Trials in Vaccinology 5 (2016) 53–60 Contents lists available at ScienceDirect Trials in Vaccinology journal homepage: www.elsevier.com/locate/trivac Review Article Development of immunization trials against Acinetobacter baumannii Tarek A. Ahmad a,b,⇑, Dina M. Tawfik b,c, Salah A. Sheweita c, Medhat Haroun c, Laila H. El-Sayed b a Scientific Support and Projects, Bibliotheca Alexandrina, Alexandria, Egypt SeptivaK Research Group, Immunology Department, Medical Research Institute, Alexandria University, Alexandria, Egypt c Biotechnology Department, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt b a r t i c l e i n f o Article history: Received 13 May 2015 Revised 23 March 2016 Accepted 31 March 2016 Keywords: Nosocomial infection Acinetobacter baumannii Vaccine Immunotherapy a b s t r a c t Acinetobacter baumannii has recently crossed all lines once considered harmless, pushing its way as a nosocomial pathogen. It had acquired resistance to almost all available chemotherapies and mainly targets intensive care residents; causing pneumonia and major outbreaks with high mortality rates. This urged the need for preventive methods, which include infection control, non-specific immune-therapy, passive, and active immunization in order to offer vulnerable immune-compromised patients a flare in the dark. Several attempts were done for constructing effective vaccines with promising results. These are precisely classified, documented, and discussed in this up-to-date review. Ó 2016 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). Contents 1. 2. 3. 4. 5. 6. 7. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Therapy and resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Control and immunostimulants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Passive immunization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Antisera against whole cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Polysaccharide antisera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Protein based antisera. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Active immunization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Whole cell vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Pure protein based vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Comment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction Hospital’s laboratories are pointing to a new emerging Gramnegative bacilli named Acinetobacter; specifically Acinetobacter baumannii. The bacterium affects different human organs particularly the lungs, causing Ventilator-Associated Pneumonia (VAP) [1] which usually develops to septicemia in intensive care unit ⇑ Corresponding author at: Bibliotheca Alexandrina, 21526 Alexandria, Egypt. E-mail addresses: (T.A. Ahmad). Tarekadnan@yahoo.com, Tarek.adnan.ahmad@gmail.com 53 54 54 54 55 55 55 55 56 56 56 57 58 58 (ICU) residents [2]. It mainly infects the peritoneal cavity then rapidly disseminates to the lungs and spleen, it replicates to produce septic shock and might lead to associated bacteremia [2,3]. Infection is characterized by a rapid onset within 36 h, and progressive symptoms that leads to a mortality rate ranging from 40% to 60% [4]. Acinetobacter spp. are Gram-negative diplococcobacilli, non-fermentative, and are strictly aerobic bacteria [5]. They are catalase-positive, oxidase negative, and hard to destain, thus sometimes misidentified as being Gram-positive. Therefore, it has undergone many changes in taxonomy from Neisseriaceae to the family Moraxellaceae [6,7]. They are non-motile due http://dx.doi.org/10.1016/j.trivac.2016.03.001 1879-4378/Ó 2016 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 54 T.A. Ahmad et al. / Trials in Vaccinology 5 (2016) 53–60 to the absence of flagella, but they rather exert a twitching movement [8]. Acinetobacter genus is pervasive in nature between soil, water, human skin, throat, and respiratory tract colonization [9]. But this was pleaded against in the case of A. baumannii and its two relatives (Acinetobacter calcoaceticus and Acinetobacter nosocomialis) collectively known as ACB complex together with Acinetobacter pittii [10] that are only isolated from hospital-setting and are outbreaks-associated [7,11,12]. Recently, two more species were added to the ACB complex, those include Acinetobacter seifertii sp. nov. [13], and Acinetobacter dijikshoornii sp. nov. [14]. Although DNA-DNA hybridization can identify Acinetobacter genus [12], further advanced techniques are required to identify A. baumannii down to the strain level. Such techniques include amplified 16S ribosomal