Vaccine using community-acquired respiratory distress syndrome toxin as an antigen against Mycoplasma pneumoniae in mice.

Mycoplasma pneumoniae (Mp) is one of the most common causes of bacterial community-acquired pneumonia in humans. Because of the frequent epidemics and the emergence of antibiotic-resistant Mp, vaccines for Mp are urgently needed to ameliorate the pneumonia and secondary complications. The community-acquired respiratory distress syndrome (CARDS) toxin produced by Mp is a pathogenic factor that induces severe inflammatory responses in lung. Although blocking CARDS toxin is expected to mitigate the severity of Mp pneumonia, the potential of CARDS toxin as a vaccine antigen has not been assessed. Here, we examined the effectiveness of vaccine using recombinant CARDS toxin (rCARDS toxin) as an antigen in mice. Immunization with rCARDS toxin induced both rCARDS toxin- and Mp-specific antibody responses, indicating that CARDS toxin is located on the surface of Mp. In addition, immunization with rCARDS toxin decreased not only lung injury, neutrophil infiltration, and the production of inflammatory cytokines but also the persistence of Mp in lung after Mp challenge. Furthermore, we elucidated that the CARDS toxin on the surface of Mp facilitates the adherence of Mp to epithelial cells. In conclusion, we have demonstrated the potential of rCARDS toxin as a vaccine antigen to ameliorate Mp pneumonia by suppressing the inflammatory responses induced by Mp and the persistence of Mp in lung. These data support the development of novel vaccines for Mp pneumonia.

An attenuated multiple genetic mutant of Mycoplasma pneumoniae imparts good immuno-protection against M. pneumoniae pneumonia in BALB/c mice.

Mycoplasma pneumoniae (M. pneumoniae) is the causative agent of both upper and lower respiratory infections that can lead to pneumonia, extrapulmonary complications and devastating sequela. With the increasing rate of macrolide-resistant strains, the severe clinical consequence of refractory mycoplasma pneumonia in children health calls for the need of vaccine research for this pathogen. In this report, the immunomodulatory effectiveness of a live attenuated M. pneumoniae vaccine was evaluated. The vaccine strain was a mutant strain of M. pneumoniae, MUT129, obtained after multiple passages of M129 strain in PPLO broth. The SNP/InDel detection results showed that mutations were present in genes encoding the adhesion organelle-associated proteins and lipoproteins of M. pneumoniae MUT129. Upon intranasal challenge of BALB/c mice with 1 × 107 CFU of MUT129, there were very small amount of Mycoplasma antigens and almost no M. pneumoniae present in the lung tissues of BALB/c mice. Besides, there was almost no inflammatory cell infiltration in the lung tissue. Results of the M. pneumoniae challenge study showed that mice immunized with MUT129 presented with less inflammation, lower detectable number of M. pneumoniae in the lungs when compared with the unimmunized mice. These results indicated that the live attenuated vaccine can efficiently prevent the proliferation of M. pneumonia in the lungs, reduce but not completely prevent the pulmonary inflammatory response.

CC16 Binding to α4ß1 Integrin Protects against Mycoplasma pneumoniae Infection.

Rationale CC16 (club cell secretory protein) is a pneumoprotein produced predominantly by pulmonary club cells. Circulating CC16 is associated with protection from the inception and progression of the two most common obstructive lung diseases (asthma and chronic obstructive pulmonary disease). Objectives Although exact mechanisms remain elusive, studies consistently suggest a causal role of CC16 in mediating antiinflammatory and antioxidant functions in the lung. We sought to determine any novel receptor systems that could participate in CC16's role in obstructive lung diseases. Methods Protein alignment of CC16 across species led to the discovery of a highly conserved sequence of amino acids, leucine-valine-aspartic acid (LVD), a known integrin-binding motif. Recombinant CC16 was generated with and without the putative integrin-binding site. A Mycoplasma pneumoniae mouse model and a fluorescent cellular adhesion assay were used to determine the impact of the LVD site regarding CC16 function during live infection and on cellular adhesion during inflammatory conditions. Measurements and Main Results CC16 bound to integrin α4ß1), also known as the adhesion molecule VLA-4 (very late antigen 4), dependent on the presence of the LVD integrin-binding motif. During infection, recombinant CC16 rescued lung function parameters both when administered to the lung and intravenously but only when the LVD integrin-binding site was intact; likewise, neutrophil recruitment during infection and leukocyte adhesion were both impacted by the loss of the LVD site. Conclusions We discovered a novel receptor for CC16, VLA-4, which has important mechanistic implications for the role of CC16 in circulation as well as in the lung compartment.

