Patent Application
20240033350
The present disclosure relates to a field of vaccine preparation; more particularly, to a lipidated FLIPr (rLF) and uses thereof in vaccine as a vaccine candidate and an adjuvant.
Staphylococcus aureus can be a harmless microorganism. It becomes an invasive pathogen when S. aureus enters the germ-free regions of the host once epithelial barriers or immune systems turn out to be compromised (Clin Microbiol Rev 2015. 28: 603-661; Lancet Infect Dis 2005. 5: 751-762; Front. Microbiol. 2018. 9:2419). Nasal S. aureus carriage is the main reservoir for Staphylococcus aureus, which is one of key risk factors resulting the pathogenic process of hospital and community acquired infections (N. Engl. J. Med. 2001. 344:11-16; Lancet 2004. 364(9435):703-705). S. aureus has evolved various approaches to escape the attack from the immune system. The approaches used by this bacterium include resistance to antimicrobial peptide, and inhibition of neutrophil recruitment, phagocytosis, killing by ROS and neutrophil (Microbial Pathogenesis 2019, 131:259-269). Thus, molecules on the surface of bacteria and products for immune evasion are potential targets for S. vaccine development (BMC Microbiology 2010, 10:173).
Formyl Peptide Receptor-like 1 Inhibitor (FLIPr) is one of the immune evasion proteins produced by S. aureus to evade immune system via binding to C1q subcomponents or C1q complex, or binding to various Fcγ receptor subclasses resulting in competitive block of IgG-ligand binding. These interaction can increase Staphylococcus aureus survival in whole human blood (Sci Rep. 2016, 6:27996.) and inhibit Fc γ receptor-mediated effector functions (J Immunol 2013. 191: 353-362). Additionally, FLIPr is detected in many infecting isolates during biofilm formation in vitro (Med Microbiol Immunol 2017, 206:11-22). Therefore, FLIPr is a potential vaccine candidate to induce immune response for reducing S. aureus virulence and biofilm formation. In this invention, a vaccine candidate to induce anti-FLIPr responses is developed to overcome FLIPr-mediated inhibition.
Inactivated virus and recombinant protein (subunit vaccines), which are noninfectious, have significant safety advantages over live or live-attenuated vaccines. However, it is known that inactivated virus and recombinant protein exhibit low immunogenicity. Thus, these two vaccine types are generally formulated with an adjuvant to induce powerful immune responses. Currently, there are rare mucosal adjuvants approved for use in human vaccines.
Accordingly, a recombinant lipidated FLIPr is prepared in the present invention, and proven to evaluate its immunogenicity. The recombinant lipidated FLIPr alone elicts potent anti-FLIPr antibody responses to overcome FLIPr-mediated inhibition of phagocytosis. Also, the recombinant lipidated FLIPr mixed with inactivated virus or recombinant protein can enhance mucosal and systemic immune response to inactivated virus or recombinant protein.
In one aspect, the present disclosure provides a vaccine composition, comprising a recombinant lipidated FLIPr (rLF).
In one embodiment, the recombinant lipidated FLIPr (rLF) comprises a lipidating sequence and a polypeptide of FLIPr.
In some embodiments, the recombinant lipidated FLIPr (rLF) is used to induce anti-FLIPr responses. In some embodiments, the recombinant lipidated FLIPr (rLF) is used to prevent phagocytosis inhibition caused by S. aureus FLIPr protein.
In some embodiments, the vaccine composition further comprises an immunogen, wherein the recombinant lipidated FLIPr (rLF) is used to enhance a vaccine immune response of the immunogen. In one embodiment, the immunogen is a recombinant subunit protein of a virus. In one embodiment, the immunogen is an inactivated virus.
In one embodiment, the immunogen is a recombinant envelope protein domain III of Zika virus (rZE3). In one embodiment, the immunogen is a recombinant mixture of the S. pneumoniae pneumococcal proteins (rAAC). In one embodiment, the immunogen is a recombinant trimeric spike protein of SARS CoV-2 (rTS).
In one embodiment, the immunogen is an inactived Zika virus (IZV). In one embodiment, the immunogen is an inactive influenza virus.
