodies against typhoid fever caused by Salmonella enterica serovar Typhi.

Description

ADJUVANT-FREE VACCINE COMPOSITIONS CONTAINING POINTS OF SAIMONELIA ENERICO SERIOVAR TYPHI FIELD OF THE INVENTION The invention relates to the use of OmpC porins and OmpF of Salmonella enterica serovar Typhi to prepare vaccine compositions, free of adjuvants, useful for conferring a long-lasting immunological response, mediated by bactericidal antibodies, against typhoid fever.

BACKGROUND OF THE INVENTION Typhoid fever continues to be a serious public health problem in many developing countries. It has been estimated that there are 33 million cases annually worldwide, resulting in 500,000 deaths (Garmory et al., 2002, FEMS Microbiol, Rev. 26, p 339-53). This infectious disease also represents a risk for visitors from endemic areas (Levine M, In: Plotkin, SA, Orenstein WA, editors, Typhoid fever vaccines, London, Saunders, 1999, p 781-814). The majority of cases occur mainly in Southeast Asia, where there is an increasing prevalence of antibiotic resistance (Parry et al., 1998, Lancet, 351, p 1289).

Salmonella is a mobile, gram-negative, rod-shaped bacterium that belongs to the Enterobacteriaceae family; the species is a close relative of Escherichia coli. This bacterium was described in 1880 by Eberth and cultivated in 1884 by Gaffky (Burrows, W., 1953. Texíbook of microbiology, 7th ed., The W. B. Saunders Co., Philadelphia, USA). The strains were differentiated based on their reaction with serum and for many decades a new serotype was given to each new species (for example, S. typhimurium, S. enteritidis, S. pullorum, and S. dublin). It is currently accepted that there is a single species of Salmonella (S. enterica) and more than 2000 serovars (McWhorter-Murlin and Hickman-Brenner, 1994, Centers for Disease Control and Prevention, Atlanta, USA); however, most researchers continue to write, for example "S. typhimurium" instead of "S. enterico serovar typhimurium" for greater convenience and continuity of the previous literature.

Salmonella is capable of causing a variety of disease syndromes: enteric fever, bacteremia, enterocolitis, and focal infections. Enterocolitis is by far the most common manifestation of the disease caused by Salmonella, but a bacteremia and "focal infections can accompany or follow enterocolitis.Intermic fever (typhoid fever) is caused mostly by S. typhi and S. paratyphi and occasionally by other serotypes While approximately 2,000 Salmonella serotypes have been associated with enterocolitis, a smaller group of approximately 10 serotypes is counted for most infections, these typically include S. typhimurium, S. enteritidis, and S. heidelberg (Centers for Disease Control and Prevention, 1994, Salmonella surveillance-annual summary-1992, Centers for Disease Control and Prevention, Atlanta, USA, and Tauxe, RV, 1996, Health Environ. Dig., 10 , p 1-4) .The incubation period is typically 6 to 48 hours and is followed by headache, abdominal pain, diarrhea, and vomiting.Diarrhea may contain blood, lymphocytes, and mucus. re, discomfort, and muscle pains are quite common. Symptoms usually resolve within a week, but Salmonella can be poured into the stool for up to 20 weeks in children older than 5 years, and up to 8 weeks in adults (Gómez, HF, and GG Cleary, 1998, Salmonella, 4th ed, vol 1. The WB Saunders Co., Philadelphia, USA). Children, especially those under one year of age, and those older than 60 are more susceptible to the disease and tend to have more severe infections (Gómez, HF, and GG Cleary, 1998, Salmonella, 4th ed, vol. The WB Saunders Co., Philadelphia, USA; Hook, EW, 1990. Salmonella species (including typhoid fever), 3rd ed. Churchill Livingstone, Inc., New York, NY; Tauxe, RV, and Pavia AT, 1998, Salmonellosis: Nontyphoidal, pp. 613-630, AS Evans, and PS Brachman (ed.), Bacterial infections of humans: epidemiology and control, 3rd ed. Plenum Medical Book Co., New York, NY, and Turnbull, PCB, 1979, Food poisoning with special reference to Salmonella-Ws epidemiology, pathogenesis and control, Clin. Gastroenterol 8, p 663-714).