[Study on the etiological characteristics and prevention and control of adult community-acquired pneumonia in hospitalized patients in a hospital in Beijing from 2015 to 2019].

Objective: To explore the distribution characteristics of pathogens in adult patients with community-acquired pneumonia (CAP) and to provide basis for the diagnosis, treatment, prevention of CAP. Methods: 1 446 inpatients with CAP were prospectively enrolled in a third-class hospital in Beijing in recent 5 years (from January 2015 to December 2019). Respiratory tract samples were collected for smear, culture, nucleic acid, antigen and antibody detection to identify the pathogen of CAP. Mann-Whitney U test was used for continuous variables and χ2 test or Fisher's exact test was used for categorical data for statistical analysis. Results: Among the 1 446 patients, 822 (56.85%) patients were infected with a single pathogen, 231 (15.98%) patients were infected with multiple pathogens, and 393 (27.18%) patients were not clear about the pathogen. Influenza virus is the first pathogen of CAP (20.95%, 303/1 446), mainly H1N1 (8.51%, 123/1 446), followed by mycoplasma pneumoniae (7.19%, 104/1 446), Mycobacterium tuberculosis (5.33%, 77/1 446) and Streptococcus pneumoniae (5.05%, 73/1 446). The outbreak of H1N1 occurred from December 2018 to February 2019, and the epidemic of mycoplasma pneumoniae pneumonia was monitored from August to November 2019. Patients under 65 years old had high detection rates of Mycoplasma pneumoniae (14.41% vs. 2.41%, χ²=74.712,P<0.001), Streptococcus pneumoniae (8.16% vs. 2.99%, χ²=18.156, P<0.001), rhinovirus (6.08% vs. 3.56%, χ²=5.025, P<0.025), Chlamydia pneumoniae (5.90% vs. 1.15%, χ²=26.542, P<0.001) and adenovirus (3.13% vs. 0.92%, χ²=9.547, P=0.002). The severe disease rate of CAP was 14.66% (212/1 446), and the average mortality rate was 3.66% (53/1 446). The severe illness rate and mortality rate of bacterial-viral co-infection were 28.97% (31/107) and 19.63% (21/107), respectively. Conclusions: Influenza virus is the primary pathogen of adult CAP. Outbreaks of Mycoplasma pneumoniae and H1N1 were detected in 2018 and 2019, respectively. The remission rate and mortality rate of virus-bacteria co-infection were significantly higher than those of single pathogen infection. Accurate etiological basis not only plays a role in clinical diagnosis and treatment, but also provides important data support for prevention and early warning.

Polydatin suppresses the development of lung inflammation and fibrosis by inhibiting activation of the NACHT domain-, leucine-rich repeat-, and pyd-containing protein 3 inflammasome and the nuclear factor-κB pathway after Mycoplasma pneumoniae infection.

