Various embodiments disclosed herein relate generally to a vaccine composition containing Bordetella pertussis (Bp) antigens and a high molecular weight polymer of glucose, more specifically for intranasal administration.
Pertussis is a bacterial, airborne disease that can be spread through coughing and sneezing. The Gram-negative bacteria invade the respiratory space, propagate, and releases bacterial toxins, causing pulmonary, and if untreated, cardiac dysfunction. Commonly known as whooping cough, pertussis can be deadly to young children or immune-compromised individuals. Currently, the standard vaccine for pertussis, is an acellular vaccine (aP; DTaP; Tdap), which presents several proteins from the B. pertussis pathogen to train the human immune system to respond with a humoral antibody response to clear the pathogen.
Despite high vaccine coverage, whooping cough has re-emerged as a major public health concern in the U.S. and the world. The incidence of pertussis has recently reached levels not seen since the 1950's. It is arguable that this increase is due to the switch from a whole cell vaccine (wP) to the currently used acellular vaccines. Originally formulated in the 1930-40's, the whole cell bacterial vaccine reduced the incidence of pertussis contraction but was associated with negative side effects. To remediate this, an acellular form of the vaccine was developed in the 1980's, which contained 2-5 proteins of B. pertussis bacteria adsorbed to alum adjuvant.
The acellular vaccines were developed to direct the immune response against the key components of the pathogen: 1) the extracellular pertussis toxin (PT), 2) the adhesion proteins (filamentous hemagglutinin (FHA) and fimbriae (FIM)), and 3) pertactin (PRN; an outer-membrane protein). aPs use aluminum hydroxide as the adjuvant to adsorb the antigens leading to a Th2 humoral response. In contrast, whole cell vaccines promote a Th1/Th17 response that activates both an IgG2a humoral response and cell mediated killing by macrophages and neutrophils. Natural immunity (due to infection) and wP immunization induced immunity lasted decades in humans.
Although acellular vaccines provide a safer alternative to whole cell vaccines, it appears that the acellular form has a shortened period of protection, resulting in a decreased efficacy in the years after immunization. This has led to a rise in the number of older children, and adolescents contracting whooping cough.
There are several hypotheses as to why whooping cough has re-emerged at such alarming rates. Data from the baboon model of pertussis has indicated that while aPs protect against the disease manifestation, the aPs do not prevent colonization or transmission of the pathogen. This increases the risk of contraction for neonates and those unable to be vaccinated. Human efficacy data also indicates that the protection wanes by as much as 35% each year after immunization. Furthermore, strains of B. pertussis are being clinically isolated do not express pertactin, which was originally characterized as one of the main virulence factors of B. pertussis. For these reasons, there is a need for a new generation of effective pertussis vaccines as returning to a whole cell vaccine is not an option due to the known risks.
Various embodiments recite a vaccine composition including a B. pertussis antigen and an effective adjuvant amount of a high molecular weight glucose polymer, wherein the composition is administered intranasally.
Various embodiments recite a vaccine composition including the B. pertussis antigens and an effective adjuvant amount of a β-glucan, wherein the β-glucan is selected from a group that includes curdlan, dextran, and baker's yeast beta-1,3/1,6-d-glucan.
Various embodiments recite a vaccine composition wherein the composition further includes an adenylate cyclase toxin antigen, such as RTX.
Various embodiments recite a vaccine composition wherein the composition induces a Th1/Th17 immune response.
Various embodiments further recite a method of immunizing a host against pertussis by administering intranasally to the host a vaccine composition including a B. pertussis antigen and an effective adjuvant amount of a high molecular weight glucose polymer.
Various embodiments further recite a method of enhancing the immune response of an intranasally administered Bordetella pertussis antigen that involves co-administering the antigen and a high molecular weight glucose polymer.
The present disclosure also describes a vaccine composition, comprising a Bordetella pertussis antigen, and an effective adjuvant amount of a high molecular weight glucose polymer, where the high molecular weight glucose polymer has a molecular weight of between 68 kDal and 680 kDal. The high molecular weight glucose polymer may be soluble or dispersible in water or aqueous base, and gellable in the presence of aqueous acid. The high molecular weight glucose polymer may be gellable in the respiratory system in the presence of acid and CO2. The high molecular weight glucose polymer may be a beta-glucan, a 1,3-beta-glucan polymer, a 1,3-beta-glucan/1,4-beta-glucan copolymer, a 1,3-beta-glucan/1,6-beta-glucan copolymer, or a mixture thereof.
