Putative novel B-cell vaccine candidates identified by reverse vaccinology and genomics approaches to control Acinetobacter baumannii serotypes.

In the last decade, Multi-drug resistance (MDR)-associated infections of Acinetobacter baumannii have grown worldwide. A cost-effective preventative strategy against this bacterium is vaccination. This study has presented five novel vaccine candidates against A. baumannii produced using the reverse vaccinology method. BLASTn was done to identify the most conserved antigens. PSORTb 3.0.2 was run to predict the subcellular localization of the proteins. The initial screening and antigenicity evaluation were performed using Vaxign. The ccSOL omics was also employed to predict protein solubility. The cross-membrane localization of the protein was predicted using PRED-TMBB. B cell epitope prediction was made for immunogenicity using the IEDB and BepiPred-2.0 database. Eventually, BLASTp was done to verify the extent of similarity to the human proteome to exclude the possibility of autoimmunity. Proteins failing to comply with the set parameters were filtered at each step. In silico, potential vaccines against 21 A. baumannii strains were identified using reverse vaccinology and subtractive genomic techniques. Based on the above criteria, out of the initial 15 A. baumannii proteins selected for screening, nine exposed/secreted/membrane proteins, i.e., Pfsr, LptE, OmpH, CarO, CsuB, CdiB, MlaA, FhuE, and were the most promising candidates. Their solubility and antigenicity were also examined and found to be more than 0.45 and 0.6, respectively. Based on the results, LptE was selected with the highest average antigenic score of 1.043 as the best protein, followed by FimF and Pfsr with scores of 1.022 and 1.014, respectively. In the end, five proteins were verified as promising candidates. Overall, the targets identified herein may be utilized in future strategies to control A. baumannii worldwide.

Production and characterization of novel monoclonal antibodies against outer membrane protein A (OmpA) of Acinetobacter baumannii.

BACKGROUND: Infection caused by Acinetobacter baumannii has emerged as a significant clinical problem with unacceptably high mortality rate due to the increase in antibiotic-resistant strains. Producing novel monoclonal antibody (MAb) against outer membrane protein A (OmpA) could be considered as a potential tool to improve treatment of A. baumannii infections. OBJECTIVES: In this study, we aimed to produce murine MAbs against OmpA peptide of A. baumannii. MATERIALS AND METHODS: BALB/c mice were immunized with 18-mer amino acid peptide as a part of the OmpA protein. Four antibody-secreting hybridomas were obtained using hybridoma technology and then characterized according to isotypes, affinity constant, reactivity in ELISA, flow cytometry, indirect immunofluorescence (IFA) and opsonophagocytic killing assays. RESULTS: All four produced MAbs (1A1-D10, 1G1-E7, 2C11-F10, and 4H2-H9) had IgG1 isotype with Kappa light chain. One of these MAbs, 1G1-E7 was purified and selected for further characterizations. 1G1-E7 showed a high reactivity with both immunogenic peptide and A. baumannii in ELISA. Our results indicated that 1G1-E7 MAb reacted with 95.3% of A. baumannii in flow cytometry as well as IFA. Moreover, the affinity of the 1G1-E7 MAb was measured 1.37 × 108 M-1. The 1G1-E7 significantly improved opsonophagocytic killing of a clinical isolate of A. baumannii. CONCLUSION: Our findings showed that the OmpA can be identified by produced MAbs. The efficacy of novel anti-OmpA antibodies in A. baumannii targeting needs to be further investigated in challenging models, and then could be subjected for genetic engineering to produce therapeutic antibody against A. baumannii.

Physicochemical and structural characterization, epitope mapping and vaccine potential investigation of a new protein containing Tetratrico Peptide Repeats of Acinetobacter baumannii: An in-silico and in-vivo approach.