Mycoplasma pneumoniae (MP) can infect both the upper and lower respiratory tracts. Polydatin (PD), a traditional Chinese medicine, is known to have anti-inflammation and antifibrosis properties. However, the protective effects of PD against MP pneumonia (MPP) remain unclear. So, the aim of this study was to describe the therapeutic effects and underlying mechanisms of PD against MPP. BALB/c mice were assigned to three groups: a normal control group, MP infection group, or PD-treated MP infection group. BEAS-2B cells transfected with or without NACHT domain-, leucine-rich repeat-, and pyd-containing protein 3 (NLRP3) were used to confirm the protective mechanisms of PD. Immunohistochemical analysis, Western blot analysis, enzyme-linked immunosorbent assay, and flow cytometry were used in this study. The results showed that PD treatment suppressed MP-induced lung injury in mice by suppressing the expression of inflammatory factors and inhibiting the development of pulmonary fibrosis. Meanwhile, PD treatment inhibited activation of the NLRP3 inflammasome and nuclear factor κB (NF-κB) pathway. Overexpression of NLRP3 reversed the protective effect of PD against MP-induced injury of BEAS-2B cells. Taken together, these results indicate that PD treatment suppressed the inflammatory response and the development of pulmonary fibrosis by inhibiting the NLRP3 inflammasome and NF-κB pathway after MP infection.

Designing of an epitope-based peptide vaccine against walking pneumonia: an immunoinformatics approach.

Mycoplasma pneumoniae is a substantial respiratory pathogen that develops not only pneumonia but also other respiratory diseases, which mimic viral respiratory syndromes. Nevertheless, vaccine development for this pathogen delays behind as immunity correlated with protection is now predominantly unknown. In the present study, an immunoinformatics pipeline is utilized for epitope-based peptide vaccine design, which can trigger a critical immune response against M. pneumoniae. A total of 105 T-cell epitopes from 12 membrane associated proteins and 7 T-cell epitopes from 5 cytadherence proteins of M. pneumoniae were obtained and validated. Thus, 18 peptides with 9-mer core sequence were identified as best T-cell epitopes by considering the number of residues with > 75% in favored region. Further, the crucial screening studies predicted three peptides with good binding affinity towards HLA molecules as best T-cell and B-cell epitopes. Based on this result, visualization, and dynamic simulation for the three epitopes (WIHGLILLF, VILLFLLLF, and LLAWMLVLF) were assessed. The predicted epitopes needs to be further validated for their adept use as vaccine. Collectively, the study opens up a new horizon with extensive therapeutic application against M. pneumoniae and its associated diseases.

Dietary Supplementation with Low-Molecular-Weight Fucoidan Enhances Innate and Adaptive Immune Responses and Protects against Mycoplasma pneumoniae Antigen Stimulation.

In this study, the low-molecular-weight (LMW) fucoidan, rich in fucose and sulfate, was extracted and purified from the edible brown seaweed, Laminaria japonica. In this study, we orally administered LMW fucoidan to mice for 6 weeks. We then examined fucoidan's effects on innate immunity, adaptive immunity, and Mycoplasma pneumoniae (MP)-antigen-stimulated immune responses. Our data showed that LMW fucoidan stimulated the innate immune system by increasing splenocyte proliferation, natural killer (NK) cell activity, and phagocytic activity. LMW fucoidan also increased interleukin (IL)-2, IL-4, and interferon (IFN)-γ secretion by splenocytes and immunoglobulin (Ig)-G and IgA content in serum, which help regulate adaptive immune cell functions, and decreased allergen-specific IgE. In MP-antigen-stimulated immune responses, the IgM and IgG content in the serum were significantly higher in the LMW fucoidan group after MP-antigen stimulation. Our study provides further information about the immunomodulatory effects of LMW fucoidan and highlights a potential role in preventing M. pneumoniae infection.

Investigations of Mycoplasma pneumoniae infections in the United States: trends in molecular typing and macrolide resistance from 2006 to 2013.

