The Protection Against Allergic Response Or Tuberculosis Induced By Repeated BCG Vaccination At Newborns

BackgroundAsthma is a chronic inflammatory disease characterized by airway infiltration of inflammatory cells,particularly eosinophils and T lymphocytes,mucous over-production,and airway hyperresponsiveness(AHR).The incidence and severity of asthma are increasing in the world and asthma is estimated to affect about 300 million persons.Currently,many anti-asthmatic medicines,including inhale corticosteroids,are available for controlling asthmatic attack.However,those treatments are directed at reducing symptoms and have little effect on inhibiting the disease progression.Therefore,preventative interventions may be a better approach to protect from asthma.Mycobacterium boris Bacillus Calmette-Guerin(BCG)is a strong inducer of the Th1 type immune response.At 1997,Shirakawa and his colleagues claimed that there was inverse relationship between tuberculin responses and atopic disorder,which cost light on the develepment of vaccine on asthma.BCG was viewed to induce Th1 type response and suppress Th2-mediated response in asthmatic model.Several studies have demonstrated that BCG can inhibit airway eosinophilia and decrease-related mucous over-production,reducing AHR in animal models of asthma.However,the relationship between BCG infection and asthma in human being is controversial.At 1997,Shirakawa and his colleagues claimed that there was inverse relationship between tuberculin responses and atopic disorder,some following studies came from Guinea,Brazil,Hungary and China also found that BCG vaccination was associated with lower asthma prevalence.A recent clinical study showed repeated BCG further improved the lung function and increased the IFN-γ/IL-4 ratio in the peripheral blood in mild to moderate asthmatic patients.But a large body of data suggested there was no protective effect of BCG vaccination against atopy.And our laboratory has demonstrated that early bacillus calmette-guerin vaccination prevents airway mucus production in mouse models of asthrna. Besides,our recent work investigates the protection induced by multiple BCG vaccination at newborn on the airway inflammation of asthmatic mice at different age. We found neonatal multiple BCG vaccination elicited long-term protection in mouse allergic responses,even at 45 week old mice.To further investigate the effect of neonatal multiple BCG vaccination on adult allergic symptoms,we tried to find the difference level of protection and T cell response induce by multiple BCG or single BCG vaccination at newborn.Tuberculosis is a global health problem and it is estimated roughly that one third of the world's population is infected with Mytobactium tuberculosis and approximately 8-10 million people become infected each year.With people's move frequently,the spread of HIV/AIDS infection and the emergence of multi-drug-resistant strains of M. tubercolusis,the risk of tubercolusis is increased.Till now,M.bovis BCG is the only available anti-tubercolusis vaccine in the world wide.However,its role in adult has been highly variable from 0-80%.A META data showed that the efficacy of BCG on tuberculosis is about 50%.The development of an improved efficacy of vaccine or new vaccination remege against tuberculosis is urgently required and the identification of immune response correlates for protection would greatly facilitate the rational design of such a vaccine.Given the safety and very extensive coverage of BCG,repeated BCG vaccination might be a clinical strategy.However,revaccination of humans with BCG is controversial,some studies had demonstrated that repeated BCG concluded that revaccination was useful,others denied the increasing protective efficacy of the primary vaccination.In addition,the protection induced by BCG was reported to last for short term.Besides,the factors which impact on the efficacy of revaccination or the reasons why revacciantion failed to protect TB were known little. Evidence from animal study indicated that the environmental situation,such as the exposure of environmental mytobacteria,warm infection would deduce the protection efficacy of BCG on tuberculosis.Becides,it was reported that the internal of primary and boost vaccination played a privotal role on the protection.The authors suggested that long internal,of vaccination reduced the protection induced by multiple BCG vaccination.We hyposisth that neonatanl mice was vaccinated and boosted with BCG at a short internal would enhance the efficacy of BCG against TB.Part 1 Neonatal multiple Bacillus Calmette-Guerin elicits a superior protection in mouse allergic responsesObjectiveTo investigate whether revaccination of neonatal mice with BCG increases the level of protection against allergic responses induced by a single BCG vaccination.MethodsC57BL/6 neonates were vaccinated with BCG for three times on days 1,7 and 14 or only vaccinated on day 1.When part of these mice grew up to 8 weeks old,IFN-γand