Immunization With Inactivated Whole Cell Mediates Rapid And Specific Protection Against Acinetobacter Baumannii Infection By Inducing Innate Immune Memory

Acinetobacter baumannii has become a major threat for public health worldwide.The types of infections caused by this pathogen included pneumonia,meningitis,bacteremia,wound infection,and urinary tract infection.It’s extremely difficult to treat infections caused by A.baumannii due to most clinical isolates are highly resistant to a wide range of antibiotics,displaying multidrug-resistant（MDR）.The mortality rates caused by MDR-A.baumannii are as high as 43%.Due to effective antibiotics are absent,it’s necessary to develop an alternative strategies to control this pathogen.Vaccination is a viable choice to prevent or treat A.baumannii infections.Hospital-acquired infections are far more common in A.baumannii infection.As time between being in hospital and being exposed to A.baumannii is short,an ideal vaccine for patients who are in high risks for A.baumannii infections should provide protective immunity as soon as possible.Currently,the immunization time of A.baumannii vaccines which have been reported effective at some levels in animal models requires more than once and usually mediates protection several weeks later.Host immune system is divided into adaptive immune and innate immune.In the classic immune theory,innate immune system responses rapidly but has no memory.Adaptive immune system is slower to response but has memory.The classic vaccine whose efficacy is dependent on adaptive immune memory limit them to rapidly control A.baumannii infections for patients.Recent researches indicated that innate immune system is able to exert adaptive characteristics and can build memory.Innate immune memory could be established within days,which means enhanced protections could be rapidly induced upon reinfection.We investigated the protection efficacy of inactivated whole cell（IWC）of A.baumamnii using different immunization route and times.The results showed IWC confered rapid protection at day 2and the immune response exhibited the feature of immune memroy at day 7 after vaccination.It’s difficult to clarify the phenomena according to the the classic theory.Thus the innate immune memory might help us to study the underlying mechanisms of the rapid protection induced by IWC and provide guideline for the design of new vaccines.Objectives:1.To evaluate the protection efficacy induced by single intranasal immunization of IWC aganist A.baumannii pneumonia and sepsis model.2.To study the role of innate immune memory in rapid protection induced by IWC of A.baumannii.3.To investigate the characteristic of IWC of A.baumannii and the influence of aging on protection efficacy induced by the vaccine.Methods:1)The evaluation of protection efficacy induced by immunization of IWC of A.baumannii1.Mice were immunized intranasally or intramuscularly with IWC three times,two times,or one time respectively and then infected intratracheally with lethal dose of LAC-4.Survival rate of mice were observed daily for 7 days to evaluate the protection efficacy of IWC.2.Mice were immunized intranasally or intramuscularly with IWC.2 days,7 days,or30 days after immunization,mice were infected intratracheally with lethal dose of LAC-4.Survival rate of mice were observed daily for 7 days to assess whether IWC mediates rapid and long-term protection.3.Mice were immunized intranasally with IWC.2 days,7 days or 30 days after immunization,mice were infected intratracheally with lethal dose of LAC-4.Bacterial counts in the lung and blood of mice were detected at 24 hours post infection（hpi）.4.Mice were immunized intranasally with IWC and were infected intratracheally with lethal dose of LAC-4 7 days after immunization.At 24 hpi,bacterial counts in the lung and blood were detected and lung histopathology were observed.Cytokine concentrations in the serum and lung were detected by ELISA kits.Chemokine mRNA expression in lungs were detected by qRT-PCR.Number of inflammatory cells were analyzed by flow cytometry to evaluate the impact of IWC on inflammatory response.5.Mice were immunized intranasally with IWC and infected intraperitoneally with lethal dose of LAC-4 2 days or 7 days after immunization.Survival rate of mice were observed daily for 7 days to evaluate the protection of IWC against A.baumannii sepsis.2)The underlying mechanism of rapid protetion induced by immunization of IWC of A.baumannii1.Mice were immunized intranasally with IWC.7 days after immunization,IgG and IgA levels in serum and BALF were detected by ELISA to assess the antibody response after IWC vaccination.2.WT and Rag1^-/-mice were immunized intranasally with IWC and infected intratracheally with lethal dose of LAC-4 7 days after immunization.Survival rate of mice were observed daily for 7 days.The genes expression in Rag1^-/-mice in transcriptional profiles were analyzed to evaluate whether protection induced by IWC is dependent on innate immune