Acinetobacter baumannii is an opportunistic multidrug-resistant pathogen that causes a significant mortality rate. The proteins containing Tetratrico Peptide Repeats (TPRs) are involved in the pathogenicity and virulence of bacteria and have different roles such as transfer of bacterial virulence factors to host cells, binding to the host cells and inhibition of phagolysosomal maturation. So, in this study, physicochemical properties of a new protein containing TPRs in A. baumannii which was named PcTPRs1 by this study were characterized and its 3D structure was predicted by in-silico tools. The protein B and T cell epitopes were mapped and its vaccine potential was in-silico and in-vivo investigated. Domain analysis indicated that the protein contains the Flp pilus assembly protein TadD domain which has three TPRs. The helix is dominant in the protein structure, and this protein is an outer membrane antigen which, is extremely conserved among A. baumannii strains; thus, has good properties to be applied as a recombinant vaccine. The best-predicted and refined model was applied in ligand-binding sites and conformational epitopes prediction. Based on epitope mapping results, several epitopes were characterized which could stimulate both immune systems. BLAST results showed the introduced epitopes are completely conserved among A. baumannii strains. The in-vivo analysis indicates that a 101 amino acid fragment of the protein which contains the best selected epitope, can produce a good protectivity against A. baumannii as well as the whole TPR protein and thus could be investigated as an effective subunit and potential vaccines.

Evaluationof the New Outer Membrane Protein A Epitope-based Vaccines for Mice Model of Acinetobacter baumannii Associated Pneumonia and Sepsis Infection.

Nosocomial infections caused by Acinetobacter baumannii (A. baumannii) nosocomial infections caused by Acinetobacter baumannii (A. baumannii) are considered as a global serious problem in hospitalized patients because of emerging antibiotic resistance. Immunotherapy approaches are promising to prevent such infections. In our previous study, five antigenic epitopes of outer membrane protein A (OmpA), as the most dangerous virulence molecule in A. baumanii, were predicted in silico. In this study, the investigators evaluated some immunological aspects of the peptides. Five peptides were separately injected into C5BL/6 mice; then the cytokine production (interleukin-4 and interferon-gamma) of splenocytes and opsonophagocytic activity of immunized serum were assessed. To identify the protective function of the peptides, animal models of sepsis and pneumonia infections were actively and passively immunized with selected peptides and pooled sera of immunized mice, respectively. Then, survival rates of them were compared with the non-infected controls. Based on the results, activated spleen cells in P127 peptide-immunized mice exhibited an increase level of IFN-γ compared with the other experimental groups, but not about the IL-4 concentration. The results of opsonophagocytic assay revealed an appropriate killing activity of produced antibodies against A. baumannii in a dose-dependent manner. Further, the survival rates of the mice under passive immunization with the immunized sera or active immunization with P127 peptide were significantly more than those in the control group. Moreover, the survival rate of the P127 peptide immunized group was considerably higher than that among the other peptide-immunized group. In conclusion, findings indicated that peptides derived from outer membrane protein-A can be used as a promising tool for designing the epitope-based vaccines against infections caused by A. baumannii.

Vaccination induces rapid protection against bacterial pneumonia via training alveolar macrophage in mice.

Vaccination strategies for rapid protection against multidrug-resistant bacterial infection are very important, especially for hospitalized patients who have high risk of exposure to these bacteria. However, few such vaccination strategies exist due to a shortage of knowledge supporting their rapid effect. Here, we demonstrated that a single intranasal immunization of inactivated whole cell of Acinetobacter baumannii elicits rapid protection against broad A. baumannii-infected pneumonia via training of innate immune response in Rag1-/- mice. Immunization-trained alveolar macrophages (AMs) showed enhanced TNF-α production upon restimulation. Adoptive transfer of immunization-trained AMs into naive mice mediated rapid protection against infection. Elevated TLR4 expression on vaccination-trained AMs contributed to rapid protection. Moreover, immunization-induced rapid protection was also seen in Pseudomonas aeruginosa and Klebsiella pneumoniae pneumonia models, but not in Staphylococcus aureus and Streptococcus pneumoniae model. Our data reveal that a single intranasal immunization induces rapid and efficient protection against certain Gram-negative bacterial pneumonia via training AMs response, which highlights the importance and the possibility of harnessing trained immunity of AMs to design rapid-effecting vaccine.

Formylglycine-Generating Enzyme-Like Proteins Constitute a Novel Family of Widespread Type VI Secretion System Immunity Proteins.