In various embodiments, the vaccine composition contains a Bordetella pertussis antigen which may be an extracellular toxin, an adhesion protein, an outer membrane protein, a receptor protein, fragments thereof, or mixtures thereof. The Bordetella pertussis antigen may be an extracellular pertussis toxin (PT), the adhesion proteins filamentous hemagglutinin (FHA) and fimbriae (FIM)), the outer membrane protein pertactin (PRN), the siderophore receptor protein FauA, the xenosiderophore receptor protein BfeA, the hemophore receptor protein BfuR, fragments thereof, or mixtures thereof. The Bordetella pertussis antigen may be the extracellular pertussis toxin (PT), the adhesion protein filamentous hemagglutinin (FHA), the siderophore receptor protein FauA, fragments thereof, or mixtures thereof.
In various embodiments, the composition is formulated for intranasal administration; for parenteral administration by subcutaneous (SC) injection, transdermal administration, intramuscular (IM) injection, or intradermal (ID) injection; or for non-parenteral administration by oral administration, intravaginal administration, pulmonary administration, ophthalmic administration, or rectal administration.
The current disclosure describes a vaccine composition for intranasal administration to a patient, including a Bordetella pertussis antigen, and an effective adjuvant amount of a high molecular weight glucose polymer, where the high molecular weight glucose polymer is configured to adhere to the airway of a patient, by forming a gel in the presence in the presence of CO2 and aqueous acid.
The current disclosure describes a method of immunizing a host against pertussis by administering a vaccine composition including a B. pertussis antigen and an effective adjuvant amount of a high molecular weight glucose polymer intranasally to the host.
The current disclosure also describes a method of enhancing the immune response of an intranasally administered B. pertussis antigen that involves co-administering the antigen and a high molecular weight glucose polymer.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
In order to better understand various embodiments, reference is made to the accompanying drawings, wherein:
To facilitate understanding, identical reference numerals have been used to designate elements having substantially the same or similar structure or substantially the same or similar function.
The description and drawings presented herein illustrate various principles. It will be appreciated that those skilled in the art will be able to devise various arrangements that, although not explicitly described or shown herein, embody these principles and are included within the scope of this disclosure. As used herein, the term, “or” refers to a non-exclusive or (i.e., and/or), unless otherwise indicated (e.g., “or else” or “or in the alternative”). Additionally, the various embodiments described herein are not necessarily mutually exclusive and may be combined to produce additional embodiments that incorporate the principles described herein.
Vaccine-induced protection in acellular pertussis vaccine (aP) immunized individuals has been associated with a robust antigen-specific IgG response to the components of the aP vaccines. Likewise, whole cell pertussis vaccine (wP) immunization also results in antigen-specific IgG responses; with the addition of a shift to a more diverse T cell response, inducing cell-mediated immunity. In the murine model, immunization through intramuscular (IM) and intraperitoneal (IP) administration has been well characterized demonstrating a Th1/Th17 response from wP immunized mice, and a Th2 with weak Th17 mediated response in aP immunized mice following B. pertussis challenge. However, these immunizations fail to induce the mucosal immune responses elicited from natural infection. Protection correlates with tissue resident memory T (TRM) cells in the lung and nasal cavity of convalescent mice, that produce interleukin-17 (IL-17) and interferon-gamma (IFN-γ), although TRM activity in pertussis is yet to be studied in humans. TRM cells have been shown to persist in the respiratory tissue and expand upon re-challenge of a convalescent mouse with B. pertussis, as well as decrease bacterial burden upon adoptive transfer to naïve mice. More recently the expansion of this population has been observed following immunization by wP, a live-attenuated wP vaccine, an outer membrane vesicle vaccine, and intranasal administration of an aP vaccine with TLR9 and stimulator of interferon genes (STING) agonists.
The induction of a mucosal immune response to B. pertussis is associated with the production of secretory IgA antibodies (sIgA) in the nasal cavity. In humans previously infected with B. pertussis, IgA antibodies have been isolated from nasal secretions. B. pertussis-specific IgA antibodies isolated from convalescent patients have been shown to inhibit adherence of B. pertussis to respiratory epithelial cells in vitro, suggesting a protective role of IgA antibodies in mucosal immunity.