response.3.The genes expression in Rag1^-/-mice in transcriptional profiles were analyzed 2days and 7 days after immunization.Mice were immunized intranasally with IWC,cytokine concentrations were detected by ELISA and neutrophil number were detected by flow cytometry from day 0 to day 7.Mice were immunized intranasally with IWC and infected intratracheally with lethal dose of LAC-4 7 days after immunization.At 0 and 4 hpi,number of inflammatory cells were analyzed by flow cytometry.At 0 and 2 hpi,cytokine concentrations in the lung homogenates were detected by ELISA and mRNA levels of chemokine in lungs were detected by qRT-PCR to assess the role of innate immune memory in rapid protection induced by IWC.4.TLR4 expression were analyzed by flow cytometry.WT and TLR4^-/-mice were immunized intranasally with IWC and were infected intratracheally with lethal dose of LAC-4 7 days after immunization.Survival rate of mice were observed daily for 7 days.At24 hpi,bacterial counts in the lung and blood of mice were detected.At 4 hpi,inflammatory cell number were analyzed by flow cytometry to evaluate the role of TLR4 in rapid protection induced by IWC.3)The characteristic of rapid protection induced by IWC of A.baumannii1.Mice were immunized intranasally with IWC（LAC-4）and were infected intratracheally with lethal dose of SJZ24 or SJZ26 7 days after immunization.Mice were immunized intranasally with IWC（ATCC17978）,IWC（SJZ09）,IWC（SJZ24）,or IWC（SJZ26）on day 0 respectively and were infected intratracheally with lethal dose of LAC-4 7 days after immunization.Survival rate of mice were observed daily for 7 days to evaluate whether IWC induced broad protection against A.baumannii infection.2.Mice were immunized intranasally with IWC and infected intratracheally with lethal dose of P.aeruginosa or S.aureus 2 days or 7 days later.Mice were immunized intranasally with IWC（P.a）,IWC（S.a）,or IWC（E.c）and infected intratracheally with lethal dose of LAC-4 2 days or 7 days later.Survival rate of mice were observed daily for 7days to evaluate whether IWC induced specific protection against A.baumannii infection..3.Mice were immunized intranasally with IWC（P.a）,IWC（K.p）,or IWC（S.a）and were infected intratracheally with lethal dose of P.aeruginosa,K.peneumoniae,or S.aureus 7 days later.Survival rate of mice were observed daily for 7 days to evaluate whether protection induced by IWC is common in other bacteria strains.4.Young and aged mice were infected with LAC-4 intratracheally.Survival rate and clinical score of mice were observed daily for 7 days.At 24 hpi,bacterial burdens in the lung,blood,and spleen were determined and lung histopathology were observed.Young and aged mice were intranasally immunized with IWC and were intratracheally challenged with lethal dose of LAC-4.Survival rate of mice were observed daily for 7 days to evaluate the impact of aging on protection efficacy induced by IWC.Results:1)The evaluation of protection efficacy induced by immunization of IWC of A.baumannii1.Mice vaccinated with IWC for three times,two times,or one time showed higher survival rate（survival rate of all vaccinated group was 100%）than control goup（0%）.The results indicated single immunization with IWC confers protection against A.baumanni infection.2.Mice vaccinated with IWC intranasally 2 days（100%）,7 days（100%）,or 30 days（100%）prior A.baumannii challenge showed higher survival rate than control group.Mice vaccinated with IWC intramuscularly 2 days prior A.baumannii infection showed similar survival rate to control group.But mice vaccinated with IWC intramuscularly 7 days or 30days prior infection showed higher survival rate than control group.Mice vaccinated with IWC intranasally had reduced bacterial burdens and lung injury than control goup.The results indicated single intranasal immunization with IWC confers more rapid protection than intramuscular immunization.3.At 24 hpi,mice vaccinated with IWC 7 days prior A.baumannii challenge had significantly lower IL-6,TNF-α,and IL-1βconcentration in the lung and lower IL-6,TNF-α,and IFN-γconcentration in the serum compared to control group.Meanwhile,neutrophil number in the lung were decreased in vaccinated group compared to control group at 24 hpi.CXCL1,CXCL2,CXCL10,CCL2,and CCL7 mRNA expression in lungs were obviously reduced in vaccinated mice compared to control group at 24 hpi.In conclusion,single intranasal immunization with IWC reduced bacterial burdens,lung damage,and decreased inflammatory response at 24 hpi.4.Mice vaccinated with IWC 2 days（100%）or 7 days（100%）prior A.baumannii challenge showed higher survival rates than control goup（0%）in sepsis model.The results indicated single intranasal immunization with IWC confers rapid protection against A.baumannii spesis.2)The underlying mechanism of rapid protetion induced by immunization of IWC of A.baumannii1.ELISA results showed IgA and IgG levels in serum and BALF of vaccinated mice have no significant difference compared to that of na?ve mice.The results