DNA restriction analysis, and high resolution fingerprint analysis [15]. However, those are complicated methods and are not present in common laboratories. Therefore, it is a hard mission to quickly identify A. baumannii in ICUs, and in times of outbreaks [16]. Moreover, A. baumannii possesses special pathogenic traits such as the ability to form biofilm, the production of siderophores, the presence of capsular polysaccharide and fimbriae, the cell to cell signals (Quorum Sensing), and the production of hydrolytic enzymes [17]. These virulence features facilitate its adherence and tolerance to desiccation on inanimate surfaces for more than 14 days [18]. All of these pathogenesis factors lead to persistent occupancy in hospitals, increase morbidity rates, and eventually lead to hospital cross transmission with persistent outbreaks [19]. 2. Epidemiology The incidence of nosocomial infection patterns are changing due to the emergence of new pathogens, along with changes in the antibiograms due to antibiotics abuse [20]. Based on the surveillance data, alerts were raised toward A. baumannii in particular due to its high persistence on inanimate objects in ICUs for months. In addition, A. baumannii rapidly acquired resistance against almost all potent antibiotics, thus adding days to ICU stay [21]. Outbreaks were correlated to seasonal factors, patient cases, and disasters times. A. baumannii was found to be more prevalent in late summer to early winter [7] rising up to 50% during the period of July to October [22] favoring humid and moist habitats [23]. A. baumannii was frequently reported in times of war, such as in Afghanistan and Iraq [24]. It was revealed that morphine used as analgesic in battlefields potentiates the growth of A. baumannii infections rather than trauma itself [25]. Not only in times of war did A. baumannii prosper, but also in natural disasters such as the 1999 Marmara earthquake aftermath [26] and Asia’s tsunami in 2004 [27]. In general, patients at risk are those who are immunocompromised, elderly, premature neonates, and patients that had recently undergone surgery or experienced a major trauma or were previously admitted to contaminated ICUs [28,29]. Smoking and alcohol abuse makes patients more prone to community acquired A. baumannii pneumonia, especially in tropical areas [30]. 3. Therapy and resistance A. baumannii exhibits a natural occurring resistance to a range of antibiotics such as Amoxicillin (penicillins), narrow-spectrum cephalosporins, Ertapenem, Trimethoprim, and Chloramphenicol [31]. A. baumannii managed to acquire resistance genes against several classes of antibiotics through the transfer of plasmids, transposons, and integrons from other Gram-negative bacteria [32]. All contributed to A. baumannii resistance and the ability to expel aminoglycosides [19], rifampicin [33], quinolones [34], fluo- roquinolones [35], tetracyclines [36], in addition to some disinfectants [37]. Therefore, different strains of multi-drug resistant (MDR) A. baumannii arouse in many countries all over the world and caused memorable outbreaks [38], with remarkable regional differences [39]. Tigecycline first showed a promising effect against MDR A. baumannii [40,41]. However, resistance to tigecycline has been reported since 2007 in Israel [42], and recently worldwide when used as monotherapy for a long time [43]. Alternatively, Colistin was found effective against Imipenem resistant A. baumannii. However, its use warranted due to its toxicity, side effects [44], cross-resistance with the host defenses that lead to the emergence of hypervirulent strains [45], reported resistance [46,47], and the worrisome hetero-resistance phenomenon [48]. Few novel classes of antibacterial agents currently show hope against A. baumannii such as peptide deformylase inhibitors (PDIs) and LpxC inhibitor, but they are still under trials with contradictory data [49–51]. The lack of effective treatment against A. baumannii introduced atypical therapeutic options [52], such as the use of bacteriophages [53], podophage [54], the intake of levamisole an anti-helminthic that is potent against Acinetobacter lwoffii [55], the use of nanoparticles generating nitric oxide [56], photodynamic therapy [57], and radio-immunotherapy. However, safety concerns still arise against their application [58]. Other methods can render MDR A. baumannii susceptible such as multidrug efflux inhibitor [59,60], anti-biofilms [61], quorum quenching [62], interference with the lipopolysaccharide (LPS) biosynthesis [63], the use of antimicrobial peptides [64,65], in addition to the use