IL-4 level were tested in splenocyte supernatants by ELISA.And others were sensitized with ovalbumin at 8 and 10 weeks of age,then challenged with allergen at 12 weeks of age.Their airway inflammation was characterized.Results8 weeks later,IFN-γwere high in BCG immunized mice,and multiple BCG vaccination increased IFN-γlevel significantly as compared with single BCG immunization(P＜0.05).IL-4 response was similar in mice with BCG immunized or without.After OVA sensitization and challenge,only multiple BCG vaccination inhibited airway hyperresponsiveness,eosinophils infiltration and mucus overproduction(P＜0.05),while single BCG vaccination on neonates had little effect on allergic responses.ConclusionOur data suggest that neonatal BCG vaccination induced predominated IFN-γresponse,which lasted to adult lifetime.And multiple BCG elicits robust IFN-γresponse.Also,multiple BCG immunization has an effect on inhibiting airway hyperresponsiveness,eosinophilia and mucus overproduction.Single BCG vaccination at newborns had no protection on the allergic response.Thus,neonatal multiple BCG vaccination has a protective effect on adult mice,which may be mediated by IFN-γresponse.Part2 Repeated Bacillus Calmette-Guérin vaccination at newborns elicits a superior but short-term protection in M.tuberculosis infected mouseObjectivesTo investigate whether revaccination of neonatal mice with BCG increases the level of protection against tuberculosis induced by a single BCG vaccination.MethodsC57BL/6 neonates were vaccinated with BCG for three times on days 1,7 and 14 or only vaccinated on day 1.When part of these mice grew up to 8 weeks old,IFN-γand IL-4 level were tested in splenocyte supernatants.And other parts of mice were challenged with M.tuberculosis H37Rv.At 3,6 and 9 weeks of post challenge,their tuberculosis burden in organs and histologic change in lung,as well as IFN-γ,IL-4 level in splenocyte supematants,frequency of IFN-γproducing cells of spleen and IFN-γ,IL-2,IL-12 and TNF-αin serum were characterized. ResultsAt 3 weeks of M.tuberculosis H37Rv challenge,BCG vaccination inhibited tuberculosis growth in lung and liver significantly(P＜0.05)and reduced lung tissue damage.Multiple BCG vaccinated mice gained lower spleen/body weight and M. tuberculosis burden as well as mild lung histological damaged in lung than those in single BCG immunized mice(P＜0.05).At week 6,however,a time-related decline of effect was appeared in both multiple and single BCG vaccinated mice.The area of lung tissue damage was enlarging and the replication of M.tuberculosis was progressing,although which were significantly different from tuberculosis mice.Till to 9 weeks of challenge,there has no any significant difference in burden of M. tuberculosis or histological change between multiple BCG vaccinated,single BCG vaccinated and infected but non-vaccinated mice.Th1 cytokines were associated with M.tuberculosis infection or BCG immunization. 8 weeks post BCG immunization,IFN-γwere upregulated,and multiple BCG vaccination increased IFN-γlevel significantly as compared to single BCG immunization(P＜0.05).3,6,9 weeks post of infection,IFN-γlevel was significantly increased in mice even infected by M.tuberculosis,compared to non-infected mice. There was no significant difference in mice with BCG immunized or without.To further assess the association of IFN-γresponse and protection against tuberculosis, the frequency IFN-γ-producing cell of spleen was detected.The data showed that there was no relationship with BCG vaccination-induced IFN-γproducing cell and protection against tuberculosis in 3 weeks of infection.Other Thl cytokines,IL-2 and IL-12 in serum didn't be affected by repeated or single BCG immunization,but the level of TNF-αwas inhibited significantly in two BCG groups at 3 weeks of infection (P＜0.05),while multiple and single BCG vaccinated mice had no significant difference in TNF-αlevel.IL-4 responses were similar in mice with BCG immunized or without before M. tuberculosis infection.At 3 weeks of infection,Th2-type cytokine in vitro were significantly up-regulated in HRV37 challenged mice as compared to saline group(P＜0.05).Th2 response was significantly lower in BCG immunized mice than in M. tuberculosis infected mice(P＜0.05).Till to 9 week of challenge,as disease processing in those BCG immunized mice and limiting in M.tuberculosis mice,IL-4 level reversed to significant high in BCG immunized mice when compared to M. tuberculosis infected mice(P＜0.05).There was no significant difference between multiple and single BCG vaccinated mice at every time point.ConclusionOur data suggest that revaccination of neonatal mice with BCG has a superior but short-term effect on tuberculosis.The time-related decline of protection may be induced by restrained TNF-αlevel induced by BCG.And the correlation between Th1 response and protection against tuberculosis was poor.Th2 response indicated to involve in the progression of tuberculosis and induced the toxic of TNF-α.