CN113855796B - Application of BCG vaccine as respiratory syncytial virus inactivated vaccine adjuvant - Google Patents
Application of BCG vaccine as respiratory syncytial virus inactivated vaccine adjuvant Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
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- A—HUMAN NECESSITIES
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55588—Adjuvants of undefined constitution
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
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Abstract
The invention discloses an application of BCG vaccine as an adjuvant of respiratory syncytial virus inactivated vaccine, BCG is used as an adjuvant of RSV inactivated vaccine, and the Th1 type cellular immune response induced by BCG can balance the too strong Th2 and Th17 type cellular immune response induced by RSV inactivated vaccine, so that a vaccinator can generate balanced immune memory in vivo, and when RSV is infected, the vaccine can provide protection and prevent vaccine-enhanced inflammatory diseases, namely 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 vaccine as an inactivated vaccine adjuvant for respiratory syncytial virus.
Background
Respiratory syncytial virus (Respiratory syncytial virus, RSV) is the most important causative agent of lower respiratory tract infection in infants and young children, almost all children are infected before age two, and in children under age 5, the number of cases of RSV infection requiring emergency treatment and hospitalization is ten times greater than that of influenza virus infection, which is the most important cause of hospitalization and death in infants; in addition, the elderly are also susceptible to RSV, with annual infection rates similar to influenza and mortality rates nearly 2 times that of influenza. The world's direct economic burden of RSV production exceeds $ 800 billion annually, and as yet no safe and effective vaccine is marketed, WHO therefore ranks RSV vaccines as the vaccine of priority.
Soon after the discovery of RSV in 1956, vaccine development work has been rapidly carried out, and unfortunately the formalin inactivated RSV vaccine (FI-RSV) developed by the company duplicon in 1960's has presented significant frustration in clinical trials: FI-RSV was approved for clinical trials in infants and young children, vaccinated with infants from two months to seven years old, and 9 months later into the RSV epidemic season, infants vaccinated with FI-RSV vaccine were not only unprotected, but rather developed more severe pulmonary inflammatory disease than placebo after RSV infection, and developed allergic-like inflammation, i.e. vaccine-enhanced disease (VED), with significant neutrophil and eosinophil infiltration and Th 2-type immune responses in the lung, leading to 80% of vaccinators requiring hospitalization, two of which die, and only 5% of placebo requiring hospitalization [1]. Failure of this vaccine severely strikes the enthusiasm for the development of RSV vaccines, increasing the commercial risk of RSV vaccine development, and "safety" becomes a prime concern for RSV vaccine researchers.
Studies in animal experiments showed that: the pathogenesis of FI-RSV vaccine-enhanced disease is mainly related to vaccine-induced unbalanced immune responses [2 ]]Especially Th2 and Th17 type cellular immune dominant responses, whereas Th1 type cellular immune responses are absent [3] 。
Adjuvants refer to non-specific immunopotentiators that are injected into the body in advance or simultaneously with an antigen, and that can enhance the immune response of the body to the antigen or alter the type of immune response. Aluminium salt adjuvant (e.g. Al (OH) 3 And aluminium phosphate etc.) are human vaccine adjuvants that have been used for nearly a hundred years, the vast majority of vaccines using adjuvants of this type.
BCG is a live vaccine made from attenuated Mycobacterium bovis suspension, and has been used for nearly a century to prevent Mycobacterium tuberculosis and leprosy. 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 also evidence that BCG can protect infants from pathogens other than mycobacterium tuberculosis, providing heterologous or non-specific cross-protection [4] . There is literature onBcg has been shown to reduce death from other respiratory infections, diarrhea or malaria; small studies from guinea shaes and brazil showed that children vaccinated with BCG vaccine had a lower rate of pneumonia; also, a recently published study based on international population and health surveys, including 15 ten thousand children from 37 countries, showed that vaccination with bcg could reduce the risk of suspected acute lower respiratory infections by 17% to 37%; BCG vaccine not only protects children from respiratory tract infection, but also reduces their severity and associated mortality.