of several botanical preparations. However, all are still under trials along with reported resistance [66]. A British report highlighted that resistance to antibiotics will definitely increase regardless of their prescription pattern, adding societal cost of treatment of around £10 billion every year. This will impact the future of invasive procedures and surgeries, that require antibiotics as a standard regimen for prophylaxis [67]. Furthermore, a study revealed that patients infected with Imipenem‐ resistant A. baumannii had longer hospital stay of about 21 days and added an amount of $334,516 to hospital charges [68], with an increase in mortality rates by 25% in general hospital population and 50% in ICUs [69]. In a survey established in European countries from 2003 to 2009, A. baumannii showed to cause mortalities ranging from 3% to as high as 67% [70]. Thus, prevention right from the start has become a necessity. 4. Control and immunostimulants The previous data declares a war whoop against superbug A. baumannii. The increased resistance of chemotherapeutics to both conventional and last resort antibiotics necessitates prevention. Infection control is an important pillar in patient care excellence especially ICUs residents, and is a key factor to decrease nosocomial infections in the society and maintain antibiotic stewardship [71]. A recent study came to a shocking conclusions that the gowns, gloves, and unwashed hands of health care providers themselves are frequently contaminated with MDR A. baumannii acting as reservoirs in the ICUs [72–74]. When A. baumannii colonizes the respiratory tract, it can disseminate horizontally by droplet infection to as far as 22-feet [75]. In addition to that, 25% of the hospital environment had persistent contamination for months even after multiple-cleaning treatments [76], due to its biofilm formation ability [77]. Although new approaches arose to control the biofilm formation of Acinetobacter [78–80], however they are all under primary trials. All these facts urge researchers to be directed towards immunotherapeutics as a lifebuoy to unmet medical needs. Immunotherapies include non-specific, passive and active immunization. T.A. Ahmad et al. / Trials in Vaccinology 5 (2016) 53–60 Non-specific immunotherapeutics include the use of synbiotics that proved to decrease the risk of A. baumannii septicemia and VAP [81]. while the use of gaseous nitric oxide probiotic patch demonstrated a significant antimicrobial efficacy against A. baumannii [82]. Furthermore, c-di-GMP (30 ,50 -cyclic diguanylic acid) showed an immuno-modulatory effect and induced chemokines associated with enhanced neutrophil recruitment in the lungs [83]. It was found that patients admitted to hospitals were colonized within the first 24 h and a significant number of them developed nosocomial respiratory infections [84]. Therefore, efficient diagnostic tools play a vital role in the early identification and control of A. baumannii [85]. Thus, this gives a rationale for nosocomial immunotherapies to be administered to patients identified under the immuno-compromised umbrella [86]. Since A. baumannii was identified in the late 1980’s, the first trial for passive immunization appeared 20 years later in India [87]. However, the emergence of MDR A. baumannii progressed at a fast pace as a result of its high persistence in the hospitals’ environment, and the inability of antibiotics to cope with the increasing resistance. That’s why starting from 2010 several individual groups in Spain [88], USA [89,90], Iran [91], Australia [92], and China [93] started to work extensively on both passive and active immunization against A. baumannii. Starting from 2013, several researchers adopted the idea of solving the A. baumannii problem by vaccination in particular [94–96]. Meanwhile others recommended the trend of ‘‘thinking out of the box” [97], by using new chemo-immuno-therapies or unraveling new epitopes for the pathogen [98]. Recent studies confirmed that the pure antigens elicit a more potent immune response [99]. Therefore, other parties concentrated on the use epitopes’ mapping techniques [92], especially the in silico computational ones [100] to reveal novel potent epitopes. They proposed phospholipase D [101], outer membrane protein BamA [102] and FilF [103], vesicle’s outer membrane proteins OmpK, FKIB and Ompp1 [104], outer membrane protein nuclease NucAb [105], Pili subunit hemaglutinin [106], and the functional exposed amino acid BauA [107] as new vaccine candidates. Simultaneously, a coupled mapping procedure that uses Western blot and mass spectrum techniques proposed OMPs as major epitopes in human sera [108]. Moreover the study of the role of the Acinetobacter surface antigen protein (SurA1) in virulence introduced its application as a vaccine candidate [109]. Another approach proposed glyco-conjugates as vaccine and diagnostic tools for A. baumannii [110]. 