CN113855796A - Application of BCG (bacille calmette-guerin) as respiratory syncytial virus inactivated vaccine adjuvant - Google Patents
Application of BCG (bacille calmette-guerin) as respiratory syncytial virus inactivated vaccine adjuvant Download PDFInfo
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Abstract
The BCG is used as an adjuvant of the RSV inactivated vaccine, and the Th1 type cellular immune response induced by the BCG can balance the over-strong Th2 and Th17 type cellular immune responses induced by the RSV inactivated vaccine, so that balanced immune memory is generated in a vaccinee body, and when RSV is infected, the vaccine is prevented from generating enhanced inflammatory diseases while protection is provided, and the vaccine is safe and effective.
Description
Technical Field
The invention relates to the technical field of vaccines and adjuvants, in particular to application of BCG (bacillus calmette guerin) as an adjuvant of a respiratory syncytial virus inactivated vaccine.
Background
Respiratory Syncytial Virus (RSV) is the most important cause of lower Respiratory tract infections in infants and young children, almost all children are infected before the age of two years, and in children under the age of 5 years, the number of cases of RSV infection requiring emergency and hospitalization is more than ten times that of influenza virus infection, which is the most important cause of hospitalization and death of infants and young children; in addition, the elderly are also susceptible to RSV, with annual infection rates similar to influenza and mortality rates almost 2 times higher than influenza. The direct economic burden of RSV production worldwide is over 800 billion dollars annually, and no safe and effective vaccine has yet been marketed, and thus, the WHO has prioritized RSV vaccines.
Vaccine development work was rapidly carried out since the discovery of RSV in 1956, unfortunately significant frustration in clinical trials has arisen with the formalin inactivated RSV vaccine (FI-RSV) developed by the pfeizu company in the 1960 s: FI-RSV is approved for clinical trials in infants, the vaccinated subjects are infants between two months and seven years, the clinical trial is 9 months later and enters the RSV epidemic season, the infants vaccinated with the FI-RSV are not only unprotected, but rather have more serious pulmonary inflammatory diseases after RSV infection than those in a placebo group, allergic-like inflammation mainly including massive neutrophil and eosinophil infiltration and Th2 type immune response is generated in the lung, namely vaccine-enhanced disease (VED), 80 percent of vaccinees need hospitalization, two cases die, and only 5 percent of the placebo group need hospitalization[1]. The failure of the vaccine seriously hits the enthusiasm for the development of the RSV vaccine, increases the commercial risk of the development of the RSV vaccine, and the safety becomes a problem which is considered by RSV vaccine researchers first.
Studies in animal experiments show that: the pathogenesis of FI-RSV vaccine enhanced disease is mainly related to the unbalanced immune response induced by the vaccine[2]In particular Th2 and Th17 type cellular immunodominant responses, but lack a Th1 type cellular immune response[3]。
An adjuvant is a non-specific immunopotentiating substance that is injected into the body, either in advance or simultaneously with an antigen, and that enhances the immune response of the body to the antigen or alters the type of immune response. Aluminium salt adjuvants (e.g. Al (OH)3And aluminum phosphate, etc.) are human vaccine adjuvants that have been used for nearly a hundred years, and the vast majority of vaccines use adjuvants of this type.
BCG vaccine (BCG) is live vaccine prepared from attenuated bovine type tubercle bacillus suspension, can prevent mycobacteria infection such as tuberculosis and leprosy, and has been used for nearly a century. In addition, BCG has the characteristics of enhancing macrophage activity and inducing Th1 type immune response, and is a good adjuvant for promoting Th1 type immune response.
There is additional evidence that BCG can protect infants from infection by pathogens other than M.tuberculosis, thereby providing heterologous or non-specific cross-protection[4]. It has been shown in the literature that bcg reduces death from other respiratory infections, diarrhea or malaria; small studies from guinea bishao and brazil showed that children vaccinated with BCG had a lower rate of pneumonia; similarly, a recently published study based on international population and health surveys that vaccination with bcg reduced the risk of suspected acute lower respiratory infections by 17% to 37%, including 15 million children from 37 countries; bcg not only protects children from respiratory infections, but also reduces their severity and associated mortality.
Reference documents:
1.Kapikian AZ,Mitchell RH,Chanock RM,Shvedoff RA,Stewart CE.An epidemiologic study of altered clinical reactivity to respiratory syncytial (RS)virus infection in children previously vaccinated with an inactivated RS virus vaccine.Am J Epidemiol 1969；89(4):405-421.
2.Ruckwardt TJ,Morabito KM,Graham BS.Immunological Lessons from Respiratory Syncytial Virus Vaccine Development.Immunity 2019；51(3):429-442.