Conventional aP or DTaP vaccine does not contain a strong pro-inflammatory adjuvant. This disclosure describes IN aP or DTaP immunization alone, or with an additional pro-inflammatory adjuvant. A suitable pro-inflammatory adjuvant is a high molecular weight glucose polymer, which may be a beta-glucan, e.g., curdlan. Vaccines containing the adjuvant curdlan, a 1,3-beta-glucan derived from Alcaligenes faecalis, were formulated. This polysaccharide has immunostimulatory properties and forms a “sticky” gel at a neutral pH, or at an acidic pH in the presence of CO2. The adjuvant has a relatively large particle size with dimensions sufficient to produce an inflammatory response. In certain embodiments, the high molecular weight glucose polymer may be a medium molecular weight glucan polymer with a molecular weight above 68 kDal, or between 68 kDal and 2MDal, or between 68 kDal and 680 kDal; this high molecular weight provides sufficient size for the inflammatory response from the soluble polymer. In various embodiments, the high molecular weight glucose polymer may be a whole glucan particle purified from a yeast. Such whole glucan particles typically have a particle size of 3 to 10 m, 3.5 to 8 μm, or 4 to 6 μm, and may include residual proteins and/or lipids from yeast cells. The whole glucan particles may include 70% to 85%, 72% to 83%, or 75% to 80% 1,3-linked glucose units; 3% to 15%, or 5% to 10%, 1,6-linked glucose units; and 3% to 15%, or 5% to 10%, 1,3/1,6-linked glucose units. Whole glucan particles have sufficient size to trigger an inflammatory response from the polymer. In various embodiments, the glucose polymer may be conjugated to a protein, such as bovine serum albumin.
In another embodiment, the adjuvant includes particles having a minimum dimension of 3 μm, such as aluminum oxide particles. In another embodiment the majority of the adjuvant particles were between 3 to 10 μm, 3.5 to 8 μm, or 4 to 6 μm.
Curdlan and other β-glucan polymers has been shown to bind to dendritic cells through the ligand Dectin-1, thereby inducing expression of NF-xB leading to a Th1/Th17 mediated immune response as well as production of antigen-specific respiratory IgA antibodies and serum IgG antibodies. Dectin-1 is a receptor for β-glucans, which binds β-glucans and mediates the production of secretion of proinflammatory cytokines. Use of a β-glucan as an adjuvant may stimulate an immune response in a patient.
As discussed in the present disclosure, the gel properties of curdlan facilitate aP or DTaP localization in the upper respiratory tract. A significant reduction in bacteria burden is found following administration of intranasally administered aP or DTaP vaccines. High serum and respiratory antibody responses were measured, following intranasal administration of aP or DtaP, with and without curdlan. Mucosal vaccination with acellular vaccine containing a beta-glucan may be a strategy for decreasing incidence of pertussis.
It has now been found that immunization with B. pertussis antigens triggers a mucosal response similar to natural B. pertussis infection. Immunization may induce production of pertussis specific immunoglobulins that may: 1) mediate complement-dependent bacterial killing, 2) prevent colonization by blocking bacterial attachment, and 3) neutralize toxins at the site of infection. In some embodiments, a vaccine composition of the invention may provide a longer lasting and more effective form of the whooping cough vaccine, leading to decreased incidence of asymptomatic carriers and contraction by immune compromised and neonatal individuals.
In various embodiments of the invention, the vaccine composition may include B. pertussis antigens selected from a group that includes extracellular pertussis toxin (PT), the adhesion proteins filamentous hemagglutinin (FHA) and fimbriae (FIM), pertactin (PRN), and combinations thereof. The vaccine composition may include fragments of B. pertussis antigens selected from a group that includes PT, FHA, FIM, pertactin, and combinations thereof.
Additional proteins targeted for peptide vaccine development include the siderophore receptor FauA, the xenosiderophore receptor BfeA, and the hemophore receptor BfuR, and fragments thereof.
Antigen proteins, including those described above, were selected for use in the vaccine composition based on the following criteria:
For example,
Acellular pertussis vaccines include PRN, PT, and FHA antigens (aP). As PRN and FHA antigens are harvested from the whole bacteria, these antigens may contain the lipooligosaccharide (LOS) endotoxin from the bacteria. The LOS endotoxin, if present, may serve as an antigen, and induce formation of antibodies against the B. pertussis bacteria, enhancing the formation of antibodies by the aP vaccine. In various embodiments, the LOS antigen may be added to the aP vaccine as a fourth antigen to enhance formation of pertussis antibodies.
The pertussis vaccine may be formulated with an adjuvant for administration. The adjuvant may be aluminum hydroxide (aP-alum), or a beta-glucan. In some cases, the beta-glucan may be a 1,3-beta-glucan (aP-beta-glucan) or curdlan (caP or aP-curdlan).