Competition is a critical aspect of bacterial life, as it enables niche establishment and facilitates the acquisition of essential nutrients. Warfare between Gram-negative bacteria is largely mediated by the type VI secretion system (T6SS), a dynamic nanoweapon that delivers toxic effector proteins from an attacking cell to adjacent bacteria in a contact-dependent manner. Effector-encoding bacteria prevent self-intoxication and kin cell killing by the expression of immunity proteins, which neutralize effector toxicity by specifically binding their cognate effector and either occluding its active site or preventing the structural rearrangements necessary for effector activation. In this study, we investigate Tsi3, a previously uncharacterized T6SS immunity protein present in multiple strains of the human pathogen Acinetobacter baumannii. We show that Tsi3 is the cognate immunity protein of an antibacterial effector of unknown function, Tse3. Our bioinformatic analyses indicate that Tsi3 homologs are widespread among Gram-negative bacteria, often encoded within T6SS effector-immunity modules. Surprisingly, we found that Tsi3 homologs are predicted to possess a characteristic formylglycine-generating enzyme (FGE) domain, which is present in various enzymatic proteins. Our data show that Tsi3-mediated immunity is dependent on Tse3-Tsi3 protein-protein interactions and that Tsi3 homologs from various bacteria do not provide immunity against nonkin Tse3. Thus, we conclude that Tsi3 homologs are unlikely to be functional enzymes. Collectively, our work identifies FGE domain-containing proteins as important mediators of immunity against T6SS attacks and indicates that the FGE domain can be coopted as a scaffold in multiple proteins to carry out diverse functions. IMPORTANCE Despite the wealth of knowledge on the diversity of biochemical activities carried out by T6SS effectors, comparably little is known about the various strategies that bacteria employ to prevent susceptibility to T6SS-dependent bacterial killing. Our work establishes a novel family of T6SS immunity proteins with a characteristic FGE domain. This domain is present in enzymatic proteins with various catalytic activities. Our characterization of Tsi3 expands the known functions carried out by FGE-like proteins to include defense during T6SS-mediated bacterial warfare. Moreover, it highlights the evolution of FGE domain-containing proteins to carry out diverse biological functions.

Sequential Vaccination With Heterologous Acinetobacter baumannii Strains Induces Broadly Reactive Antibody Responses.

Antibody therapy may be an alternative treatment option for infections caused by the multi-drug resistant (MDR) bacterium Acinetobacter baumannii. As A. baumannii has multiple capsular serotypes, a universal antibody therapy would need to target conserved protein antigens rather than the capsular polysaccharides. We have immunized mice with single or multiple A. baumannii strains to induce antibody responses to protein antigens, and then assessed whether these responses provide cross-protection against a collection of genetically diverse clinical A. baumannii isolates. Immunized mice developed antibody responses to multiple protein antigens. Flow cytometry IgG binding assays and immunoblots demonstrated improved recognition of both homologous and heterologous clinical strains in sera from mice immunized with multiple strains compared to a single strain. The capsule partially inhibited bacterial recognition by IgG and the promotion of phagocytosis by human neutrophils. However, after immunization with multiple strains, serum antibodies to protein antigens promoted neutrophil phagocytosis of heterologous A. baumannii strains. In an infection model, mice immunized with multiple strains had lower bacterial counts in the spleen and liver following challenge with a heterologous strain. These data demonstrate that antibodies targeting protein antigens can improve immune recognition and protection against diverse A. baumannii strains, providing support for their use as an antibody therapy.

Monoclonal Antibody Therapy against Acinetobacter baumannii.

Extremely drug-resistant (XDR) Acinetobacter baumannii is a notorious and frequently encountered pathogen demanding novel therapeutic interventions. An initial monoclonal antibody (MAb), C8, raised against A. baumannii capsule, proved a highly effective treatment against a minority of clinical isolates. To overcome this limitation, we broadened coverage by developing a second antibody for use in a combination regimen. We sought to develop an additional anti-A. baumannii MAb through hybridoma technology by immunizing mice with sublethal inocula of virulent, XDR clinical isolates not bound by MAb C8. We identified a new antibacterial MAb, 65, which bound to strains in a pattern distinct from and complementary to that of MAb C8. MAb 65 enhanced macrophage opsonophagocytosis of targeted strains and markedly improved survival in lethal bacteremic sepsis and aspiration pneumonia murine models of A. baumannii infection. MAb 65 was also synergistic with colistin, substantially enhancing protection compared to monotherapy. Treatment with MAb 65 significantly reduced blood bacterial density, ameliorated cytokine production (interleukin-1ß [IL-1ß], IL-6, IL-10, and tumor necrosis factor), and sepsis biomarkers. We describe a novel MAb targeting A. baumannii that broadens immunotherapeutic strain coverage, is highly potent and effective, and synergistically improves outcomes in combination with antibiotics.