3.Zhang L,Li H,Hai Y,Yin W,Li W,Zheng B,Ruihong Zeng.CpG in Combination with an Inhibitor of Notch Signaling Suppresses Formalin-Inactivated Respiratory Syncytial Virus-Enhanced Airway Hyperresponsiveness and Inflammation by Inhibiting Th17 Memory Responses and Promoting Tissue-Resident Memory Cells in Lungs.J Virol 2017；91(10).
4.Arts RJW,Moorlag S,Novakovic B,Li Y,Wang SY,Oosting M,et al. BCG Vaccination Protects against Experimental Viral Infection in Humans through the Induction of Cytokines Associated with Trained Immunity.Cell Host Microbe 2018；23(1):89-100e105
in the prior art, no related reports and researches of BCG serving as a respiratory syncytial virus inactivated vaccine adjuvant exist.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the application of BCG as an adjuvant of an inactivated vaccine for respiratory syncytial virus, BCG is used as an adjuvant of an RSV inactivated vaccine, and the Th1 type cellular immune response induced by BCG can balance the over-strong Th2 and Th17 type cellular immune responses induced by the RSV inactivated vaccine, so that balanced immune memory is generated in the body of a vaccine vaccinee, and when RSV is infected, the protective effect is provided and the vaccine is prevented from enhancing inflammatory diseases, namely, the safety and the effectiveness are realized.
In order to achieve the technical purpose and achieve the technical effect, the invention is realized by the following technical scheme:
the BCG vaccine is used as the respiratory syncytial virus inactivated vaccine adjuvant.
Further, the inactivated respiratory syncytial virus vaccine is with or without an aluminum salt adjuvant;
further, the inactivated vaccine is a formalin inactivated RSV vaccine (FI-RSV), a beta-propiolactone inactivated RSV vaccine, and RSV inactivated by other inactivators or inactivation methods.
Further, the application is that BCG and RSV inactivated vaccine (with or without aluminum salt adjuvant) are injected into the body in advance or simultaneously to induce balanced immunological memory.
Further, the application is that the adjuvant BCG is injected only once or the same times as the inactivated RSV vaccine.
The invention has the beneficial effects that:
the BCG is used as an adjuvant of the RSV inactivated vaccine, and the Th1 type cellular immune response induced by the BCG can balance the over-strong Th2 and Th17 type cellular immune responses induced by the RSV inactivated vaccine, so that balanced immune memory is generated in a vaccinee body, and when RSV is infected, the vaccine is prevented from enhancing inflammatory diseases while protection is provided, and safety and effectiveness are realized.
Of course, it is not necessary for any product in which the invention is practiced to achieve all of the above-described advantages at the same time.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the pathological changes of lung tissues of groups after the RSV inactivated vaccine plus adjuvant is used for immunizing suckling mice and is attacked by RSV according to the embodiment of the invention; A. PBS; B. PBS/RSV; C. FI-RSV + Al (OH)3； D、FI-RSV+BCG(two)；E、FI-RSV+BCG(one)；F、 FI-RSV+Al(OH)3+BCG(one)；
FIG. 2 shows the RSV inactivated vaccine plus adjuvant immunized suckling mice and real-time quantitative PCR detection of RSV-N protein and cytokine mRNA expression in each group of lung tissues after RSV challenge (p <0.05, p <0.01, p < 0.001);
FIG. 3 shows lung tissue pathology of each group of RSV inactivated vaccine and adjuvant immunized adult mice after RSV challenge according to the present inventionVariation (H)&E staining 100 ×); A. PBS; B. PBS/RSV; C. FI-RSV + Al (OH)3；D、FI-RSV+BCG；E、FI-RSV+Al(OH) 3+BCG；
FIG. 4 shows RSV inactivated vaccine plus adjuvant immunized adult mice and real-time quantitative PCR detection of RSV-N protein and cytokine mRNA expression in each lung tissue group after RSV challenge (p <0.05, p <0.01, p < 0.001);
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Experimental study on immunization of suckling mice with RSV inactivated vaccine plus adjuvant
1.1 grouping suckling mice:
a PBS group;
② PBS/RSV group;
③FI-RSV+Al(OH)3group (d);
FI-RSV + BCG (two) group;
⑤FI-RSV+BCG(one)；
⑥FI-RSV+Al(OH)3+ BCG (one). 6-10 pieces/group
1.2 vaccination and RSV challenge protocol in suckling mice:
note: i.m. intramuscular injection, s.c. subcutaneous injection.