5. Passive immunization Different antisera preparations from bacterial cell components were evaluated for their potency to neutralize the invading pathogen; those might be life-saving in case of acute infections or in time of outbreaks. 5.1. Antisera against whole cells In 2010 McConnell et al. [88] started to apply whole cell multiantigen preparations to raise protective polyclonal hyperimmune serum against A. baumannii. The formalin Inactivated A. baumannii Whole Cell (IWC) combined with an adjuvant was used to vaccinate mice. The produced antiserum was able to reduce the bacterial load in the spleen post-infection by 1000-fold, which was sufficient to protect the infected mice, prevent A. baumannii’s dissemination, and increase the mice’s survival rate. The Outer Membrane Complexes (OMC) was found to be a valuable complex of antigens, composed of surface proteins on the outer membrane of A. baumannii. One year later, the same research group [111] proved that the polyclonal hyperimmune serum raised against 55 sonicated OMC prevented the rise of escape mutants, and eliminated A. baumannii from all the immunized mice. Simultaneously, Huang et al. [93] demonstrated the eminent role of antisera raised against outer membrane vesicle (OMV) to control Acinetobacter. 5.2. Polysaccharide antisera Poly-N-acetyl-b-(1-6)-glucosamine (PNAG) is a conserved surface associated exopolysaccharides that are crucial in the maintenance of biofilms integrity under stressful conditions and protects A. baumannii against innate host defenses [112]. A passive immunization attempt was done with a synthetic oligosaccharide that mimics PNAG conjugated to tetanus toxoid (TT). The raised antisera were able to induce opsonization against several clinical isolates of MDR A. baumannii, and reduced burdens in infected tissue especially in cases of pneumonia. However, Anti-PNAG sera showed to be highly specific to only PNAG positive strains even in minute amounts [90]. PNAG based immunization was named the ‘‘magic bullets” of the 21st century as PNAG is a basic component of biofilm in MDR bacteria involved in both community and nosocomial infections such as Staphylococcus aureus, Escherichia coli and A. baumannii. With these promising facts, a monoclonal preparation against PNAG is already in phase II studies to be marketed soon [113,114]. Although, passive immunization might be considered a golden treatment in the case of acute infection when outbreaks rise, polysaccharide active vaccination is not preferred due to its ability to induce T-cell independent response, and its inability to induce memory cells unless conjugated to a protein [114,115]. The K1 capsular polysaccharide (CPS), is rich in polysaccharides of trisaccharide repeated units, consisting of three pyranose residues. The K1 capsule proved its importance only recently; when A. baumannii mutants were used to unravel its role in evading the host immune defenses by protecting it against complement, opsonization, and phagocytosis [115]. Its high polysaccharide content helps in the host-adherence process, helps in the delay of bacterial clearance from the host tissues, and is a requisite for survival within the host soft tissues during infection [116]. The seroprevalence of the K1 capsule in different A. baumannii strains reached only 13% [115]. The monoclonal antibodies (mAb) raised against K1 capsule of AB307-0294 strain in rat initiated opsonization with a marked increase in neutrophil-mediated bactericidal activity, along with a significant 4-logdecrease in the bacterial load of the infected soft tissues. The use of K1 capsule in immunization is safe, as it offers an immunogenic response with no clues of crossreactivity with human epitopes. However, K1 capsule antiserum has a drawback of being serotype specific [115]. 