Chimeric vaccine designs against Acinetobacter baumannii using pan genome and reverse vaccinology approaches.

Acinetobacter baumannii (A. baumannii), an opportunistic, gram-negative pathogen, has evoked the interest of the medical community throughout the world because of its ability to cause nosocomial infections, majorly infecting those in intensive care units. It has also drawn the attention of researchers due to its evolving immune evasion strategies and increased drug resistance. The emergence of multi-drug-resistant-strains has urged the need to explore novel therapeutic options as an alternative to antibiotics. Due to the upsurge in antibiotic resistance mechanisms exhibited by A. baumannii, the current therapeutic strategies are rendered less effective. The aim of this study is to explore novel therapeutic alternatives against A. baumannii to control the ailed infection. In this study, a computational framework is employed involving, pan genomics, subtractive proteomics and reverse vaccinology strategies to identify core promiscuous vaccine candidates. Two chimeric vaccine constructs having B-cell derived T-cell epitopes from prioritized vaccine candidates; APN, AdeK and AdeI have been designed and checked for their possible interactions with host BCR, TLRs and HLA Class I and II Superfamily alleles. These vaccine candidates can be experimentally validated and thus contribute to vaccine development against A. baumannii infections.

Designing Multi-Antigen Vaccines Against Acinetobacter baumannii Using Systemic Approaches.

Vaccines and monoclonal antibodies are promising approaches for preventing and treating infections caused by multidrug resistant Acinetobacter baumannii. However, only partial protection has been achieved with many previously tested protein antigens, which suggests that vaccines incorporating multiple antigens may be necessary in order to obtain high levels of protection. Several aspects that use the wealth of omic data available for A. baumannii have not been fully exploited for antigen identification. In this study, the use of fractionated proteomic and computational data from ~4,200 genomes increased the number of proteins potentially accessible to the humoral response to 8,824 non-redundant proteins in the A. baumannii panproteome. Among them, 59% carried predicted B-cell epitopes and T-cell epitopes recognized by two or more alleles of the HLA class II DP supertype. Potential cross-reactivity with human proteins was detected for 8.9% of antigens at the protein level and 2.7% at the B-cell epitope level. Individual antigens were associated with different infection types by genomic, transcriptomic or functional analyses. High intra-clonal genome density permitted the identification of international clone II as a "vaccitype", in which 20% of identified antigens were specific to this clone. Network-based centrality measurements were used to identify multiple immunologic nodes. Data were formatted, unified and stored in a data warehouse database, which was subsequently used to identify synergistic antigen combinations for different vaccination strategies. This study supports the idea that integration of multi-omic data and fundamental knowledge of the pathobiology of drug-resistant bacteria can facilitate the development of effective multi-antigen vaccines against these challenging infections.

Monoclonal Antibody Requires Immunomodulation for Efficacy Against Acinetobacter baumannii Infection.

Monoclonal antibodies (mAbs) are gaining significant momentum as novel therapeutics for infections caused by antibiotic-resistant bacteria. We evaluated the mechanism by which antibacterial mAb therapy protects against Acinetobacter baumannii infections. Anticapsular mAb enhanced macrophage opsonophagocytosis and rescued mice from lethal infections by harnessing complement, macrophages, and neutrophils; however, the degree of bacterial burden did not correlate with survival. Furthermore, mAb therapy reduced proinflammatory (interleukin-1ß [IL-1ß], IL-6, tumor necrosis factor-α [TNF-α]) and anti-inflammatory (IL-10) cytokines, which correlated inversely with survival. Although disrupting IL-10 abrogated the survival advantage conferred by the mAb, IL-10-knockout mice treated with mAb could still survive if TNF-α production was suppressed directly (via anti-TNF-α neutralizing antibody) or indirectly (via macrophage depletion). Thus, even for a mAb that enhances microbial clearance via opsonophagocytosis, clinical efficacy required modulation of pro- and anti-inflammatory cytokines. These findings may inform future mAb development targeting bacteria that trigger the sepsis cascade.

Immunoprotectivity of Valine-glycine repeat protein G, a potent mediator of pathogenicity, against Acinetobacter baumannii.