indicated IWC didn’t induce antibody response on 7 days after single intranasal immunization.2.Single intranasal immunization with IWC confered 100%protection in WT mice and 75%protection in Rag1^-/-mice.RNA-seq analysis showed the majority genes associated with inflammatory response were obviously downregulated in vaccinated Rag1^-/-mice.The results indicated protection induced by single intranasal immunization with IWC is depedent on innate immune response.3.RNA-seq analysis showed the genes associated with inflammatory response reduced to baseline level in vaccinated Rag1^-/-mice at day 7 after immunization.The levels of TNF-αand IL-6 in the lung and serum and neutrophil number in the lung of vaccinated mice declined to baseline level after 7 days after vaccination.The results indicated the innate immune status returns to a basal state.Mice vaccinated with IWC had significantly increased neutrophil and monocyte number compared to control group at 4 hpi.Mice vaccinated with IWC had significantly increasd IFN-γand TNF-αconcentrations in the lung compared to control group at 2 hpi.mRNA expression of CXCL1,CXCL2,CXCL5,CXCL10,and CCL2 were obviously higher in vaccinated mice compared to control group at 2 hpi.In conclusion,single immunization with IWC increased innate immune response at2 and 4 hpi.The results indicated IWC mediates rapid protection by inducing innate immune memory4.TLR4 expression is significantly higher after 7 days after vaccination compared to control group.Single intranasal immunization with IWC confered 100%protection in WT mice and 0%protection in TLR4^-/-mice.Vaccinated TLR4^-/-mice had significantly higher bacterial counts in the lung and blood compared to vaccinated WT mice at 24 hpi.Vaccinated TLR4^-/-mice had significantly lower number of neutrophil and monocyte in the lung compared to vaccinated WT mice at 4 hpi.The results indicated rapid protection induced by single immunization with IWC is dependent on TLR4.3)The characteristic of rapid protection induced by IWC of A.baumannii1.When mice were infected with SJZ24,mice vaccinated with IWC（100%）showed higher survival rate compared to control goup（0%）.When mice were infected with SJZ26,mice vaccinated with IWC（80%）showed increased survival rate compared to control group（20%）.Mice vaccinated with IWC（ATCC17978）,IWC（SJZ09）,IWC（SJZ24）,or IWC（SJZ26）showed higher survival rate（100%,100%,90%,100%respectively）than control group（0%）.The results indicate IWC induced broad protection against A.bauamnnii clinical strains2.When mice were infected with P.aeruginosa,IWC of A.baumannii immunized 2days and 7 days prior infection confered 80%and 20%protection respectively.When mice were infected with S.aureus,IWC of A.baumannii immunized 2 days and 7 days prior infection confered 100%and 40%protection respectively.IWC（P.a）,IWC（S.a）or IWC（E.c）immunized 2 days prior infection confered 100%,100%,75%protection respectively.IWC（P.a）,IWC（S.a）or IWC（E.c）immunized 7 days prior infection confered 0%,0%,25%protection respectively.The results indicated protection induced by IWC of A.baumannii is specific at day 7 after vaccination3.IWC（P.a）confered 100%protection against P.aeruginosa infection.IWC（K.p）confered 80%protection against K.peneumoniae infection.IWC（S.a）confered 20%protection against S.aureus infection.The results indicated protection induced by single immunization of IWC might be common in gram-negative strains.4.Aged mice had higher mortality,increased bacterial burdens and more severe lung injury after A.baumannii infection.The results indicated aging increase susceptibility to A.baumannii infection and we bulit an aged pneumonia mouse model sucessfully.When mice were vaccinated with 1×10⁷ CFU IWC for one time,IWC confered 100%protection in young mice and 0%protetion in aged mice.When mice were vaccinated with 1×10⁸ CFU IWC for one time,IWC confered 100%protection in young mice and 40%protection in aged mice.When mice were vaccinated with IWC for three times,IWC confered 100%protection in young mice and 80%protection in aged mice.The results indicated aging reduces the rapid protection efficacy of IWC.Increased immunization dose and repeated immunization could improve the efficacy of IWC in aged mice.Conclusions:1.Single intranasal immunization with IWC confers rapid protection against A.baumanni pneumonia and spesis.2.IWC of A.baumannii meidateds rapid protection by inducing innate immune memory and rapid protection is dependent on TLR4.3.IWC of A.baumannii induced broad protection against A.baumannii clinical strains and protection induced by IWC is specific at day 7 after IWC vaccination.Rapid protection induced by IWC might be common in gram-negative strains.Aging reduces the rapid protection efficacy induced by IWC.Significance:In this study,single intranasal immunization with IWC confers rapaid and specific infection against A.baumannii infection.The protection is dependent on innate immune memory.The study could be used for design of novel bacterial vaccine.