Reference is made to:
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.JVirol 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 report and research on BCG vaccine serving as an inactivated respiratory syncytial virus vaccine adjuvant exist.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the application of BCG vaccine serving as an adjuvant of respiratory syncytial virus inactivated vaccine, BCG serving as an adjuvant of RSV inactivated vaccine, and the Th1 type cell immune response induced by BCG can balance the excessively strong Th2 and Th17 type cell immune response induced by the RSV inactivated vaccine, so that a balanced immune memory is generated in a vaccinator, and when the RSV is infected, the vaccine can provide protection and prevent the occurrence of vaccine-enhanced inflammatory diseases, thereby realizing safety and effectiveness.
In order to achieve the technical purpose and the technical effect, the invention is realized by the following technical scheme:
use of BCG as an adjuvant for the preparation of an inactivated vaccine for the treatment or prophylaxis of respiratory syncytial virus (Respiratory syncytial virus, RSV), the inactivated vaccine being a formalin inactivated RSV vaccine FI-RSV.
Further, the inactivated vaccine may or may not contain an aluminum salt adjuvant.
Furthermore, the application is to inject BCG into a human body in advance or simultaneously with the RSV inactivated vaccine to induce balanced immune memory.
Further, the use is that the adjuvant BCG is injected only once or the same number of times as the inactivated RSV vaccine.
The invention has the beneficial effects that:
the invention provides application of BCG vaccine as an adjuvant of respiratory syncytial virus inactivated vaccine, BCG is used as the adjuvant of RSV inactivated vaccine, and the Th1 type cellular immune response induced by BCG can balance the excessively strong Th2 and Th17 type cellular immune response induced by RSV inactivated vaccine, so that the vaccination person generates balanced immune memory in vivo, and when RSV is infected, protection is provided, and meanwhile, the occurrence of vaccine enhanced inflammatory diseases is prevented, namely, the safety and effectiveness are realized.
Of course, it is not necessary for any one product to practice the invention to achieve all of the advantages set forth above at the same time.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the pathological changes of lung tissues of groups of mice immunized with an RSV inactivated vaccine and an adjuvant according to the embodiment of the present 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 expression of RSV-N protein and cytokine mRNA in lung tissue of various groups tested by real-time quantitative PCR after RSV challenge in a vaccine+adjuvant immunized mice according to the present invention (p < 0.05; p < 0.01; p < 0.001);
FIG. 3 shows the pathological changes of lung tissue of adult mice immunized with RSV inactivated vaccine and adjuvant according to the present invention (H)&E staining 100X); 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 the expression of RSV-N protein and cytokine mRNA in lung tissue of adult mice immunized with RSV vaccine plus adjuvant and challenged with RSV using real-time quantitative PCR according to the example of the invention (p < 0.05; p < 0.01; p < 0.001);
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Experimental study of immunization of milk mice with RSV inactivated vaccine + adjuvant
1.1 groups of rats:
(1) a PBS group;
(2) PBS/RSV group;
③FI-RSV+Al(OH) 3 a group;
(4) FI-rsv+bcg (two) group;
⑤FI-RSV+BCG(one)；
⑥FI-RSV+Al(OH) 3 +bcg (one). 6-10 animals/group
1.2 vaccination and RSV challenge protocol for milk mice:
note that: i.m. intramuscular injection, s.c. subcutaneous injection.
After receiving BALB/c pregnant mice and producing the pregnant mice, the pregnant mice were born with 1 day of birth and subcutaneously vaccinated with BCG ((4) - (6) groups), and 1 day of birth were intramuscular injected with PBS ((1) and (2) groups), FI-RSV ((4) and (5) groups) or FI-RSV+Al (OH) 3 ((3) and (6)) groups 7 (4) were again subcutaneously vaccinated with BCG and 8 were again intramuscular injected with PBS, FI-RSV or FI-RSV+Al (OH) 3 Day 21 was challenged with 30 μl of PBS ((1) or RSV ((2) - (6)) at the nasal drops, sacrificed after 5 days, lung tissue was harvested, paraffin embedded, sectioned, H&E, dyeing; and taking part of lung tissues, extracting RNA, and carrying out real-time quantitative PCR detection.
Example 2
Experimental study of immunization of adult mice with RSV inactivated vaccine + adjuvant
2.1 grouping of adult mice
(1) PBS group, (2) PBS/RSV group, (3 FI-RSV+Al (OH) 3 Group (4) FI-RSV+BCG group (5) FI-RSV+Al (OH) 3 + BCG group. 6/group
2.2 adult pestilence vaccination and RSV challenge protocol:
note that: i.m. is intramuscular injection and s.c. is subcutaneous injection.