Type VI Secretion System (T6SS) contributes to both virulence and antimicrobial resistance in Acinetobacter baumannii. Valine-glycine repeat protein G (VgrG) is the core component of T6SS that exists in many bacterial pathogens that have emerged as a potent mediator of pathogenicity in A. baumannii. Two conserved sequences of vgrG 1263-2295 and vgrG1263-1608 were identified antigenic in various strains of Acinetobacter baumannii. The vgrg1263-1608 sequence was implanted in the Loopless C lobe (LCL) from N. meningitidis for surface display and exposure to functional epitopes. The VgrG and LCL-VgrG were expressed and purified. Groups of BALB/c mice were immunized with these proteins and challenged with A. baumannii. Specific IgG titers, whole-cell ELISA, animal survival rates in active and passive immunizations, the bacterial burden in mice tissues, and cytotoxicity of the proteins were determined. The specific IgG suppressed bacterial burdens in the organs, and increased survival rates were noted in the immunized mice. LCL-VgrG immunization provided better protection against A. baumannii infection than the VgrG immunization. The conserved region of VgrG is probably a safe immunogen to effective vaccine development or an antiserum to control A. baumannii infections.

A Multiepitope Peptide, rOmp22, Encapsulated in Chitosan-PLGA Nanoparticles as a Candidate Vaccine Against Acinetobacter baumannii Infection.

BACKGROUND: The development of vaccines is a promising and cost-effective strategy to prevent emerging multidrug-resistant (MDR) Acinetobacter baumannii (A. baumannii) infections. The purpose of this study was to prepare a multiepitope peptide nanovaccine and evaluate its immunogenicity and protective effect in BALB/c mice. METHODS: The B-cell and T-cell epitopes of Omp22 from A. baumannii were predicted using bioinformatics methods and identified by immunological experiments. The optimal epitopes were conjugated in series by 6-aminocaproic acid and chemically synthesized multiepitope polypeptide rOmp22. Then, rOmp22 was encapsulated by chitosan (CS) and poly (lactic-co-glycolic) acid (PLGA) to prepare CS-PLGA-rOmp22 nanoparticles (NPs). The immunogenicity and immunoprotective efficacy of the vaccine were evaluated in BALB/c mice. RESULTS: CS-PLGA-rOmp22 NPs were small (mean size of 272.83 nm) with apparently spherical structures, positively charged (4.39 mV) and nontoxic to A549 cells. A high encapsulation efficiency (54.94%) and a continuous slow release pattern were achieved. Compared with nonencapsulated rOmp22, CS-PLGA-rOmp22 immunized BALB/c mice induced higher levels of rOmp22-specific IgG in serum and IFN-γ in splenocyte supernatant. Additionally, lung injury and bacterial burdens in the lung and blood were suppressed, and potent protection (57.14%-83.3%) against acute lethal intratracheal A. baumannii challenge was observed in BALB/c mice vaccinated with CS-PLGA-rOmp22. CONCLUSION: CS-PLGA-rOmp22 NPs elicited specific IgG antibodies, Th1 cellular immunity and protection against acute lethal intratracheal A. baumannii challenge. Our results indicate that this nanovaccine is a desirable candidate for preventing A. baumannii infection.

Cross-reactivity and immunotherapeutic potential of BamA recombinant protein from Acinetobacter baumannii.

Acinetobacter baumannii is an important nosocomial pathogen. BamA is a protein that belongs to a complex responsible for organizing the proteins on the bacterial outer membrane. In this work, we aimed to evaluate murine immune responses to BamA recombinant protein (rAbBamA) from A. baumannii in an animal model of infection, and to assess cross-reactivity of this target for the development of anti-A. baumannii vaccines or diagnostics. Immunization of mice with rAbBamA elicited high antibody titers and antibody recognition of native A. baumannii BamA. Immunofluorescence also detected binding to the bacterial surface. After challenge, immunized mice demonstrated a 40% survival increase and better bacterial clearance in kidneys. Immunoblot of anti-rAbBamA against other medically relevant bacteria showed binding to proteins of approximately 35 kDa in Klebsiella pneumoniae and Escherichia coli lysates, primarily identified as OmpA and OmpC, respectively. Altogether, our data show that anti-rAbBamA antibodies provide a protective response against A. baumannii infection in mice. However, the response elicited by immunization with rAbBamA is not completely specific to A. baumannii. Although a broad-spectrum vaccine that protects against various pathogens is an appealing strategy, antibody reactivity against the human microbiota is undesired. In fact, immunization with rAbBamA produced noticeable effects on the gut microbiota. However, the changes elicited were small and non-specific, given that no significant changes in the abundance of Proteobacteria were observed. Overall, rAbBamA is a promising target, but specificity must be considered in the development of immunological tools against A. baumannii.