5.3. Protein based antisera Acinetobactertrimeric autotransporter (Ata) is a surface protein composed of a long peptide with an N-terminal, a surface-exposed passenger domain, and a C-terminal domain encoding for a 4-b strands found in the outer membrane of A. baumannii. Ata has a role in biofilm formation, and enhances the bacterial adhesion to the host’s cells. Therefore, it facilitates survival, and enhance virulence of the pathogen [117]. The anti-recombinant Ata were able to impair Ata’s binding to collagen type IV of the host’s cells in both immunocompetent and immunocompromised neutropenic mice. It promoted complement-dependent bactericidal killing activity, enhanced both phagocyte-dependent and -independent killing, protected the challenged mice from A. baumannii pneumonia, and increased survival rates [89]. Despite anti-Ata proved to decrease the virulence and colonization, the murine model remains a poor experimental host for A. baumannii [118]. Although, Ata is a well-defined protein [111], in-depth analysis revealed that the 56 T.A. Ahmad et al. / Trials in Vaccinology 5 (2016) 53–60 anti-Ata bactericidal efficiency highly depends upon a certain threshold level of Ata expression, which is a limitation against low-producing Ata strains [89]. Unlike other proteins, the Ata epitope is conserved among several strains, which makes it a valuable candidate [89,119]. The Iron Regulated Outer Membrane Proteins (IROMP) are low molecular weight catechol siderophores, having high affinity to chelate iron. They are composed of four outer membrane proteins, with molecular weights ranging from 77 kDa to 88 kDa. They have a vital role in A. baumannii virulence and proliferation, and in iron acquisition specifically in iron deficient environments [17]. AntiIROMPs are promising against homologous A. baumannii in vitro. Immunization was achieved through the blocking of siderophoremediated iron acquisition; thereby inhibiting growth, which in turn demonstrated both bactericidal effect ranging from 80% to 90%, and increased complement-mediated opsono-phagocytic activity reaching up to 6–8-fold [87]. However this study was criticized as the researchers did not measure the direct bactericidal effect in tissues, but rather the in vitro uptake of bacteria by opsonization in poly-morpho-nuclear cells (PMNs) [89]. Added to that, IROMPs only develop in iron-depleted environments which might be a limiting factor as a broad spectrum passive immunization approach due to its iron-dependency. The candidate outer membrane protein A (OmpA) is a part of the bacterial surface outer membrane, referred to as Omp36, having a major role in pathogenesis. It adheres to the host cells in early infection stages and activates surface cell death receptors. Then, it localizes in the mitochondria inducing pro-apoptotic molecules that triggers a cascade of reactions leading to eventual cell death [120]. When perspective A. baumannii antigens were screened in a natural systemic infection, OmpA was identified as a primary epitope target of humoral immune response. Resistance of A. baumannii strains to the host’s complement system in serum, were linked mainly to OmpA when it binds to factor H (a main regulator to the serum alternative complement-pathway) [121]. Its structure is more than 99% conserved among clinical strains from different infection sites and P89 conserved within other A. baumannii strains, with low homology compared to the human proteome. Raised anti-recombinant OmpA antibodies conferred protection through enhanced opsono-phagocytosis but not the complement mediated killing against MDR A. baumannii, since anti-OmpA was not able to restore the complement system activity against A. baumannii [119]. Further research on antisera against OmpW [122], Omp22 [123], and outer membrane nuclease (NucAb) [105] of Acinetobacter demonstrated their role to improve survival in experimental animals. 6. Active immunization The use of different antigens as vaccine candidates in healthy models paved its way to prevent infections due to A. baumannii, which include the followings. 6.1. Whole cell vaccines Mice were injected by formalin Inactivated Whole A. baumannii Cells (IWC), formulated with aluminum phosphate as adjuvant (Adjuphos). The intramuscular injection of the IWC preparation resulted in a rapid robust of antibody titers. The immunized mice had less bacterial burdens in the infected tissues, and a reduced production of pro-inflammatory cytokine serum levels of IL-1b, TNF-a, and IL-6 that are normally associated with sepsis. Thus, it helped to secure high survival rates in vaccinated mice [88]. A more recent study proved that the vaccine could be administered intranasally as well [124] and that the antibiotic exposed bacterial cells produce a potent vaccine [125]. IWC are easy to prepare, inexpensive, and does not require expensive denaturation of antigens, that may induce conformational changes of the epitopes. Added to that, it was shown that A. baumannii while acquiring resistance; has the ability to down-regulate the surface expression of certain surface