Getting BALB/c pregnant mouse, and getting pregnant mouseAfter delivery, the day of birth of the suckling mice is recorded as 0 day, BCG (the fourth to sixth groups) is inoculated subcutaneously, PBS (the first and the second groups), FI-RSV (the fourth and the fifth groups) or FI-RSV + Al (OH) is injected intramuscularly after 1 day of birth3(iii and (iv) group (s)), group(s) on day 7(s) was subcutaneously inoculated with BCG(s), group(s) on day 8(s) was intramuscular injected with PBS, FI-RSV or FI-RSV + Al (OH)3On day 21, the patient was attacked by 30. mu.l of PBS (group) ((ii)) or RSV (group) ((ii) -sixth) by nasal drip, and after 5 days, the patient was sacrificed, lung tissue was collected, paraffin-embedded, sectioned, and H&E, dyeing; taking part of lung tissue, extracting RNA, and performing real-time quantitative PCR detection.
Example 2
Experimental study of immunization of adult mice with RSV inactivated vaccine plus adjuvant
2.1 grouping of adult mice
PBS group, PBS/RSV group, FI-RSV + Al (OH)3Group, FI-RSV + BCG group, FI-RSV + Al (OH)3+ BCG group. 6 pieces/group
2.2 adult mouse vaccination and RSV challenge protocol:
6 weeks female BALB/c mice were picked up, inoculated subcutaneously with BCG (group (r) and (v)), and 1 day later, intramuscularly injected with PBS (group (r) and (r)), FI-RSV (group (r)) or FI-RSV + Al (OH)3(group III and V), intramuscular injection of PBS, FI-RSV or FI-RSV + Al (OH) again on days 11 and 213On day 35, the patient was attacked by nasal drip with 50. mu.l of PBS (group) ((ii) or RSV (group) ((ii) -v)), and after 5 days, the patient was sacrificed, lung tissues were collected, paraffin-embedded, sectioned, and H&E, dyeing; taking part of lung tissue, extracting RNA, and performing real-time quantitative PCR detection.
Statistical analysis of results
Lung tissue pathology, RSV-N protein and cytokine expression in groups of suckling mice after RSV challenge
Note: PBS group: neither vaccination nor infection with RSV challenge(ii) a PBS/RSV group: no vaccination, but infection with RSV challenge; FI-RSV + BCG (two) group: BCG injection is carried out twice; FI-RSV + BCG (one) and FI-RSV + Al (OH)3+ BCG (one) group: BCG was injected only once.
Pathological section of lung tissue, H&E staining results (as in fig. 1): A. PBS group: in the normal group, the alveolar structure is intact, the alveolar space is normal, and the bronchial columnar epithelial cells are smooth. B. PBS/RSV group: inflammatory cell infiltration exists locally, part of alveoli are fused, the alveoli become bigger, and inflammatory cell infiltration occurs around the bronchus. C. FI-RSV + Al (OH)3Group (2): the periphery of the bronchus is obviously infiltrated by inflammatory cells, the alveolar space is thickened, the alveoli are fused, and columnar cells of the bronchus protrude inwards, so that the obvious vaccine-enhanced pulmonary inflammation is shown. D. FI-RSV + BCG (two): the infiltration of inflammatory cells is reduced to some extent, the alveolar space tends to be normal, the infiltration of inflammatory cells exists around the bronchus, obvious hyperemia and edema exist, part of alveoli merge, the alveoli enlarge and the inflammation is more FI-RSV + Al (OH)3The group was significantly reduced, but compared to FI-RSV + BCG (one), FI-RSV + Al (OH)3+ BCG (one) is associated with more severe inflammatory symptoms. E. FI-RSV + BCG (one): the infiltration of inflammatory cells is obviously reduced, but obvious edema still exists, the alveolar space tends to be normal, part of alveoli is fused, and the alveoli become bigger, but the inflammation is more FI-RSV + Al (OH)3The group was significantly reduced. F. FI-RSV + Al (OH)3+ BCG (one): has no obvious inflammatory cell infiltration, no obvious hyperemia and edema, complete alveolar structure, relatively normal alveolar interval and relatively normal bronchial columnar epithelial form.
Pathological section result suggestion: FI-RSV + Al (OH)3The panel showed significant vaccine-enhanced inflammation, while FI-RSV + Al (OH)3+ BCG (one) group showed significant suppression of inflammation as an optimal immunization schedule.
As shown in fig. 2
And FI-RSV + Al (OH)3Group phase FI-RSV + Al (OH)3+ BCG (one) group of lung tissues in RSV-N (housekeeping gene-N gene of RSV virus) significantly reduced expression levels, indicating FI-RSV + Al (OH)3The immunization schedule of the + bcg (one) group provided excellent protection;
furthermore, FI-RSV + Al (OH)3+ BCG (one) groups of lung tissues expressed at significantly lower levels of eotaxin, Gro-alpha, MCP-1, TNF-alpha, IL-17, Th17, IL-6 and IL-10(Th 2) cytokines than FI-RSV + Al (OH)3Group, and FI-RSV + Al (OH)3+ BCG (one) lung tissue IFN-gamma (Th1 type cytokine capable of activating macrophage and lymphocyte and enhancing pathogen killing ability) expression level is obviously higher than that of PBS and PBS/RSV group, and FI-RSV + Al (OH)3There were no statistical differences between groups. These results show that: FI-RSV + Al (OH)3+ BCG (one) immunization of suckling mice provided significant protection and significantly reduced levels of Th2 and Th17 cytokines and pro-inflammatory factors in the lungs.