In one aspect, the present disclosure provides a method for enhancing a vaccine immune response, comprising immunizing a subject with a vaccine comprising a recombinant lipidated FLIPr (rLF) as an adjuvant. In one embodiment, the vaccine immune response is an antigen-induced antibody response. In one embodiment, the antigen-induced antibody response is a systemic and/or mucosal antibody response. In one embodiment, the vaccine immune response is a T cell response.
In one embodiment, the vaccine comprises an antigen (or antigens) adjuvanted with the recombinant lipidated FLIPr (rLF).
In some embodiments, the vaccine is a subunit vaccine. In one embodiment, the vaccine comprises a recombinant envelope protein domain III of Zika virus (rZE3) adjuvanted with the recombinant lapidated FLIPr.
In one embodiment, the vaccine comprises one or more S. pneumoniae pneumococcal proteins (rAAC) adjuvanted with the recombinant lipidated FLIPr (rLF).
In one embodiment, the vaccine comprises a recombinant trimeric spike protein of SARS CoV-2 (rTS) adjuvanted with the recombinant lipidated FLIPr (rLF).
In some embodiments, the vaccine is an inactivated virus vaccine. In one embodiment, the vaccine comprises an inactived Zika virus (IZV) adjuvanted with recombinant lipidated FLIPr (rLF). In one embodiment, the vaccine comprises an inactive influenza virus adjuvanted with the recombinant lipidated FLIPr (rLF).
The present disclosure is described in detail in the following sections. Other characteristics, purposes and advantages of the present disclosure can be found in the detailed description and claims.
The present disclosure can be more readily understood by reference to the following detailed description of various embodiments of the disclosure, the examples, and the chemical drawings and tables with their relevant descriptions. It is to be understood that unless otherwise specifically indicated by the claims, the disclosure is not limited to specific preparation methods, carriers or formulations, or to particular modes of formulating the extract of the disclosure into products or compositions intended for topical, oral or parenteral administration, because as one of ordinary skill in the relevant arts is well aware, such things can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meaning:
Often, ranges are expressed herein as from “about” one particular value and/or to “about” another particular value. When such a range is expressed, an embodiment includes the range from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the word “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to and independently of the other endpoint.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.
As used herein, the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.
As used herein, the term “lipidated FLIPr (rLF)” refers to a polypeptide of FLIPr fused with a lipidating sequence at its N-terminus. The term “lipidating sequence” used herein refers to a non-naturally occurring amino acid sequence that facilitates lipidation in E. coli of a polypeptide carrying the lipidating sequence at its N-terminus.
The following examples are provided to aid those skilled in the art in practicing the present disclosure.
To express the recombinant lipidated FLIPr (rLF), the E. coli C43(DE3) strain is transformed with plasmid pLF. The transformed cells are cultured with LB broth at 37-C overnight. The overnight culture is scaled up 50 times of the original volume in a 2 L-shake flask and incubated at 37° C. until the OD600 reach to 0.6. Protein expression is induced (OD600=0.6) by adding 1 mM IPTG, followed by incubation at 20° C. for 24 h. rLF is purified by disrupting the harvested cells in a French press (Constant Systems, Daventry, UK) at 27 Kpsi in homogenization buffer [20 mM Tris (pH 8.0), 40 mM sucrose, 400 mM NaCl and 10% glycerol]. The cell lysate is clarified by centrifugation with 32,000 rpm for 40 min in 4° C. Most of the rLF is present in inclusion bodies.
rLF is then solubilized with extraction buffer [10 mM Na2HPO4 (pH9.0) and 1% TritonX-100]. The extracted fraction is loaded onto immobilized metal affinity chromatography (IMAC) columns (BIO-RAD, Hercules, CA, USA, 2.5 cm i.d.×10.0 cm) containing 20 ml Ni-NTA resin (Qiagen, San Diego, CA, USA) to purify rLF. The column washed with the extraction buffer and the same buffer containing 20 mM imidazole. Then, rLF is eluted with the homogenization buffer containing 500 mM imidazole. The eluted rLF is dialyzed to 20 mM Tris (pH 8.0) three times for at least 6 h each time. After dialysis, the rLF is loaded onto a 20-ml Q Sepharose fast flow column (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The column washed with dialysis buffer containing 200 mM NaCl, and then washed with a 100-fold column volume of dialysis buffer containing 0.1% Triton X-114 to remove the lipopolysaccharide. Next, the column is washed without 0.1% Triton X-114 to remove the residual detergent and rLF is eluted with elution buffer [10 mM Na2HPO4 (pH9.0), 300 mM NaCl and 8M Urea]. The eluted rLF is dialyzed to 10 mM Na2HPO4 (pH9.0) three times for at least 6 h each time.