As noted above fragments of B. pertussis proteins may be used as antigens. For example, extracellular regions of the proteins may be prepared and used as antigens. A bioinformatics pipeline may be used to identify the most immunogenic regions of proteins to be used as antigens for vaccination. A 3D protein structure analysis is performed. The structure analysis may use known crystal structures for a proposed antigen protein. Alternatively, the structure analysis may use a computational study to predict the protein structure. Structures of the xenosiderophore receptor BfeA and the hemophore receptor BfuR are shown in
The extracellular regions of the proposed antigen proteins are identified, and their immunogenicity is predicted based on their hydrophobicity and B cell epitope predictions. Using this approach, the sequences of various potential immunogenic regions were identified. Antigenic fragments based on these sequences were prepared. The antigen fragment peptides may then be modified by adding a cysteine residue on the N-terminus, and by conjugating them to a carrier protein. The carrier protein may be, but is not limited to, Keyhole Limpet Hemocyanin (KLH), diphtheria toxoid Cross-Reactive Material 197 (CRM197), or recombinant tetanus toxoid (rTTHc). Based on this approach, multiple potential antigenic peptide fragments based on FauA, BfeA, and BhuR were identified. These peptide fragments are presented in Table 1.
In one embodiment of the invention, the vaccine composition includes a B. pertussis antigen and an effective adjuvant amount of a high molecular weight polymer of glucose, such as β-glucan, dextran and the like. Preferred β-glucans include curdlan and baker's yeast beta-1,3/1,6-d-glucan. Curdlan is a bacterial and fungal β-1,3-glucan that binds to Dectin-1 receptors which are expressed on macrophages and dendritic cells.
The term “effective adjuvant amount” will be well understood by those skilled in the art, and includes an amount of a high molecular weight glucose polymer which is capable of stimulating the immune response to nasally administered antigens, i.e. an amount that increases the immune response of a nasally administered antigen composition.
In another embodiment, the vaccine composition of the invention may further be supplemented with an adenylate cyclase toxoid (ACT) which may improve efficacy of the vaccine composition by 1) generating anti-toxin antibodies against ACT, and 2) slowing vaccine-driven strain evolution. Suitable adenylate cyclase toxin antigens include purified repeats in the toxin domain (RTX antigen).
The vaccine composition of the invention may also contain additional adjuvants such as aluminum hydroxide.
The vaccine composition of the invention may be used as part of a prime-boost vaccine regimen. Conventional acellular pertussis vaccine (DTaP) is administered to human patients in five prime vaccinations at the following ages: 2 months, 4 months, 6 months, 15 to 18 months, 4 to 6 years. Periodic acellular pertussis vaccine boosts (TDaP) may be administered at age 11, and subsequently as needed. In a murine model, mice may be vaccinated with an aP vaccine as a prime, and then be given a boost vaccine 21 days later.
In one embodiment, the vaccine composition of the invention may be formulated for intranasal administration.
In other embodiments, the vaccine composition of the invention may be administered using alternative routes of administration including, without limitation, parenteral administration methods, such as subcutaneous (SC) injection, transdermal, intramuscular (IM), intradermal (ID), as well as non-parenteral, e.g., oral, intravaginal, pulmonary, ophthalmic and or rectal administration.
Pertussis toxin (PT) is an essential virulence factor, responsible for multiple factors in the pathogenesis of B. pertussis. PT facilitates infection by aiding in adherence to ciliated airway epithelial cells and through disruption of host innate immune cell recruitment to the site of infection. In numerous studies it has been demonstrated that neutralization of PT alone ablates symptoms of the disease. Neutralization of pertussis toxin at the site of infection may inhibit the systemic long-range activity of PT before colonization of the respiratory tract. Intranasal immunization with pertussis antigens may prime a protective systemic and mucosal immune response. Furthermore, the gel-like properties of curdlan may have a beneficial role in increasing antigen uptake.
The vaccines administered were prepared no longer than 1 h before administration. In Examples 1-3, INFANRIX (GSK) human vaccine (DTaP), and the National Institute for Biological Standards and Control WHO whole-cell pertussis vaccine (NIBSC code 94/532) were used as the aP acellular pertussis vaccine. The aP vaccine used in Examples 1-3 contains DTaP with formaldehyde killed pertussis toxoid. In Examples 4-8, genetically detoxified pertussis toxoid was used. The aP vaccine in Examples 4-8 contained PRN and PT antigens obtained from List Biologicals and FHA antigen obtained from ENZO bio. As the PRN and FHA antigens are harvested from B. pertussis bacteria, they may also contain the lipooligosaccharide (LOS) endotoxin from the bacteria.
Vaccines administered with curdlan were diluted with PBS to 1/12th of the human dose (based on total antigen content). Curdlan adjuvant was administered at 200 μg per mouse. A vaccine dose of 1/12th the human dose was the highest concentration of vaccine that was possible to use due to volume of vaccine required for the solubility of curdlan.