Where are we and how far is there to go in the development of an Acinetobacter vaccine?

INTRODUCTION: Healthcare-associated infections caused by multidrug-resistant Acinetobacter baumannii are becoming alarming worldwide. However, the pipeline of new antibiotics is very limited. Vaccination is one of the most cost effective and promising strategies to prevent infections and can play an important role in combat multidrug resistance A. baumannii and prevent the development of new drug resistance. AREA COVERED: This review gives an overview of the research and development of A. baumannii vaccines during the past five years (2015-2020), discusses the key progresses and current challenges of the field, and speculates on the future of A. baumannii vaccine development. EXPERT OPINION: Moderate progresses have been made in the research and development of A. baumannii vaccine in the last five years, in particular in the areas of identification of new protein targets, development of multicomponent vaccines, and use of vaccines and antibodies as adjuncts for antibiotics therapies. However, substantial scientific and logistic challenges, such as selection of lead vaccine candidates and formulation, vaccine clinical trials and targeted population, and financial incentives, remain. Thus, innovative strategies will be needed before an A. baumannii vaccine candidate can be brought into late stage of preclinical development in next five years.

Protective response against Acinetobacter baumannii with ferric iron receptors HemTR-BauA in a murine sepsis model.

Aim: Iron uptake and metabolism pathways are promising targets in vaccine development as an alternative strategy for antibiotics. Methods & methods: HemTR, a putative heme receptor of Acinetobacter baumannii, was expressed and its protectivity against A. baumannii was determined singly or in combination with the siderophore receptor, BauA, in mice. Results: High level of IgG was elicited. There was a delay in mice mortality with reduced bacterial loads in internal organs in the sublethal challenge. Protection was better in the HemTR-BauA group in both lethal and sublethal challenges. Passive transfer of anti-HemTR and anti-BauA partially protected mice against A. baumannii infection. Conclusion: HemTR in combination with other iron receptors could contribute to the development of protective vaccines against A. baumannii.

Specific egg yolk immunoglobulin as a promising non-antibiotic biotherapeutic product against Acinetobacter baumannii pneumonia infection.

Acinetobacter baumannii is a serious health threat with a high mortality rate. We have already reported prophylactic effects of IgYs raised against OmpA and Omp34 as well as against inactivated whole-cell (IWC) of A. baumannii in a murine pneumonia model. However, the infection was exacerbated in the mice group that received IgYs raised against the combination of OmpA and Omp34. The current study was conducted to propose reasons for the observed antibody-dependent enhancement (ADE) in addition to the therapeutic effect of specific IgYs in the murine pneumonia model. This phenomenon was hypothetically attributed to topologically inaccessible similar epitopes of OmpA and Omp34 sharing similarity with peptides of mice proteins. In silico analyses revealed that some inaccessible peptides of OmpA shared similarity with peptides of Omp34 and Mus musculus. Specific anti-OmpA and anti-Omp34 IgYs cross-reacted with Omp34 and OmpA respectively. Specific IgYs showed different protectivity against A. baumannii AbI101 in the murine pneumonia model. IgYs triggered against OmpA or IWC of A. baumannii were the most protective antibodies. IgY triggered against Omp34 is ranked next after those against OmpA. The lowest protection was observed in mice received IgYs raised against the combination of rOmpA and rOmp34. In conclusion, specific IgYs against OmpA, Omp34, and IWC of A. baumannii could serve as novel biotherapeutics against A. baumannii pneumonia.

Analysis of Immune Responses in Acinetobacter baumannii-Infected Klotho Knockout Mice: A Mouse Model of Acinetobacter baumannii Infection in Aged Hosts.