The endotoxin levels of the purified rLF are determined by the Limulus amebocyte lysate (LAL) assay (Associates of Cape Cod, Inc., Cape Cod, MA), and the resulting endotoxin levels are <30 EU/mg. After dialysis the rLF is lyophilized and stored at −20° C. The fractions from each step are analyzed by SDS-PAGE and immunoblotted with anti-FLIPr and anti-Histag antibodies.
Antibody Responses Induced by Recombinant Lipidated FLIPr (rLF)
In order to validate that the lipidated FLIPr is a vaccine candidate, groups of mice are intranasally immunized three times with PBS, rFLIPr or rLF. PBS-immunized mice are served as the control. The titers of anti-rFLIPr IgG antibodies in the serum before immunization (W0) or 6 weeks after immunization (W6) are determined by ELISA.
As showed in
Recombinant Lipidated FLIPr (rLF)-Immunized Serum Overcome the FLIPr-Mediated Inhibition of Phagocytosis by Human Neutrophils
The serum from immunized mice collected at 6-weeks post-immunization are used to opsonize phagocytosis of fluorescent staphylococci by human neutrophils. The 30 μg/ml rFLIPr are used to inhibit the phagocytosis. Phagocytosis is determined as the percentage of cells with fluorescent bacteria, or the relative geometric mean fluorescence intensity of cells with fluorescent bacteria.
As showed in
In summary, the lipidated FLIPr can be used as a vaccine to elicit antibodies against FLIPr, and also to prevent phagocytosis inhibition caused by S. aureus FLIPr protein.
In this example, the role of the lipidated FLIPr in a vaccine as an adjuvant to enhance immune responses is identified by the immunizations of rLF with other immunogens, including recombinant proteins or inactivated vaccines.
Antibody Responses Induced by Recombinant Ovalbumin (rOVA) Adjuvanted with Recombinant Lipidated FLIPr (rLF).
Groups of mice intranasally immunized three times with PBS, rOVA or rOVA adjuvanted with rLF. PBS-immunized mice are served as the control. Serum samples are collected as indicated time after the first immunization. The titers of anti-rOVA IgG antibodies in the serum are determined by ELISA.
As showed in
T Cell Responses Induced by rOVA Adjuvanted with rLF
To further validate the functions of with rLF in the enhancement of T-cell immune responses, groups of mice intranasally immunized three times with PBS, rOVA or rOVA adjuvanted with rLF. PBS-immunized mice are served as the control. Splenocytes from immunized mice are cultured and stimulated with rOVA, OT-1 peptide, or OT-2 peptide for 2-3 days. Stimulation with BSA, control peptides, or medium alone served as controls. Frequencies of IFN-γ producing cells are evaluated by ELISPOT. The supernatants are collected to evaluate levels of IFN-γ, IL-5, IL-13, and IL-17A by ELISA.
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Antibody Responses Induced by Recombinant Envelope Protein Domain III of Zika Virus (rZE3) Adjuvanted with Recombinant Lipidated FLIPr (rLF)
Groups of mice intranasally immunized twice with PBS, rZE3 or rZE3 adjuvanted with rLF. PBS-immunized mice are served as the control. The titers of anti-rZE3 IgG and IgA antibodies in the serum are determined by ELISA. The Zika virus-neutralizing antibody titer of the serum samples is determined by FRNT.
As showed in
Cytokine Production Profiles Induced by rZE3 Adjuvanted with rLF
Groups of mice are intranasally immunized twice with PBS, rZE3 or rZE3 adjuvanted with rLF (rZE3+rLF). PBS-immunized mice are served as the control. Splenocytes isolated from the immunized mice are cultured and stimulated with rZE3 for 4 days. Stimulation with BSA or medium alone served as controls. The supernatants are collected to evaluate levels of Th1-type cytokines (IFN-γ and IL-2), Th2-type cytokines (IL-5 and IL-13), and Th17-type cytokines (IL-17A) by ELISA.