Vaccination is an effective tool for the prevention of infections with Salmonella (Mastroeni, et al., 2001, Vet. J., 161, p 132-164) .. The vaccines currently available against salmonellosis can be divided into three groups principal: a) killed whole cell vaccines, they consist of heat inactivated bacterial cells or acetone and are administered parenterally; in humans, killed vaccines present good antibody responses and confer a moderate degree of protection (Levine, et al., 1989, Rev. Infect. Dis., 11, p S552-S567). This type of vaccine is reactogenic and induces poor cellular immunity (Coliins, F., 1974, Bacterial, Rev. 38, p 371-402, Harrison, et al., 1997, Immunology, 90, p 618-625). ); b) subunit vaccines, such as those based on the Vi polysaccharide of S. typhi, are safe, immunogenic and are currently licensed for use in humans. Vi vaccines provide between 55 and 75% protection against typhoid fever in endemic areas (Acharya et al., 1987, N. Engl. J. Med. 317, p 101-104, and Klugman et al., 1996 , Vaccine, 14, p 435-438). The immunogenicity and protective ability of Vi increases when the latter is linked to carrier proteins (Kossaczka et al., 1999, Infecí.Immun.67, p 5806-5810, Lin et al., 2001, N. Engl. J. Med 344, p 1263-1269, Singh et al., 1999, Microbiol, Immunol 43, p 535-542, Szu et al., 1987, J. Exp. Med. 166, p 1510-1524). Other vaccine subunits, such as those based on detoxified LPS, cell extracts, porins, O-polysaccharides and O-conjugates have been tested in experimental models and have had less efficacy. AND c) live attenuated vaccines; The potential for superiority of live attenuated vaccines compared to inactivated preparations has recently been a challenge of research towards the development of Salmonella mutants so that they can be used in human and veterinary medicine. To date, the development of live attenuated vaccines against Salmonella infections has been based mainly on empirical criteria. The availability of the complete sequence of the genome of S. typhi and S. typhimurium and the advanced methods to identify virulence genes expressed in vivo are useful tools for the generation of attenuated Salmonella mutants (McClelland et al., 2001, Nature, 413 , p 852-856, Parkhill et al., 2001, Nature, 413, p 848-852, Shea st ai., 1996, Proc. Nati, Acad. Sci. USA, 23, p 25S3-259.7; al., 1994, Methods Enzymol., 235, p 481-492). However, the rational design and selection of Salmonella strains to be used as potential vaccines should be based on knowledge of the pathogenesis and immunobiology of the infection. Indeed, the profile and anatomical location of the immunological response induced by the vaccine have an enormous influence on whether a long and solid acquired resistance to the pathogen will be established in the vaccinated individual.

Various typhoid vaccines are currently available (Garmory et al., 2002, FEMS Microbiql Rev. 26, p 339-53). The live oral vaccine attenuated ga / E mutant Ty21a is effective in endemic areas, but there is no license for use in children under 6 years, and this requires three to four doses (Levine et al., 1999, vaccine, 17 , p S22-7, Murphy et al., 1991, Infect. Immun 59, p 4291-3). The polysaccharide vaccine, Vi, is licensed for use in children over 2 years of age. An injection of such vaccine provides protection similar to the Ty21a vaccine, but is effective only for 2 to 3 years. Thus, the main disadvantage of this vaccine is the lack of induction of long-term immunity (Klugman et al., 1996, Vaccine, 14, p 435-8). A new typhoid vaccine, based on the Vi polysaccharide conjugated with the non-toxic recombinant exotoxin A of Pseudomonas aeruginosa (Vi-rEPA), has been presented to be safe and protective even in children aged 2 to 5 years, and is currently in the Phase 3 clinical trials (Lin et al., 2001, N. Engl. J. Med., 344, 1263-9). However, the emergence of strains of enteric Salmonella serovar typhi negative to the Vi antigen has been reported in epidemic cases of typhoid fever in Calcutta (Sana et al., 2000, Indian Nati Mea, J. India, 13, p 164) which leads to the need for the development of new vaccines.