Mycoplasma pneumoniae is a leading cause of respiratory infections, including community-acquired pneumonia (CAP). Currently, pathogen-specific testing is not routinely performed in the primary care setting, and the United States lacks a systematic surveillance program for M. pneumoniae. Documentation of individual cases and clusters typically occurs only when severe illness and/or failure to improve with empirical antibiotic therapy is observed. Outbreaks, some lasting for extended periods and involving a large number of cases, occur regularly. However, many more likely go unrecognized due to the lack of diagnostic testing and structured reporting. We reviewed data from 17 investigations of cases, small clusters, and outbreaks of M. pneumoniae infections that were supported by the Centers for Disease Control and Prevention (CDC) between 2006 and 2013. We examined 199 M. pneumoniae-positive specimens collected during this time period in order to identify trends in antimicrobial resistance and circulating types. Overall, macrolide resistance was identified in approximately 10% of M. pneumoniae infections occurring during this time period. Typing of strains revealed cocirculation of multiple multilocus variable-number tandem-repeat analysis (MLVA) and P1 types throughout this period, including diversity in types detected within individual outbreaks. Three MLVA types (4572, 3562, and 3662) accounted for 97% of the infections during the study period. A systematic surveillance program is necessary to understand the burden of M. pneumoniae disease in the United States, facilitate case and outbreak identification, and inform appropriate therapeutic and infection control strategies.

Challenges of pastoral cattle production in a sub-humid zone of Nigeria.

PURPOSE: More than 80 % of the 20 million heads of Nigerian cattle are kept by pastoral households. As such, optimal herd management is important in maintaining human nutrition, livelihoods and socio-cultural balance. This study was conducted to contribute to discussions on emerging challenges of the Nigerian livestock sector and to estimate herd prevalence, relative incidence, case fatality and impact on livelihood of cattle diseases in pastoral areas. METHODS: Participatory epidemiological approaches: listing; pairwise ranking; proportional piling; matrix scoring and probing were used to collect data through focus group interviews with Fulani herdsmen from selected pastoral areas of the country. RESULTS: The main cattle production problems were as follows: conversion of land used for cattle routes into crop fields, cattle rustling and water scarcity with median scores for impact on livelihood being 19, 17 and 16%, respectively. Animal diseases were fourth in the list of problems, and diseases reported to have significant impacts on livelihood were trypanosomiasis (25%), contagious bovine pleuropneumonia (15%), foot and mouth disease (13%) and fascioliasis (13%). High relative incidence rates were reported for trypanosomiasis (27%), fascioliasis (24%) and foot and mouth disease (19%). CONCLUSION: Change of land use and rustling indicate weaknesses in the producers' institutional environments. Water scarcity, limited access to veterinary services and substandard drugs supplied by vendors were identified as key factors contributing to persistence and frequent outbreaks of diseases. The paper revealed a greater importance of land constraints and rustling relative to disease and highlighted policy issues on management of natural resources and livestock development given challenges associated with pastoralism and insecurity in Nigeria.

Design of a novel multiepitope vaccine with CTLA-4 extracellular domain against Mycoplasma pneumoniae: A vaccine-immunoinformatics approach.

BACKGROUND: Community-acquired pneumonia often stems from the macrolide-resistant strain of Mycoplasma pneumoniae, yet no effective vaccine exists against it. METHODS: This study proposes a vaccine-immunoinformatics strategy for Mycoplasma pneumoniae and other pathogenic microbes. Specifically, dominant B and T cell epitopes of the Mycoplasma pneumoniae P30 adhesion protein were identified through immunoinformatics method. The vaccine sequence was then constructed by coupling with CTLA-4 extracellular region, a novel molecular adjuvant for antigen-presenting cells. Subsequently, the vaccine's physicochemical properties, antigenicity, and allergenicity were verified. Molecular dynamics modeling was employed to confirm interaction with TLR-2, TLR-4, B7-1, and B7-2. Finally, the vaccine underwent in silico cloning for expression. RESULTS: The vaccine exhibited both antigenicity and non-allergenicity. Molecular dynamics simulation, post-docking with TLR-2, TLR-4, B7-1, and B7-2, demonstrated stable interaction between the vaccine and these molecules. In silico cloning confirmed effective expression of the vaccine gene in insect baculovirus vectors. CONCLUSION: This vaccine-immunoinformatics approach holds promise for the development of vaccines against Mycoplasma pneumoniae and other pathogenic non-viral and non-bacterial microbes.