membrane protein. IWC vaccine was able to generate hyper-immune antibodies against its multi-antigenic components, mostly those belonging to the outer membrane, thus providing diverse protection against several A. baumannii strains. However, safety remains a concern with IWC use as incomplete inactivation of the bacterial cell which might initiate infection or the possibility of being contaminated with pyrogenic endotoxin during injection [88]. Researchers overcame this obstacle by the production of an IWC vaccine based on LPS deficient A. baumannii strain. The vaccine proved to induce a protective level of immunoglobulins, and reduced the pro-inflammatory cytokines [126]. The sonicated Outer Membrane Complex (OMC) was formulated with Adjuphos, and used as a vaccine. The OMC vaccine was confirmed to be highly reproducible, and can limit post infection pro-inflammatory cytokines associated with septic shock. It was able to increase IFN-c, to be highly potent and heterologous, and reduced the bacterial burdens in tissues by 105-fold. The OMC preparation elicited a rapid IgG and IgM response against the bacterial OMP only (not the other bacterial components such as the LPS). The humoral response was able to provide a protective immunity with just a single vaccine dose in as few as 6 days postimmunization and was maintained for 21 days. This aspect can be life-saving in case of outbreaks or critical conditions. However, safety concerns still arise about the possible endotoxin contamination [111]. Outer Membrane Vesicles (OMV) are secreted by A. baumannii during growth. They are spherical, non-viable, and acellular nanovesicles that contribute to colonization during infection [127]. OMV has a role in spreading antibiotic resistance genes and quorum sensing ability [17]. A hundred and thirteen different proteins were identified and found to be packed within OMV, majorly consisting of OmpA, other outer membrane proteins, CarO protein, tissue degrading enzymes, as well as, LPS and nucleic acids [128]. Mice were vaccinated with OMV formulated with Adjuphos, then boosted after 3 weeks by the same preparation. It was found that the IgG increased by 60-fold, bacterial tissue burdens were reduced by 106-fold, and the pro-inflammatory cytokines IL-6 and IL-1b decreased post-infection [129]. OMV vaccine offered a full heterologous protection to immunized mice against several A. baumannii strains including pan-resistant isolates, which is a perk compared to subunit vaccines that can become ineffective in the case of proteins down-regulation observed in A. baumannii. The vaccine conferred protection in both pneumonia and sepsis models [93]. The extraction process of OMV involves the use of detergents to reduce the endotoxin content. Although the detergents might deplete its active soluble antigens, it still holds the benefit of rapid production requiring only filtration compared with the manufacture of recombinant single antigen vaccines. OMV being acellular, has a competitive advantage over inactivated whole cell as it is much safer and produces less adverse toxic events, although endotoxin is still present and might trigger safety issues [129]. This may be counted as a defect for all other whole cell vaccines, since the components are not standard between lots (81). 6.2. Pure protein based vaccines In 2011, a mixture of pure proteins majorly OMPs and fimbrae proteins was patented as effective vaccine against A. baumannii [130]. Later on, in silico mapping confirmed the potency of OmpA type 1 in particular [131]. The preparation of recombinant OmpA (rOmpA) with aluminum hydroxide as adjuvant in a dose of T.A. Ahmad et al. / Trials in Vaccinology 5 (2016) 53–60 3 lg/kg was used to immunize mice. Immunization marked an increase in mice survival rates by decreasing bacterial tissue burdens, and induced high anti-OmpA IgG antibodies titers that soared by increasing the dose up to 30-fold [132]. Although, it was proposed that the pure OmpA is more favorable than the complex IWC, the broad spectrum immune responses to other proteins within the IWC cannot be denied [133]. More recently, a vaccine was developed by being formulated with nano-chitosan as adjuvant, and proved good efficacy [134]. OmpA vaccines are very promising as they are highly reproducible, easily to be manufactured commercially, and safer than the whole complex preparations [132]. However, when it is in a purer OmpA form it becomes insoluble making its routes of delivery hard [135]. Further research on the Omps such as Omp22 [123], OmpW [122], as well as the epitope mapped outer membrane nuclease (NucAb) [105] and