Therefore, there is a continuing need for safer and more effective vaccines against Salmonella, which ideally do not have to be administered in large doses.

The antibody response is important to achieve protection against infection with Salmonella. We have previously reported that serum, inactivated by heat, specific for plasma membrane proteins of Salmonella enterica serovar typhi can protect mice against the challenge of 50 to 100 lethal doses of virulent bacteria (Isibasi et al., 1988, Infect Immun., 56, p 2953-9). In humans, high titers of specific antibodies against surface antigens of Salmonella enterica serovar typhi, such as the Vi polysaccharide, correlates with protection against disease (Robbins and Schneerson, 2004, Ann. NY Acad. Sci., 1038, p. 49-59). These studies reflect that the antibody response is essential to achieve protection against enteric Salmonella serovar typhi. In mice, the contribution of the antibody-mediated immune response during infection with Salmonella has been studied in animals deficient in B cells (Maestroeni et al., 2000, Infect. Immun., 68, p 46-53; Ugrinovic et al. , 2003, Infect, Immun., 71, p 6808-19, Mittrucket et al., 2000, J. Immunol., 164, p 16848-52). These experiments establish that the protective immunity against S. typhimurium depends on the combined action of specific antibodies, B cells and an acquired immune response of T cells (Maestroeni et al., 2000, Infect, Immun., 68, p 46-53; Ugrinovic et al., 2003, Infect. Immun., 71, p 6808-19, Mittrucket et al., 2000, J. Immunol., 164, p 16848-52). In addition, antibodies against lipopolysaccharides, porin-lipopolysaccharide complexes, and to a lesser extent, native proteins, are important in the acquired resistance against infection with S. typhimurium (Singh et al., 1996, Microbial, Pathogenesis, 21, p 249 -63).

Due to the ability of the porins to awaken an immunological response and in this way a protection of the host against infection, our group and others have studied the porins of Salmonella enterica serovar typhi as candidates for a vaccine against typhoid fever (Salazar-González et al., 2004, Immunol-Lett., 93, p 115-22, Singh et al., 1999, Microbiol. Immunol., 43, p 535-42). With an estimate of above 100,000 molecules per cell, porins are the most abundant proteins found in the plasmatic membrane of Gram-negative bacteria; the porins are assembled in trimer and form stable pores that allow the passive transport of nutrients (Nikaido H, 996, Cellula and Molecular Biology, Washington DC, ASM Press, 29-47). The first evidence of porin immunogenicity was that serum from patients in the acute and convalescent phases of typhoid fever induced IgM and IgG antibodies that mainly recognize those proteins (Calderón et al., 1986, Infect. Immun. 52, p. 209-12, Ortiz et al., 1989, J. Clin. Microbiol., 27, p 1640-5, Verdugo-Rodriguez et al., 993, Eur. J. Clin. Microbiol. Infect. Dis., 12, p248 -54). In addition, the porins of various pathogens have been described as activators of innate and adaptive immune responses (Galdiero et al., 1998, Immunol, 94, 5-13; Wetzier et al., 1996, J. Exp. Ed., 183, p 151 -9). These proteins are agonists for the Toll-like receptor 2 (TLR2) and induce the expression of costimulatory molecules, upregulate the class II molecules of the Histocompatibility Main Complex, release of cytokines by macrophages, and increase the production of antibodies by B cells (Massari et al., 2002, J. Immunol., 168, p 1533-7, Galdiero et al., 2004, Infect. Immun., 72, p 1204-9, Ray and Biswas, 2005, Immunology, 14, p 94-100). Immunization with purified porridins can protect mice against the challenge with virulent strains of Salmonella (Muthukkumar et al., 1993, Infect. Immun., 61, p 3017-25, Singh et al., 1999, J. Med. Microbiol. , 48, p 79-88). During the immunological response, the porins of S. enterica serovar typhi induce a response by T cells and anti-porin antibodies (Salazar-Gonzalez et al., 2004, Immunol.Lett., 93, p.115-22; Diaz-Quiñonez et al., 2004, Infect. Immun., 72, p 3059-62).