Breed-linked polymorphisms of porcine toll-like receptor 2 (TLR2) and TLR4 and the primary investigation on their relationship with prevention against Mycoplasma pneumoniae and bacterial LPS challenge.

Toll-like receptors (TLRs) play a crucial role in innate immunity, serving as pattern-recognition receptors and the first barrier in host defense against microbial infections. Genetic variations of TLR2 and TLR4 are closely associated with a variety of infectious diseases, particularly lung diseases. In this study, we detected six and four single nucleotide polymorphisms (SNPs) in the coding sequences of porcine TLR2 and TLR4 genes, respectively. Only SNP 1027C>A of TLR4 was shown to be markedly biased in Western and Oriental pig populations. Hence, the susceptibility of pigs with different genotype at position 1027C>A to Mycoplasma hyopneumoniae (Mhp) infection was investigated, and changes to the expression of TLR2, TLR4, TNF-α and IL-1ß were monitored. The results showed that there was no significant difference in susceptibility to Mhp infection between AA and CC individuals despite expression levels for all detected genes of the challenge groups being significantly higher than the corresponding control groups. Furthermore, porcine alveolar macrophages of different genotype were collected and stimulated by lipopolysaccharide. We found that the expression of TLR2, TLR4, TNF-α and IL-1ß genes were enhanced to different levels by lipopolysaccharide stimulation. TLR2 and TLR4 gene expressions and their rates of increase of 1027CC pigs were significantly higher than for 1027AC pigs (P < 0.01), while TNF-α and IL-1ß expressions were significantly lower than for 1027AC pigs (P < 0.01). We predict that allele C at position 1027 of the TLR4 gene contributes to the pig's immune response to gram-negative bacterial infections.

Development of protective anti-Mycoplasma pneumoniae antibodies after immunization of guinea pigs with the combination of a P1-P30 chimeric recombinant protein and chitosan.

The attachment organelle of the human respiratory tract pathogen Mycoplasma pneumoniae is essential for colonization of the host mucosa. Furthermore, adherence-related proteins such as the major adhesin P1 and protein P30 represent vaccine candidates. Using the chimeric recombinant protein HP14/30, which combines surface-localized and adherence-involved regions of both proteins, we developed an optimized strategy to immunize guinea pigs. The vaccination protocol includes subcutaneous prime immunization followed by presentation of the antigen directly to the respiratory mucosa by two intranasal (i.n.) administrations and combination of antigen with the mucosal adjuvant chitosan. The immunization scheme induced high, consistent and long-lasting IgA levels in respiratory tract samples (BAL, nasal and throat washing fluid) from the animals. In comparison with a preimmune serum, incubation of M. pneumoniae cells with sera from these animals reduced the mean adhesion of bacteria to HeLa cells to 6%. After i.n. infection, immunized animals showed significantly decreased numbers of M. pneumoniae-specific genome copies, especially in the upper respiratory tract, in comparison with the control group. The results demonstrated that optimized immunization with the chimeric protein HP14/30 is promising for further vaccination efforts to prevent host colonization with M. pneumoniae.

Vaccination of BALB/c mice with an avirulent Mycoplasma pneumoniae P30 mutant results in disease exacerbation upon challenge with a virulent strain.