the selected Omps [104] proved their potency as vaccine candidates. The candidate Bap protein is a high molecular weight 854 kDa and one of the most acidic proteins, found on the surface of A. baumannii. It was identified as an important key regulator of biofilm maturation and maintenance of the biofilm structure, thickness, and volume. The Bap’s presence increases the bacterial cell hydrophobicity which enhances its adherence to the host cells and helps the bacteria to dodge phagocytes [136]. Active immunization with Bap targets the most virulent character of A. baumannii and deprives it from biofilm formation, with an added benefit of being 43% conserved among isolates. Recombinant Bap subunit was prepared, emulsified with complete Freund’s adjuvant, and used to vaccinate mice. Immunized mice exhibited high IgG antibody titers, with a complete bacterial clearance from the infected tissues, which in turn increased mice survival rates. Bap subunit vaccine has around 20 antigenic determinants and 55 discontinuous B-cell epitopes, hence demonstrated immune-dominancy [91]. Further research on the combination of Bap with OMV or OmpA of Acinetobacter proved an augmented potency versus the individual components [137]. More recently, the previously mapped acinetobactin (BauA) [107] proved its importance as a prophylactic agent against Acinetobacter [138]. 7. Comment A. baumannii has risen as an opportunistic resistant superbug causing outbreaks worldwide, which resulted in high morbidity and mortality rates especially to prone patients. A. baumannii is not easily identified, but rather requires sophisticated and timeconsuming equipment, when time is of the essence to save lives. A. baumannii is also hard to manage due to its fast acquisition of antimicrobial resistance; already putting an end to the antibiotic golden era. New antibiotics inventory became depleted against A. baumannii invasion, while all alternative therapies are still under development, requiring years to be available on the market. Thus, breaking the vicious cycle right from the start makes an ideal rationale side-by-side with infection control. Indeed the pioneer work of the Australian group to use in silico techniques to screen the antigenic determinants of A. baumannii was the right step to find the effective epitopes, but was rather an incomplete one, since A. baumannii has some immune-dominant antigens that may overshadow other effective ones in the pathogen. Moreover, in silico based methods only predict the protein epitopes, but never the polysaccharide ones. Therefore, the bare application of those proposed epitopes may be of limited value, and further investigation by binding techniques are necessary to design a potent vaccine against A. baumannii. It is clear nowadays that the epitope screening protocols are crucial, since miss-guided empirical vaccine production is a trial and error challenge. 57 In general the ideal immunotherapy candidate antigen against A. baumannii should possess important qualities. These qualities include being protective for the host, highly prevalent, conserved among different strains of A. baumannii to deliver broad spectrum protection (heterologous), highly reproducible, cheap, and surfaceexposed. As well as being able to remain stable and soluble after preparation or in its recombinant form, and induce a rapid robust humoral antibody titers yet having a sustained cellular response. They should also reduce the incidence of septic shock, and can alleviate the pro-inflammatory cytokines. It is important as well that the ideal antigen is able to protect immunocompromised candidate, to be of well-defined components in term of quantity and identity, and last but not least to be safe for human use. Moreover, that candidate should have a proven efficacy for both passive and active immunization, which is an additional privilege. That’s why the evaluation of any immune-preparation should fulfill these criteria. Several experimental attempts against whole cell, polysaccharides or protein preparations generated therapeutic antiserum against A. baumannii. They all conferred protection to laboratory animals; the majority increased opsonization, reduced the bacterial burden in tissues, and prevented its dissemination. The use of K1-CPS to produce mAb against A. baumannii was expensive and limited to only 13% of CPS-positive isolates. The use of the anti-IROMP was not practical since it exhibited a limited homologous protection for the pathogen grown in iron depleted environments. Although, the antiserum raised against r-Ata was the only preparation that