Acinetobacter baumannii is an important opportunistic pathogen that primarily afflicts elderly people. To clarify the pathogenicity of A. baumannii in the elderly, we investigated immune responses to A. baumannii ATCC 19606 infection in klotho knockout (KO) mice, the mouse model of aging. Following intravenous inoculation, the mice seldom displayed severe symptoms. However, the survival rate was 56% at 7 days post-infection. Bacteria were detected in the lungs of klotho KO mice but not klotho wildtype (WT) mice at 7 days post-infection. Neutrophils, eosinophils, interstitial macrophages, and monocyte/dendritic cell subset in the lungs of klotho KO mice were transiently induced after infection with A. baumannii. The number of alveolar macrophages in klotho KO mice was lower than that in klotho WT mice, except for 1 day post-infection. CD11b expression on neutrophils and alveolar macrophages in the lungs of klotho KO mice was seldom upregulated by the infection. These results suggested that immune functions eliminating bacteria in the lungs of klotho KO mice were insufficient. CD11blow conventional DC cells hardly increased in klotho KO mice infected with A. baumannii. Additionally, the production of interleukin (IL)-10 in the sera of klotho KO mice was significantly higher than that in klotho WT mice, whereas that production of interferon-gamma was not detected in the sera of klotho KO mice. These results suggested that acquired immune responses were hardly induced in klotho KO mice. IL-1ß, CXCL1, CXCL2, and CCL2 expression was significantly higher in the lungs of klotho KO mice infected with A. baumannii than in those of klotho WT mice at 1 day post-infection. These results suggested that pulmonary inflammation was elicited in klotho KO mice during early infection. The expression levels of proinflammatory cytokines significantly correlated with TLR9 expression in the lungs of klotho KO mice. The collective results demonstrate an A. baumannii infection state in aged hosts and suggest that pulmonary inflammation and bacterial burden should be noted in aged hosts even in the absence of severe symptoms of A. baumannii infection.

Antigen Epitope Developed Based on Acinetobacter baumannii MacB Protein Can Provide Partial Immune Protection in Mice.

Acinetobacter baumannii (A. baumannii) is an important opportunistic pathogen widely present in medical environment. Given its complex drug resistance, A. baumannii poses a serious threat to the safety of critically ill patients. Given the limited alternative antibiotics, nonantibiotic-based functional anti-A. baumannii infection proteins must be developed. In this study, we firstly used a series of biological software to predict potential epitopes in the MacB protein sequence and verified them by antibody recognition and lymphocyte proliferation tests. We finally screened out B cell epitope 2, CD8+ T cell epitope 7, and CD4+ T cell epitope 11 and connected them to construct a recombinant antigen epitope (RAE). The determination of IgG in the serum of immunised mice and cytokines in the supernatant of lymphocytes showed that the constructed epitope induced an immune response mediated by Th-1 cells. Finally, the challenge experiment of A. baumannii infection in mice confirmed that the epitope developed based on MacB, especially RAE, provided incomplete immune protection for mice.

The current state of immunization against Gram-negative bacteria in children: a review of the literature.

PURPOSE OF REVIEW: Gram-negative bacteria (GNB) are a major cause of infection worldwide and multidrug resistance in infants and children. The major pathogens include Klebsiella pneumoniae, Escherichia coli, Enterobacter spp., Pseudomonas aeruginosa and Acinetobacter baumannii. With new antibiotic options limited, immunization is likely to play a critical role in prevention. This review discusses their epidemiology, the current state of vaccine research and potential immunization strategies to protect children. A comprehensive review of the literature, conference abstracts along with web searches was performed to identify current and investigational vaccines against the major GNB in children. RECENT FINDINGS: Phase I--III vaccine trials have been undertaken for the major Gram-negative bacteria but not in infants or children. E. coli is a common infection in immune-competent children, including neonatal sepsis. Several vaccines are in late-phase clinical trials, with some already licensed for recurrent urinary tract infections in women. Klebsiella spp. causes community-acquired and hospital-acquired infections, including sepsis in neonates and immunocompromised children although no vaccine trials have extended beyond early phase 2 trials. P. aeruginosa is a common pathogen in patients with cystic fibrosis. Phase 1--3 vaccine and monoclonal antibody trials are in progress, although candidates provide limited coverage against pathogenic strains. Enterobacter spp. and A. baumannii largely cause hospital-acquired infections with experimental vaccines limited to phase 1 research. SUMMARY: The current immunization pipelines for the most prevalent GNB are years away from licensure. Similar to incentives for new antibiotics, global efforts are warranted to expedite the development of effective vaccines.