The results of this section suggest: for neonates, FI-RSV + Al (OH)3+ BCG (one) is an optimal immunization regimen, both safe and effective.
Mouse lung tissue pathology, RSV-N protein and cytokine expression in each component year following RSV challenge
Pathological section of lung tissue, H&E staining results (as in fig. 3), PBS group: in the normal group, the alveolar structure is intact, the alveolar space is normal, and the bronchial columnar epithelial cells are smooth. B. PBS/RSV group: inflammatory cell infiltration exists locally, part of alveoli are fused, the alveoli become bigger, and inflammatory cell infiltration occurs around the bronchus. C. FI-RSV + Al (OH)3Group (2): the periphery of the bronchus is obviously infiltrated by inflammatory cells, the alveolar space is thickened, the alveoli are fused, columnar cells of the bronchus protrude inwards, and the obvious vaccine enhanced pulmonary inflammation is shown. D. FI-RSV + BCG group: no obvious inflammatory cell infiltration is seen, the alveolar space is normal, and occasionally, alveoli are fused. E. FI-RSV + Al (OH)3+ BCG group: has more obvious inflammatory cell infiltration, thickened alveolar space, alveolar fusion and inflammatory cell infiltration around bronchi, but is more FI-RSV + Al (OH)3The group had reduced inflammation, with more severe symptoms of inflammation compared to the FI-RSV + BCG group. The above results suggest: in adult mice, the FI-RSV + BCG immunization regimen can significantly reduceLung inflammation pathology.
Real-time quantitative PCR detection of RSV-N protein and cytokine mRNA expression in adult mouse lung tissue: FIG. 4 results show FI-RSV + Al (OH) compared to the PBS/RSV group3FI-RSV + BCG and FI-RSV + Al (OH)3+ the RSV-N gene (RSV virus housekeeping gene) expression after the challenge of BCG group was significantly reduced, and in addition, FI-RSV + BCG and FI-RSV + Al (OH)3+ BCG group showed significantly lower amounts of RSV-N than FI-RSV + Al (OH)3Groups, indicating FI-RSV + BCG and FI-RSV + Al (OH)3The + BCG group provided significant protection;
eotaxin (chemotactic eosinophilic cytokine) or FI-RSV + Al (OH) of the FI-RSV + BCG group3+ BCG group eotaxin, Gro-alpha, MCP-1, FI-RSV + Al (OH)3The group comparison is obviously reduced; IL-17 and IL-23 (Th17 type cytokine) and FI-RSV + Al (OH) of the FI-RSV + BCG group3IL-23 of the + BCG group was also significantly lower than FI-RSV + Al (OH)3Group (d); and FI-RSV + Al (OH)3Group comparisons FI-RSV + BCG and FI-RSV + Al (OH)3The IL-13(Th2 type cytokine) was significantly reduced in the + BCG group, while IFN-. gamma. (Th1 type cytokine) was significantly increased.
The results of this section suggest: for the adult population, FI-RSV + BCG is the optimal immunization scheme, and is safe and effective.
All the above experimental results show that: BCG as FI-RSV (with or without Al (OH))3) The adjuvant can obviously improve FI-RSV or FI-RSV + Al (OH)3Protection and security.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims (6)
1. The application of bacillus Calmette-guerin vaccine (BCG) as an adjuvant of Respiratory Syncytial Virus (RSV) inactivated vaccine.
2. The use of claim 1, wherein: the inactivated vaccine may or may not contain an aluminium salt adjuvant.
3. The use of claim 1, wherein: the inactivated vaccine is a formalin inactivated RSV vaccine (FI-RSV), a beta-propiolactone inactivated RSV and other RSV inactivated vaccines inactivated by an inactivating agent or an inactivating method.
4. The use of claim 1, wherein: the application is that BCG and RSV inactivated vaccine (with or without aluminum salt adjuvant) are injected into body in advance or simultaneously to induce balanced immunological memory.
5. The use of claim 4, wherein: the application is that the adjuvant BCG is injected only once or the injection is injected for the same times as the inactivated RSV vaccine.
6. The application of BCG vaccine as the adjuvant of the inactivated vaccine of preventive or therapeutic respiratory syncytial virus.
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