Previous studies have shown that Salmonella porins induce lgG1 production in BALB / c mice, predominantly, after 30 days (Nanda Kumar et al., 1999, Scand. J. Immunol., 50, p 188-94). We have recently reported that human volunteers vaccinated with porcine S. enterica serovar typhi produce IgG and IgG2 bactericidal antibodies two weeks after immunization (Salazar-Gonzalez et al., 2004, Immunol.Lett., 93, p.115-22 ). These studies have shown as a whole that porin are good immunogens.

High-affinity and long-lasting protective antibodies are maintained after an infection or immunization (Gourley et al., 2004, Semin. Immunol., 16, p 323-33; Zinkernagel RM, 2003, Annu., Rev. Immunol., 21, p 515-46). However, only a few purified antigens have been studied and found to induce a persistent, long-lasting antibody response in the absence of adjuvant (Zinkernagel RM, 2003, Annu, Rev. Immunol., 21, p 515-46). For this reason, the characterization of new antigens with the ability to induce a memory response of bactericidal or neutralizing antibodies constitutes an important effort in the understanding of protective immunological memory.

In this sense, our invention is indicative that the OmpC and OmpF porins of S. enterica serovar typhi are responsible, in the absence of adjuvants, for inducing a long-lasting immune response and high-affinity and long-lasting protective antibodies.

SUMMARY OF THE INVENTION According to the present invention, vaccine compositions are provided, free of adjuvants, based on porins of Salmonella enterica serovar Typhi, useful for conferring a long-lasting immunological response, mediated by bactericidal antibodies, against typhoid fever caused by Salmonella enterica serovar Typhi.

In another aspect of the invention, the use of Porcine Salmonella enteric serovar Typhi is provided to prepare vaccine compositions, free of adjuvants, useful for conferring a long-lasting immunological response, mediated by bactericidal antibodies, against typhoid fever caused by Salmonella enteric serovar Typhi.

DETAILED DESCRIPTION OF THE INVENTION The vaccine compositions, free of adjuvant, can be prepared according to methods known in the state of the art. The present compositions comprise an immunogenic amount of the OmpC porins and OmpF of Salmonella enterica serovar Typhi, usually combined with a pharmaceutically acceptable carrier. I5 Pharmaceutically acceptable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slow metabolizing macromolecules, such as proteins, polysaccharides, polylactic acids, polyglyclic acids, polymeric amino acids, amino acid copolymers; and inactive virus particles. Such carriers are widely known to those skilled in the art.

Vaccine compositions can be administered by oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, mucosal, air, rectal, vaginal and topical (including buccal and sublingual). The route of administration will depend on the composition of a particular therapeutic preparation of the invention, and in some cases, on the intended site of the action.

The invention will now be described with reference to specific examples. They are merely for illustrative purposes: they do not attempt in any way to limit in any way the purpose of the invention described. Said examples up to now constitute the best method currently contemplated for the practice of the invention.