Curdlan (Invivogen, tlrl-curd) was prepared by dissolving 50 mg in 2.5 ml sterile purified water. Curdlan preparation was brought into solution by adding 100 μl 1N NaOH and vortexing. The curdlan suspension (20 mg/ml) was then sonicated for 10 mins and placed in 37° C. water bath until administration. At the time of vaccination, the vaccines containing curdlan were administered in a liquid form to reduce risk of choking due to excessive gel formation in the airway. IN administered mice recovered at the same rate as IP immunized mice. These experiments were conducted in accordance with the National Institutes of Health Guide for the care and use of laboratory animals. The protocols used were approved by West Virginia University Institutional Animal Care and Use Committees (WVU-ACUC protocol 1602000797). Curdlan, as used herein is a medium molecular weight glucan polymer, i.e., a polymer with a molecular weight of between 68 kDal and 680 kDal. The 1,3-β-glucan adjuvant, as used herein, is a whole glucan particle adjuvant having a particle size of 3 to 4 μm.
As comparison models, a group of mice were mock vaccinated with phosphate buffered saline (PBS). The mock vaccinated group was divided into a control group (Control or NVNC) which was not infected with B. pertussis, and a group which was infected with B. pertussis (Mock Vac). A third group of mice included unvaccinated mice recovering from B. pertussis infection (Convalescent)
aP vaccine lots are validated based on using high doses such as ⅕th human dose with intranasal B. pertussis challenge. However, those doses are not physiologically relevant to a mouse. Consequently, high doses of DTaP or Tdap antigens do not afford the ability to determine if addition of new antigens to the “base” vaccine can improve protection. In order to circumvent these issues, an approach was designed that could identify antigens that synergistically improve a vaccine. In these studies, 30 variables of data per mouse are collected to determine the correlates of protection. Lung IL-6 levels are an indication of 1) inflammation due to presence of pathogen and 2) ACT activity is known to activate IL-6 secretion. The vaccine minimum protective dose was identified as 1/40th based on the viable bacteria (
Inclusion of RTX Antigen into the aP
Mice immunized with RTX alone with aluminum hydroxide are not protected against Bp challenge. However, when the 1/80th aP vaccine was supplemented with RTX antigen (5 μg), a significant decrease in bacterial burden was observed at day 3 (
For the studies, curdlan was selected as an adjuvant in order to induce a Th1/Th17 response which would mimic the wP or convalescent immunity. By interacting with the Dectin-1 receptor, curdlan primes naïve CD4+ T cells to differentiate to Th1 and Th17 T cells. In addition to its excellent adjuvant properties, curdlan can form a gel in the presence of carbon dioxide and at acidic pH. Thus, intranasal immunization with curdlan allows the antigen to adhere to the airway to allow uptake of the antigens by antigen presenting cells.
In the model presented in
In order to observe if curdlan facilitates the localization of the vaccine to the upper airway, DTaP was fluorescently labeled with Alexa fluor 660 dye (
IN-caP Intranasal Immunization Protects Against B. pertussis Challenge in Mice
Mice were immunized with curdlan alone (200 μg/dose) with no antigens added. When mice were challenged with Bp, no protection was observed (
The IN-caP vaccine contained curdlan and aluminum hydroxide adjuvants. To determine the T-cell response, Elispot analysis was performed on splenocytes of the immunized mice. Splenocytes were stimulated with PT and FHA antigens and it was observed that mice immunized with IN-caP induced significantly more IL-17-producing splenocytes than wP immunized mice (
To determine the antibody response triggered by IN-caP immunization, IgG production against FHA, PT, and lipooligosaccharide (LOS) antigens were measured in each vaccine group by ELISA. As seen in Table 2, both IP-aP and IN-caP induced production of FHA and PT IgGs, but did not induce production of LOS IgG. Intraperitoneally administered whole cell pertussis vaccine (IP-wP) and an intranasal whole cell pertussis vaccine containing curdlan (IN-cwP) induced production of FHA and LOS IgGs, but did not induce production of detectable anti-PT. Additionally, the immune response against FHA induced by IN-caP was stronger than the immune response against FHA induced by IN-cwP.
CD-1 (outbred; strain code 022) mice aged four weeks were obtained from Charles River Laboratories. At five weeks mice were anesthetized with 77 mg/kg ketamine and 7.7 mg/kg xylazine. Mice were administered 50 μl of vaccine or control, with 25 μl into each nostril (IN).