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Antibody Responses Induced by Intranasal Immunization of Recombinant Trimeric Spike Protein of SARS CoV-2 (rTS) Adjuvanted with Recombinant Lipidated FLIPr (rLF)
Groups of mice are vaccinated twice with PBS, rTS, or rTS adjuvanted with rLF (rTS+rLF). The titers of anti-rTS IgG and IgA antibodies in the serum are determined by ELISA. The titers of anti-rTS IgA antibodies in mucosal tissue are determined by ELISA, including nasal wash and bronchoalveolar lavage fluid (BALF). Vaccine-induced neutralizing antibody against SARS-CoV-2 is evaluated by neutralization assay.
As showed in
Cytokine Production Profiles Induced by rTS Adjuvanted with rLF
Groups of mice are vaccinated twice with PBS, rTS, or rTS adjuvanted with rLF. PBS-immunized mice are alone served as control. Splenocytes are cultured and stimulated with rTS for 4 days. Stimulation with BSA or medium alone served as controls. The supernatants are collected to evaluate levels of the Th1-type cytokines (IFN-γ and IL-2), Th2-type cytokines (IL-5 and IL-13), and Th17-type cytokines (IL-17A) by ELISA.
As showed in
Systemic and Mucosal Antibody Responses Induced by Recombinant Mixture of the S. pneumoniae Pneumococcal Proteins (rAAC) Adjuvanted with Recombinant Lipidated FLIPr (rLF)
The mixture protein rAAC including pneumococcal surface adhesin A (rPsaA), pneumococcal surface protein A (rPspA), and pneumococcal surface protein C (rPspC). Groups of mice intranasally immunized three times with PBS, rAAC or rAAC adjuvanted with rLF (rAAC+rLF). The titers of anti-rPsaA, PspA, and PspC IgG in the serum are determined by ELISA.
Furthermore, the titers of anti-rPsaA, PspA, and PspC IgA in the nasal wash or BALF are determined by ELISA.
As showed in
Cytokine Production Profiles Induced by rAAC Adjuvanted with rLF.
Groups of mice are vaccinated twice with PBS, rAAC, or rAAC adjuvanted with rLF (rAAC+rLF). PBS-immunized mice are alone served as control. Splenocytes are cultured and stimulated with rAAC for 4 days. Stimulation with BSA or medium alone served as controls. The supernatants are collected to evaluate levels of Th1-type cytokines (IFN-γ), Th2-type cytokines (IL-13), and Th17-type cytokines (IL-17A) by ELISA.
As showed in
The Protective Effect Against S. pneumoniae Induced by rAAC Adjuvanted with rLF
Groups of mice intranasally immunized three times with PBS, rAAC or rAAC adjuvanted with rLF (rAAC+rLF). The percent survival of mice after challenge by intranasal inoculation with 2×105 CFU of the S. pneumoniae D39 strain.
As showed in
Antibody Responses Induced by Intranasal Immunization of Inactived Zika Virus (IZV) Adjuvanted with Recombinant Lipidated FLIPr (rLF)
Groups of mice intranasally immunized twice with PBS, IZV, or IZV adjuvanted with rLF (IZV+rLF). The titers of anti-IZV IgG antibodies in the serum are determined by ELISA. The titers of anti-rZE3 IgG antibodies in the serum are determined by ELISA.
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Antibody Responses Induced by Intranasal Immunization of Inactive Influenza Viruses H7N9 (IH7N9) Adjuvanted with Recombinant Lipidated FLIPr (rLF)
Groups of mice are vaccinated twice with IH7N9, or IH7N9 adjuvanted with rLF (IH7N9+rLF). The titers of anti-IH7N9 IgG antibodies in the serum are determined by ELISA. The titers of anti-IH7N9 IgA antibodies in mucosal tissue are determined by ELISA, including nasal wash and bronchoalveolar lavage fluid (BALF).
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As the results, the lipidated FLIPr can be widely used in vaccines with different antigens, including recombinant proteins and inactivated virus vaccines to enhance specific antibody and T-cell immune responses.
While the present disclosure has been described in conjunction with the specific embodiments set forth above, many alternatives thereto and modifications and variations thereof will be apparent to those of ordinary skill in the art. All such alternatives, modifications and variations are regarded as falling within the scope of the present disclosure.
This application claims priority of U.S. Provisional Application No. 63/394,132 filed on Aug. 1, 2022 under 35 U.S.C. § 119(e), the entire contents of all of which are hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63394132 | Aug 2022 | US |