Mycoplasma pneumoniae is a significant human respiratory pathogen that causes high morbidity worldwide. No vaccine to prevent M. pneumoniae infection currently exists, since the mechanisms of pathogenesis are poorly understood. To this end, we constructed a P30 cytadhesin mutant (P-130) with a drastically reduced capacity for binding to erythrocytes and an inability to glide on glass substrates. This mutant was determined to be avirulent and cannot survive in the lungs of BALB/c mice. We also ascertained that the previously identified P30 gliding motility mutant II-3R is avirulent and also cannot be recovered from the lungs of mice after infection. Mutant P130 was then assessed for its efficacy as a live attenuated vaccine candidate in mice after challenge with wild-type M. pneumoniae. After vaccination with the P-130 P30 mutant, mice showed evidence of exacerbated disease upon subsequent challenge with the wild-type strain PI1428, which appears to be driven by a Th17 response and corresponding eosinophilia. Our results are in accordance with other reports of vaccine-induced disease exacerbation in rodents and emphasize the need to better understand the basic mechanisms of M. pneumoniae pathogenesis.

Mycoplasma pneumoniae respiratory illness - two rural counties, West Virginia, 2011.

On October 28, 2011, the West Virginia Department of Health and Human Resources notified CDC of an increase in pneumonia cases among school-aged children in two rural counties. Mycoplasma pneumoniae was the suspected cause, based on epidemiology, clinical presentation, and testing of specimens sent to CDC. Three of six nasopharyngeal swabs were positive for M. pneumoniae in testing by quantitative real-time polymerase chain reaction (qPCR). The West Virginia Department of Health and Human Resources and CDC conducted an outbreak investigation to confirm the etiology of the outbreak, establish active case surveillance, and provide recommendations for treatment and containment. The investigation confirmed M. pneumoniae as the cause and identified 125 cases, including two caused by macrolide-resistant isolates. The outbreak was contained with public health interventions that included communicating to the public the importance of respiratory hygiene, providing hand sanitizer in schools, and informing health-care providers about macrolide resistance; antibiotic prophylaxis was not used. Despite the large number of cases and macrolide-resistant strains, no severe extrapulmonary manifestations (e.g., erythema multiforme) were reported.

Protective immune responses in mice induced by intramuscular and intranasal immunization with a Mycoplasma pneumoniae P1C DNA vaccine.

Mycoplasma pneumoniae is an important causative agent of atypical pneumonia. This study was to determine the ability of a DNA expression vector, which encodes the carboxy terminal region of the M. pneumoniae P1 protein (P1C), to induce humoral and cellular immune responses and to protect against M. pneumoniae infection in BALB/c mice. Mice were immunized with pcDNA3.1/P1C by either intramuscular injection (i.m.) or intranasal inoculation (i.n.). Our results showed that p1c DNA immunization generates detectable antibodies specific to M. pneumoniae, and elicits high levels of IgG1, IgG2a, and IgG2b isotypes (P < 0.01). The levels of IFN-γ and IL-4 in spleen cells of the immunized mice were significantly elevated by immunization via both the i.m. and i.n. methods. Moreover, p1c DNA-immunized mice exhibited detectable protection against M. pneumoniae infection. The lung tissue inflammation was relieved and the histopathologic score (HPS) of pcDNA3.1/P1C-immunized mice was significantly decreased than those in phosphate-buffed saline (PBS) or vaccine-vector-immunized mice (P < 0.01), whereas there were no significant differences in HPS between i.m. and i.n. vaccination (P > 0.05). Our results suggest that pcDNA3.1/P1C could be useful for developing a vaccine against M. pneumoniae infection.

Protective efficacy of a Mycoplasma pneumoniae P1C DNA vaccine fused with the B subunit of Escherichia coli heat-labile enterotoxin.