DTaP vaccine particles were labeled using the Alexa Fluor 660 Protein Labeling Kit (Molecular Probes). Briefly, 0.5 ml of DTaP vaccine was added to 50 μl of 1 M sodium bicarbonate, then added to Alexa Fluor 660 dye stock. The mixture was incubated for 1 h at room temperature with agitation. The solution was concentrated by dialysis in phosphate-buffered saline overnight to remove unlabeled dye. Vaccine particles were examined using the Cy5 channel on an EVOS FL microscope. Particles were mounted on a slide and visualized using a 100× objective. Particle diameter was measured using ImageJ (version 1.52a) with the line segment tool in proportion to the scale bar. Four fields of view were measured to determine particle size and standard deviation. Labeled vaccine was used to immunize mice. At 0, 6, 12 and 24 h post vaccination, fluorescent signal was measured using an IVIS Spectrum (Xenogen), as shown in
At 6, 12, 24, and 48 h post vaccination, animals were euthanized to quantify DTaP in lungs by flow cytometry analysis, as shown in
Detection of DTaP particles in the lung and nasal cavity were confirmed using confocal imaging. Mock vaccinated and challenged mice were euthanized at 6 h post challenge, as shown in
Skulls were removed from mouse, and the lower jawbone discarded. The skulls were fixed in formalin for 12 h at 4° C., then de-calcified at room temperature for 24 h, before samples were embedded in paraffin. Sectioned samples were de-paraffinized and rehydrated using xylene, and washes with decreasing ethanol concentrations (100 to 70%). An antigen retrieval step was performed using citrate buffer, where samples were heated to 98° C. for 20 mins. Samples were then stained as mentioned above. Samples were analyzed for DTaP particles in tissue and airway mucus using a Nikon AiR confocal microscope. Images were acquired using DAPI, FITC, and Cy5 channels using a 100× oil immersion lens (100×/1.40 Nikon Plan Apo). DTaP particles were quantified using ImageJ. Briefly, the threshold tool was used to select only the fluorescent particles above background levels. Then, the threshold adjusted area was quantified using the analyze particles tool. Thus, the data is represented as the percentage of fluorescent particles per area of the total image field. Three image fields per sample were quantified and averaged per mouse.
B. pertussis Strains and Growth Conditions
B. pertussis strain UT25Sm1 was used for murine challenge in all experiments. UT25Sm1 was cultured on Bordet Gengou agar plus 15% defibrinated sheep's blood (Remel) with streptomycin 100 mg/ml. B. pertussis was incubated at 36° C. for 48 h, then transferred to modified Stainer-Scholte liquid medium, without the cyclodextrin, heptakis. Liquid cultures were incubated for 24 h at 36° C., with shaking until reaching an OD600 of ˜0.6, at which time cultures were diluted for challenge dose.
Vaccination and B. pertussis Challenge
IN immunized mice received 50 μl of vaccine as described above. IP immunized mice received 200 μl of vaccine injected into the peritoneal cavity. IN and IP immunized mice received the same antigen dose of 1/12th. Mice received a boost of the vaccines with the same concentrations twenty-one days after initial immunization. At thirty-five days post initial vaccination, mice were challenged with 2×107 CFU B. pertussis administered in 20 μl through nostrils. At days 1 and 3 post challenge (pc), mice were euthanized, blood and respiratory tissue were isolated as previously described.
Serological Analysis of B. pertussis Specific Antibodies
Serological responses specific to B. pertussis antigens were quantified by ELISA. High-binding microtiter plates were coated with PT (50 ng/well)(LIST Biologicals) and FHA (50 ng/well) (Enzo Life Sciences), as described in Boehm et al. Serological responses against UT25Sm1 were cultured to an OD600 of 0.24 and microtiter plates coated with 50 μl of bacteria per well. Bound antibodies were detected using goat anti-mouse IgG, IgA, IgG2a, and IgG1 antibody conjugated to alkaline phosphatase (Southern Biotech). Positive antibody titers were determined using a baseline set at two times the average of blanks.
To determine cell types infiltrating the lung and leukocytes present in peripheral blood, single cell suspensions from the tissues mentioned above were prepared. Briefly, lung tissue was homogenized by Dounce homogenizers, filtered with a 100 μm filter, and red blood cells were lysed for 2 min at 37° C. (Pharmlyse). Single cell populations were blocked by initial incubation with Fc Block (BD, 553141) for 15 min at 4° C. Cell populations were incubated in the dark with antibodies to cell surface markers for 1 h at 4° C. Neutrophil populations were identified using: PE-conjugated GR-1 (BD, 553128), Alexa Fluor 700-conjugated CD11b (Biolegend, 101222). Neutrophils were classified as CD11b+Gr-1hi single, live cells. TRM populations were determined using: APC-Cy7-conjugated CD4 (Biolegend, 100526), BB515-conjugated CD44 (BD, 564587), APC-conjugated CD62L (BD, 553152), and BV421-conjugated CD69 (BD, 562920).
To quantify inflammatory cytokines at the site of infection, lung homogenate supernatant was prepared and stored at −80° C., as described in prior work. Quantitative analysis of cytokines was performed using Meso Scale Discovery cytokine kits: V-PLEX pro-inflammatory panel (K15048D) and IL-17A V-PLEX (K152RFD), per the manufacturer's instructions.