6 week female BALB/c mice were picked up, inoculated subcutaneously with BCG ((4) and (5)) and after 1 day injected intramuscularly with PBS ((1) and (2), FI-RSV ((4)) or FI-RSV+Al (OH) 3 (groups (3) and (5)), day 11 and day 21 again by intramuscular injection of PBS, FI-RSV or FI-RSV+Al (OH) 3 Day 35 use 5Nasal drip challenge with 0 μl PBS ((1) or RSV ((2) - (5)) and 5 days later, sacrifice, lung tissue, paraffin embedding, slicing, H&E, dyeing; and taking part of lung tissues, extracting RNA, and carrying out real-time quantitative PCR detection.
Statistical result analysis
Lung histopathology in groups of suckling mice following RSV challenge, RSV-N protein and cytokine expression
Note that: PBS group: neither vaccinating nor challenge infection with RSV; PBS/RSV group: vaccinated but challenged with RSV; FI-rsv+bcg (two) group: BCG was injected 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: the normal group can see that the alveoli are intact in structure, the alveoli are normal in interval, and the bronchial columnar epithelial cells are smooth. B. PBS/RSV group: local inflammatory cell infiltration exists, partial alveoli are fused, the alveoli become large, and inflammatory cell infiltration exists around bronchi. C. FI-RSV+Al (OH) 3 Group: the bronchus is obviously infiltrated by inflammatory cells, alveolar space is thickened, alveoli are fused, and bronchus columnar cells are protruded inwards, so that obvious vaccine-enhanced lung inflammation is shown. D. FI-rsv+bcg (two): inflammatory cell infiltration is reduced, alveolar space is normal, inflammatory cell infiltration exists around bronchus, obvious congestion edema exists, partial alveoli are fused, alveoli become larger, and inflammation is higher than that of FI-RSV+Al (OH) 3 The group was significantly reduced but with FI-RSV+BCG (one), FI-RSV+Al (OH) 3 The symptoms of inflammation were heavier than +bcg (one). E. FI-rsv+bcg (one): inflammatory cell infiltration is obviously reduced, but obvious edema still exists, alveolar space is normal, partial alveoli are fused, and the alveoli become larger, but inflammation is higher than that of FI-RSV+Al (OH) 3 The group was significantly reduced. F. FI-RSV+Al (OH) 3 +bcg (one): no obvious inflammatory cell infiltration, no obvious congestion and edema, complete alveolar structure, relatively normal alveolar space and relatively normal bronchial columnar epithelium morphology.
Pathological section result prompts: FI-RSV+Al (OH) 3 The group showed significant vaccine-enhanced inflammation, whereas FInflammation in the I-RSV+Al (OH) 3+BCG (one) group was significantly inhibited, and was the optimal immunization regimen.
As shown in fig. 2
With FI-RSV+Al (OH) 3 Group comparative FI-RSV+Al (OH) 3 The expression level of RSV-N (housekeeping gene of RSV virus-N gene) in lung tissue of +BCG (one) group was significantly reduced, indicating FI-RSV+Al (OH) 3 Vaccination regimens in the +bcg (one) group can provide good protection;
in addition, FI-RSV+Al (OH) 3 The expression level of eotaxin (eosinophil chemokine), gro-alpha (neutrophil chemokine), MCP-1 (monocyte chemokine), TNF-alpha (pro-inflammatory cytokine), IL-17 (Th 17 type inflammatory cytokine), IL-6 and IL-10 (Th 2 type cytokine) in lung tissue of +BCG (one) group is significantly lower than that of FI-RSV+Al (OH) 3 Group, FI-RSV+Al (OH) 3 IFN-gamma (Th 1 type cytokine, which can activate macrophage and lymphocyte and enhance its ability to kill pathogen) expression level in lung tissue of +BCG (one) group is obviously higher than PBS and PBS/RSV group, and FI-RSV+Al (OH) 3 There was no statistical difference in group comparisons. These results indicate that: FI-RSV+Al (OH) 3 The +BCG (one) vaccinated rats provided significant protection and significantly reduced levels of Th2 and Th17 cytokines and pro-inflammatory factors in the lungs.