In the present study, we investigated the immunomodulatory responses of a DNA vaccine constructed by fusing Mycoplasma pneumoniae P1 protein carboxy terminal region (P1C) with the Escherichia coli heat-labile toxin B subunit (LTB). BALB/c mice were immunized by intranasal inoculation with control DNAs, the P1C DNA vaccine or the LTB-P1C fusion DNA vaccine. Levels of the anti-M. pneumoniae antibodies and levels of interferon-γ and IL-4 in mice were increased significantly upon inoculation of the LTB-P1C fusion DNA vaccine when compared with the inoculation with P1C DNA vaccine. The LTB-P1C fusion DNA vaccine efficiently enhanced the M. pneumoniae-specific IgA and IgG levels. The IgG2a/IgG1 ratio was significantly higher in bronchoalveolar lavages fluid and sera from mice fusion with LTB and P1C than mice receiving P1C alone. When the mice were challenged intranasally with 10(7) CFU M. pneumoniae strain (M129), the LTB-P1C fusion DNA vaccine conferred significantly better protection than P1C DNA vaccine (P < 0.05), as suggested by the results, such as less inflammation, lower histopathological score values, lower detectable number of M. pneumoniae strain, and lower mortality of challenging from 5 × 10(8) CFU M. pneumoniae. These results indicated that the LTB-P1C fusion DNA vaccine efficiently improved protective efficacy against M. pneumoniae infection and effectively attenuated development of M. pneumoniae in mice.

Rational design of multimeric based subunit vaccine against Mycoplasma pneumonia: Subtractive proteomics with immunoinformatics framework.

Mycoplasma pneumoniae is the prevalent cause of acquired respiratory infections around the globe. A multi-epitope vaccine (MEV) must be developed to combat infections of M. pneumoniae because there is no specific disease-modifying treatment or vaccination is present. The objective of this research is to design a vaccine that targets M. pneumoniae top five highly antigenic proteins using a combination of immunological techniques and molecular docking. T-cell (HTL & CTL), B-cell, and IFN-γ of target proteins were forecasted and highly conservative epitopes were chosen for further study. For designing of final vaccine, 4LBL, 7CTL, and 5HTL epitopes were joined by linkers of KK, AAY, and GPGPG. The N-end of the vaccine was linked to an adjuvant (Cholera enterotoxin subunit B) with a linker named EAAAK to enhance immunogenicity. After the addition of adjuvants and linkers, the size of the construct was 395 amino acids. The epitopes of IFN-γ and B-cells illustrate that the model construct is optimized for cell-mediated immune or humoral responses. To ensure that the final design is safer and immunogenic, properties like non-allergens, antigenicity, and various physicochemical properties were evaluated. Molecular docking of the vaccine with the toll-like receptor 4 (TLR4) was conducted to check the compatibility of the vaccine with the receptor. Besides, in-silico cloning was utilized for validation of the credibility and proper expression of the vaccine. Furthermore, to confirm that the multi-epitope vaccine created is protective and immunogenic, this research requires experimental validation.

T-B cell epitope peptides induce protective immunity against Mycoplasma pneumoniae respiratory tract infection in BALB/c mice.

Mycoplasma pneumoniae is the most common pathogen of community-acquired pneumonia in humans. Due to its high rates of antibiotic resistance, vaccination has become the best method to control the dissemination of M. pneumoniae. The recombinant carboxyl terminus of the P1 (P1C) protein is an immunodominant antigen, but it has negative effects such as poor stability and lower purity. In the current study, T-B epitopes of the P1C protein were predicted according to bioinformatics analysis and assessed for efficacy in peptide vaccination. BALB/c mice were subcutaneously inoculated with the T-B epitope peptides four times and then infected with M. pneumoniae through the respiratory tract. The results showed that the T-B epitope peptides of the P1C protein (P1C103-117, P1C155-169, P1C224-238 and P1C244-258) induced strong antigen-specific serum antibody responses and cellular immune responses with high levels of serum IgG, IgA antibodies and Th1-biased (IFN-γ and IL-2) cytokines. Immunization with T-B epitope peptides significantly reduced the M. pneumoniae burden and the degree of inflammation in the challenged mice. Furthermore, the levels of IFN-γ and TNF-α in the supernatants of lung homogenates were observably reduced compared to those in the PBS group. Overall, our findings demonstrate that T-B epitopes (P1C103-117, P1C155-169, P1C224-238 and P1C244-258) play significant roles in the P1C protein and can be used to induce powerful humoral and cellular immune responses to provide significant protection against M. pneumoniae pulmonary infection, which provides new insight into the design of potential multiepitope vaccines to prevent host infection by M. pneumoniae.