Experiments in the study were performed with 3 to 8 biological replicates. Data were analyzed using GraphPad Prism 7. ROUT method was used to removed outliers. Comparisons between groups were performed using one-way analysis of variance (ANOVA) with Dunnett's and Tukey's post hoc tests. Comparisons between groups with or without curdlan were analyzed by two-tailed unpaired t-test, when applicable multiple T-tests with Holm-Sidak post hoc test were applied to curdlan inclusion groups.
Acellular Pertussis Vaccine was Retained in the Upper and Lower Respiratory Tract when Administered by Intranasal Administration.
To determine if use of curdlan would increase vaccine retention in the respiratory system, CD-1 mice were intranasally (IN) vaccinated with commercially available DTaP (IN-aP), DTaP with curdlan (IN-caP), or phosphate-buffered saline (PBS; mock vaccinated) and the vaccine for up to 48 h after vaccination. The protocol is illustrated in
To quantify DTaP particles that were bound to innate immune cells flow cytometry was utilized. Single-cell suspensions were prepared from homogenized lung tissue and antigen presenting cells (APCs) bound to DTaP were quantified as live, single cells positive for CD11b+DTaP+ (
To visualize the deposition of DTaP particles, sections from the lung and nasal cavity were imaged using confocal microscopy. Vaccinated mice were euthanized after 6 h. Lung tissue was flash frozen, and skulls were embedded in paraffin for sectioning. Sections from the lung and nasal cavity were counterstained with NucBlue and ActinGreen to visualize epithelial tissue and fluorescent DTaP particles (
Vaccination of Mice for with Genetically Detoxified Pertussis Toxoid
This example, and all subsequent examples, were carried out with genetically detoxified pertussis toxoid vaccines. PRN and PT antigens were obtained from List Biologicals and FHA antigen was obtained from ENZO bio.
The following vaccines were formulated:
In aP-alum, the aluminum hydroxide adjuvant is an aluminum hydroxide wet gel suspension. The aluminum hydroxide induces a Th2 response by improving the attraction and uptake of antigen by antigen-presenting cells (APCs). Aluminum hydroxide particles have a net positive electrical charge at pH 5-7, and are attracted to negatively charged antigens.
CD-1 mice aged weeks were anesthetized with 77 mg/kg ketamine and 7.7 mg/kg xylazine. Mice were administered 50 μl of vaccine, with 25 μl into each nostril (IN). Four groups of mice (n=4) were vaccinated, with each group being vaccinated with one of the aP, aP-alum, aP-curdlan, and aP-β-glucan vaccines. Each vaccinated group of mice was given a boost vaccination 3 weeks later. At 10 weeks of age, the mice were challenged by exposure to B. pertussis. A fifth group of mice was mock-vaccinated with PBS, then challenged by exposure to B. pertussis. A sixth group of mice (NVNC) was mock-vaccinated with PBS, without being challenged by B. pertussis. Finally, a group of unvaccinated mice recovering from exposure to pertussis was examined (Convalescent mice).
To determine if an IN-delivered pertussis vaccine would induce a systemic immune response, enzyme-linked immunosorbent assays (ELISAs) with serum from vaccinated and challenged mice against B. pertussis antigens found in the vaccine were performed. Pertussis toxin (PT) and filamentous hemagglutinin (FHA) antigens were tested. ELISAs were not performed against pertactin antigen, as 85% of current clinical isolates in the US do not express the protein. Serum anti-PT IgG titers were similar between mice immunized with IP-aP and those immunized through IN administration, as no significant differences were determined between IP-aP, IN-aP or IN-caP (
IgA antibodies and a local mucosal immune response in the lung and nasal cavity are important to B. pertussis immunization. The presence of IgA antibodies in the murine respiratory tissue due to IN immunization was measured. Using ELISAs, B. pertussis-specific IgA titers in homogenized lung tissue supernatant and nasal lavage fluid were measured. A robust IgA response was observed in the lung only when mice were immunized through the IN route (
Neutralization of PT and inhibition of bacterial adhesins associated with DTaP protection leads to a reduced pro-inflammatory environment at the site of infection when compared to a natural infection. Conversely, challenge in whole-cell protected animals resulted in a severe pro-inflammatory response, similar to the natural infection of B. pertussis. To determine whether the immunostimulatory properties of curdlan would induce a more pro-inflammatory response following challenge with B. pertussis compared to IP-aP, cytokine concentrations were determined from supernatant of lung homogenate at day 3 post challenge. There is a significant reduction of IL-6 in the lungs of either IN-aP or IN-caP immunized mice when compared to mock vaccinated or IN-curdlan control mice, as shown in