The experimental results in this section suggest that: FI-rsv+al (OH) 3+bcg (one) is the optimal immunization regimen for newborns, both safe and effective.
Lung histopathology and RSV-N protein and cytokine expression in mice of each constituent year after RSV challenge
Pathological section of lung tissue, H&E staining results (see fig. 3), PBS group: the normal group can see that the alveoli are intact in structure, the alveoli are normal in interval, and the bronchial columnar epithelial cells are smooth. B. PBS/RSV group: local inflammatory cell infiltration exists, partial alveoli are fused, the alveoli become large, and inflammatory cell infiltration exists around bronchi. C. FI-RSV+Al (OH) 3 Group: the bronchus is obviously infiltrated by inflammatory cells, alveolar space is thickened, alveoli are fused, and bronchus columnar cells are protruded inwards, so that obvious vaccine-enhanced lung inflammation is shown. D.FI-rsv+bcg group: no obvious inflammatory cell infiltration was seen, the alveolar spaces were normal, and fusion was occasioned in alveoli. E. FI-RSV+Al (OH) 3 +bcg group: has obvious inflammatory cell infiltration, alveolar space thickening, alveolar fusion, and inflammatory cell infiltration around bronchus, but is more than FI-RSV+Al (OH) 3 Group inflammation was reduced and symptoms of inflammation were heavier compared to FI-rsv+bcg group. The above results suggest: in adult mice, FI-rsv+bcg vaccination regimen significantly reduced lung inflammatory pathology.
Real-time quantitative PCR detection of expression of RSV-N protein and cytokine mRNA in adult mouse lung tissue: FIG. 4 shows the results of FI-RSV+Al (OH) in comparison to the PBS/RSV group 3 FI-RSV+BCG and FI-RSV+Al (OH) 3 The expression of RSV-N gene (RSV housekeeping gene) after challenge in +BCG group was significantly reduced, and FI-RSV+BCG and FI-RSV+Al (OH) 3 The amount of RSV-N in the +BCG group is significantly lower than that of FI-RSV+Al (OH) 3 Groups, indicating FI-RSV+BCG and FI-RSV+Al (OH) 3 The +bcg group provides significant protection;
eotaxin (chemotactic eosinophil factor) or FI-RSV+Al (OH) of the FI-RSV+BCG group 3 Eotaxin (chemotactic eosinophil factor), gro-alpha (chemotactic neutrophil factor), MCP-1 (chemotactic monocyte chemotactic protein 1 factor) and FI-RSV+Al (OH) of the +BCG group 3 The group ratio was significantly reduced; IL-17 and IL-23 (Th 17 type cytokines) of FI-RSV+BCG group, FI-RSV+Al (OH) 3 IL-23 in +BCG group was also significantly lower than FI-RSV +Al (OH) 3 A group; with FI-RSV+Al (OH) 3 FI-RSV+BCG and FI-RSV+Al (OH) in comparison to the group 3 IL-13 (Th 2 type cytokine) was significantly reduced in +BCG group, while IFN-gamma (Th 1 type cytokine) was significantly increased.
The experimental results in this section suggest that: FI-rsv+bcg is the optimal immunization regimen for the adult population, both safe and effective.
All the experimental results above show that: BCG as FI-RSV (with or without Al (OH) 3 ) Can obviously improve FI-RSV or FI-RSV+Al (OH) 3 Is provided for protection and safety.
The preferred embodiments of the invention disclosed above are intended only to assist in the explanation of the invention. The preferred embodiments are not exhaustive or to limit the invention to the precise form 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 understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.
Claims (4)
1. Use of BCG as an adjuvant for the preparation of an inactivated vaccine for the treatment or prevention of respiratory syncytial virus (Respiratory syncytialvirus, RSV), characterized in that: the inactivated vaccine is a formalin inactivated RSV vaccine FI-RSV.
2. The use according to claim 1, characterized in that: the inactivated vaccine contains or does not contain an aluminum salt adjuvant.
3. The use according to claim 1, characterized in that: the application is that BCG is injected into a human body in advance or simultaneously with the RSV inactivated vaccine to induce balanced immune memory.
4. A use according to claim 3, wherein: the use is that the adjuvant BCG is injected only once or the same number of times as the inactivated RSV vaccine.
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