EXAMPLES Example 1. The porins of S. enterico serovar typhi induce a long-lasting antibody response. BALB / c mice were immunized intraperitoneally with 10 flg of native porins on day zero and given an identical challenge on day 15. These antigens were diluted in physiological saline as a carrier vehicle, and they were injected in the absence of any adjuvant. Blood samples from the retro-orbital sinus were collected at different times of the immunization. From the serum obtained from each of the blood samples, the titer and isotype of antibodies were determined using the ELISA technique. Briefly, 96-well polystyrene plates were covered with 10 Dg / ml of porins, OmpC, OmpF, or lipopolysaccharide in 0.1 M carbonate-bicarbonate buffer, pH 9.5. The plates were incubated for 1 hour at 37 ° C and subsequently at 4 ° C overnight. The plates were then washed 3 times in phosphate sa.ir.c solution (P3S), pH 7.2 containing 0.05% Tween-20 (PBS-T). Nonspecific junctions were blocked using a 5% solution of fat-free milk in PBS for 2 hours at 37 ° C. After washing the plates, serum diluted 1: 40 in PBS with 5% fat-free milk and serial dilutions was added to the wells. The plates were incubated for 2 hours at 37 ° C, followed by 6 washes with PBS-T. Subsequently, an optimal dilution, 1000: 1, of rabbit anti-IgM antibody or mouse anti-IgG conjugated to the enzyme peroxidase was added. Then it was incubated for 1 hour at 37CC. Subsequently, the plates were washed 8 times using PBS-T. O-Phenylenediamine (0.5 mg / ml) was used in 0.1 M citrate buffer, pH 5.6, containing 30% hydrogen peroxide, as the enzyme substrate. The reaction was stopped using 1.25 M H2SO4, and then the optical density was determined at 490 nm using an automatic ELISA plate reader. During the primary antibody response, a 16-fold increase in IgM and IgG antibody titers was observed on day 4. After the 15-day challenge, a higher antibody titer was observed, around day 30, with a 1024-fold increase in the IgG titer over the control values. On day 90, the IgG titers decreased to half their maximum values, and continued to decrease slightly until reaching levels similar to those of day 4. The antibody titre was maintained 16 times higher than the control levels until The last mouse died, apparently from natural causes, 476 days after the first immunization. Although the IgM antibody titer was lower, a long-lasting IgM response was observed, with a rise of 4 times a day 476. To examine whether the route of administration influenced the antibody response, the mice were immunized intravenously and subcutaneous The kinetics of the antibody titer was similar in these groups with respect to the animals immunized intraperitoneally. These results indicate that the porins induce a long-lasting antibody response in the absence of an exogenous adjuvant, regardless of the route of administration.

Example 2. Porins induce a long-lasting bactericidal antibody memory response. Because an efficient memory of B cells depends on the ability of the antibodies to achieve a neutralizing or bactericidal activity, an antibody-dependent bactericidal assay was used to examine whether anti-porin antibodies could mediate bacteriostasis of S. enterica serovar typhi . Briefly, anti-porin serum, anti-OmpC, and anti-OmpF were heat inactivated for 30 minutes at 56 ° C. Polystyrene microtiter plates of 96 U-bottom wells were used. Duplicates of heat inactivated serum were pre-diluted 1: 40 in PBS and doubly serial dilutions were added to the wells containing 200 +/- 50 colony forming units. S. enteric serovar typhi wild or the isogenic VALE39 strain (deficient in porins), and a source of complement (90% guinea pig serum (v / v)) Controls included samples containing: (1) a known positive sample , (2) bacterium and complement-inactivated anti-porin serum, (3) guinea pig bacteria and whey, and (4) bacteria, anti-porin serum, and guinea pig serum inactivated by heat. for 18 hours at 37 ° C and the colony forming units were counted, although a weak bactericidal response was observed at day 4 after immunization, by day 10, the bactericidal titres increased rapidly to 128 times above the level of the During the secondary response, the bactericidal activity showed an increase of 512 times on day 25, and remained at that level until day 476, even though antibody titres markedly decreased after day 150. The bactericidal activity of these sera was directed against the porins, since the sera of the immunized mice could not induce bacteriolysis of the VALE strain39. These data indicate that the low titers of antibodies measured by ELISA were as efficient as the high titers of anti-porin antibodies observed during the secondary response in killing the bacteria.

Claims (4)

1. The use of at least one porin selected from the group of OmpC and OmpF of Salmonella enterica serovar Typhi to prepare a vaccine composition, free of adjuvant, useful for conferring a long-lasting immune response, mediated by bactericidal antibodies, against typhoid fever caused by Salmonella enterica serovar Typhi.

2. A vaccine composition, free of adjuvant, useful for conferring a long-lasting immune response, mediated by bactericidal antibodies, against typhoid fever caused by Salmonella enterica serovar Typhi, characterized in that it contains at least one porin selected from the OmpC and OmpF group of Salmonella enteric serovar Typhi.

3. The vaccine composition according to claim 2, whn the vaccine composition can be therapeutic or prophylactic.

4. The vaccine composition according to claims 2-3, characterized in that it additionally contains pharmaceutically acceptable excipients.