Vaccination using inactivated Mycoplasma pneumoniae induces detrimental infiltration of neutrophils after subsequent infection in mice.

Mycoplasma pneumoniae (Mp) is one of the most common causes of community-acquired pneumonia. Given the emergence and high rates of antibiotic-resistant Mp strains, vaccines that prevent the pneumonia and secondary complications due to Mp infection are urgently needed. Although several studies have shown the protective efficacy of Mp vaccines in human clinical trials, some reports suggest that vaccination against Mp exacerbates disease upon subsequent Mp challenge. Therefore, to develop optimal vaccines against Mp, understanding the immune responses that contribute to post-vaccination exacerbation of inflammation is crucial. Here we examined whether Mp vaccination might exacerbate pneumonia after subsequent Mp infection in mice. We found that vaccination with inactivated Mp plus aluminum salts as an adjuvant induced Mp-specific IgG, Th1 cells, and Th17 cells. Toll-like receptor 2 signaling contributed to the induction of an Mp-specific IgG response and was necessary for Mp-specific Th17-cell-but not Th1-cell-responses in vaccinated mice. In addition, vaccination with inactivated Mp plus aluminum salts suppressed the number of Mp organisms in the bronchoalveolar lavage fluid, indicating that vaccination can reduce Mp infection. However, the numbers of total immune cells and neutrophils in bronchoalveolar lavage fluid after Mp challenge did not differ between vaccinated mice and non-vaccinated control mice. Furthermore, depletion of CD4+ T cells prior to Mp challenge decreased pulmonary neutrophil infiltration in vaccinated mice, suggesting that Th1 or Th17 cells (or both) are responsible for the vaccination-induced neutrophil infiltration. These results suggest that, despite reducing Mp infection, vaccination of mice by using inactivated Mp fails to suppress inflammation, such as neutrophil infiltration into the lung, after subsequent Mp infection.

Strategy to create chimeric proteins derived from functional adhesin regions of Mycoplasma pneumoniae for vaccine development.

The cell wall-less bacterium Mycoplasma pneumoniae is one of the most common agents of respiratory tract diseases in humans. Adhesin-mediated binding of the bacteria to host cells is a crucial step in colonization and subsequent pathogenesis. For the first time, we expressed 16 recombinant proteins covering almost the whole major adhesin P1 and the adherence-associated protein P30 to characterize these proteins immunologically and functionally. We describe a new in vitro assay using several human cell lines in combination with fluorescence-activated cell sorting analysis to screen antisera raised against the recombinant proteins quantitatively for adherence inhibition activity. The protein derived from the nearly C-terminal part of the P1 adhesin (amino acids [aa] 1288 to 1518) and the protein P30 (aa 17 to 274) especially showed prominent immunoreactivity with sera from M. pneumoniae-immunized guinea pigs as well as with M. pneumoniae-positive patient sera. We demonstrate that the same protein regions are involved in mediating cytadherence since antibodies against these adhesin regions decrease mycoplasma adhesion to human cells significantly. For further vaccine studies, we optimized the immunogenic and adherence-mediating properties of the antigen by combining both the P1 and the P30 regions in a novel chimeric protein. Antibodies against this protein show an increased reduction of M. pneumoniae adherence to human bronchial epithelial cells by 95%, which is comparable to results with polyspecific anti-M. pneumoniae animal serum. Our strategy results in a promising defined antigen candidate for reducing or even preventing M. pneumoniae colonization of the respiratory tract in future vaccination studies.