IN administration of curdlan, and moreover, vaccine administration through the IN route regardless of adjuvant has been shown to induce an increased IL-17 response. Levels of IL-17 in mice immunized with IN-caP were observed and compared to those in mice immunized with IN-aP or IP-aP. IL-17A in the lung supernatant was quantified, and significant increases of IL-17A with the addition of curdlan in IP-caP and IN-caP immunized mice were observed. When compared to IP-aP and IN-aP groups, IL-17A levels increased 4-fold and 14.9-fold, respectively (
Natural infection with B. pertussis causes severe leukocytosis, which can be measured by elevated neutrophils in the peripheral blood. Following B. pertussis challenge, all vaccinated groups showed ameliorated symptoms of leukocytosis by day 3 pc; however, only the administration of DTaP either by IP or IN administration significantly reduced CD11b+Gr-1hi neutrophils in the peripheral blood (
To determine if IN-aP or IN-caP could induce the expansion of a TRM population, population, CD4 T cells were isolated from the lung at day 3 pc, and were identified as TRM cells based on expression of surface markers: CD4+CD62L-CD44+CD69+. We did not observe a statistical difference of this population following challenge with either IP or IN administered vaccines. However, we did observe a slight increase in IP-wP, IP-caP, IN-aP and IN-caP compared to mock vaccinated mice (
Lastly, the clearance of B. pertussis from the respiratory tract following IN immunization was examined. At days 1 and 3 pc, viable bacterial burden was quantified by counting of CFU in the lung, trachea, and nasal lavage fluid. A significant reduction in viable bacteria recovered from the lung was observed in all immunized groups by day 3 pc; however, these changes were not observed at day 1 pc (
Pertussis patients and mice immunized with FauA peptides have anti-FauA antibodies. Mice were vaccinated with a set of six FauA-derived peptides (n=4), specifically SEQ ID NOS. 1 to 6 of Table 1; the peptides were conjugated to CRM197, a non-toxic mutant of diphtheria toxin. A set of unvaccinated control mice (n=4) was also tested.
CD-1 (outbred; strain code 022) mice aged four weeks were anesthetized with 77 mg/kg ketamine and 7.7 mg/kg xylazine. Mice were administered 50 μl of PBS control or an aP vaccine with PT, FHA, and PRN antigens, with 25 μl into each nostril (IN). After 21 days, a similar boost vaccine was administered into each nostril. Mice were divided into the following groups (n=4 for each group):
Three days post challenge, the respiratory track bacterial burden was measured for each group of mice except the unchallenged NVNC mice. Burden was measured by nasal lavage, and in the lungs and trachea, using techniques described above. Results are shown in
Three days post challenge, total IgG serum titers to B. pertussis and IgG serum titers to the PT and FHA vaccine antigens were measured, and found to be significantly elevated after challenge in mice vaccinated intranasally with aP, aP-alum, aP-curdlan, and aP-β-glucan vaccines. Results are shown in
Three days post challenge, production of the cytokine IL-6 in mice vaccinated intranasally with aP, aP-alum, aP-curdlan, and aP-β-glucan vaccines was comparable to IL-6 production in the unchallenged NVNC mice, as shown in
A protocol for testing long-term protection against pertussis by intranasal vaccination is shown in
As shown in
Six months after the initial prime vaccination, mice in each group except convalescent mice and NVNC mice were challenged by B. pertussis infection. Three days post challenge, mice were euthanized, and the effect of the vaccine was tested, e.g., by flow cytometry or serology. As shown in
As seen in
Antibody secreting cells (ASCs) are differentiated cells of the humoral immune response. ASCs differentiate from activated B cells in lymph nodes. Most of the circulating ASCs undergo apoptosis, but some ASCs migrate to the bone marrow (BM) and eventually mature into long-lived plasma cells (LLPCs). Accordingly, the bone marrow of mice vaccinated as in Example 6 was examined following euthanasia for the presence of ASCs which secrete B. pertussis antibodies.
An enzyme-linked immune absorbent spot (ELISpot) analysis was used for detecting antibody-secreting cells in bone marrow tissue, in response to B. pertussis infection. Data were recorded 3 days (
Although the various embodiments have been described in detail with particular reference to certain aspects thereof, it should be understood that the invention is capable of other embodiments and its details are capable of modifications in various obvious respects. As is readily apparent to those skilled in the art, variations and modifications can be affected while remaining within the spirit and scope of the invention. Accordingly, the foregoing disclosure, description, and figures are for illustrative purposes only and do not in any way limit the invention, which is defined only by the claims.
Number | Date | Country | |
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62824730 | Mar 2019 | US |