prevented the bacterial adherence, the response was highly dependent on the Ata level expressed by the bacterial cells. Yet, the anti-Ata showed to be active in both immunecompetent and immune-compromised models, the applied murine model is a poor experimental host. Despite PNAG antiserum is the only preparation that went through clinical trials, it did not confer heterologous protection and required a complicated conjugation step with a protein to elicit a remarkable antibody titer as being a hapten. This increases the production cost and may alter the immunogenicity of PNAG. The OmpA is a conserved antigen that generates a sufficient amount of antibodies, as being a protein in nature. The anti-OmpA was safe and provided a heterologous protection. However, the facts that A. baumannii undergoes protein down-regulation (such as all other Omp-related preparations), and that r-OmpA is not sufficiently soluble thus limiting the attractiveness of anti-OmpA as a powerful immunotherapeutic preparation. Moreover, none of the above antisera protected the host from septic shock, such as the effect conferred by the polyclonal preparation raised by the whole cell preparations. The IWC, OMV and OMC preparations are the cheapest and their immunogenic determinants do not undergo any denaturation processes during preparation. However, LPS toxicity remains a concern during antibody generation, which may be resolved by a mild acid hydrolysis detoxification. While the use of the whole cell isolates vaccine that lack LPS may be useless, since the whole cell antisera will lose the potency to protect the host from the septic shock. All the up-to-date vaccine formulations conferred high cellular and humoral immune response, protection from both homologous and heterologous bacterial challenges, and hence increased survival rates among experimental animals. The cost of production of recombinant Omps and Bap is relatively higher, and the preparations might lose their potency in case the pathogen develops protein down regulation mechanisms. Since direct extraction is not practical, cloning techniques were adapted to produce the Omps and Bap. Unfortunately; these cloning and refolding techniques may alter the conformational structure of the produced proteins and render the preparation insoluble. This will affect the immune 58 T.A. Ahmad et al. / Trials in Vaccinology 5 (2016) 53–60 response potency which is highly dependent on the native 3dimentional structure of the produced protein. On the other hand, the whole cell vaccine preparations are very cheap to prepare, broad spectrum, and do not require any intervention during production that might denature the structure of the proteins. Added to that, the whole cell vaccines reduce the post inflammatory cytokines, generated antibodies against the LPS, and hence avoid the septic shock cascade. Although, IWC are less safe when used in human beings, OMC and OMV overcame this problem as being acellular. In addition, OMC possesses a unique role in eliciting a rapid protective humoral immune response in less than six days from administering the first dose. This privilege favors’ OMC as the best choice to be used in the case of outbreaks; especially that the antiserum raised against it, has a proven potency to control active bacterial infections. Moreover, OMC can induce the production of the IFN-c that enhances non-specific NK cell activity, which is an advantage for immunocompromised patients residing ICUs. However, the OMC is not powerful enough to elicit antibodies against the LPS, due to the overshadowing effect. Whereas, OMV has the potency to highly increase the immunoglobulin titers against A. baumannii, and remarkably reduced the bacterial burden from infected tissues. Therefore, a combined vaccine between OMV and OMC would be an ideal choice. However, concerns regarding the endotoxic impurities remain as safety barriers. It is well known that the toxicity of the LPS is exclusively due to the lipid A portion in the molecule. However, the majority of the classical methods used to remove endotoxins depend on detergents that fully remove the LPS molecule and may also deplete the vaccine from its other soluble active antigens. Simultaneously, other researches depend on the use of LPS-deficient isolates. Thus the presence of the polysaccharide portion of the LPS in the vaccine induces a broad protective spectrum against the pathogen’s endotoxin, lowers the pro-inflammatory cytokines, and in turn reduces the mortality due to septic shock. This point is a highly critical point, since LPS is a necessary component in a powerful vaccine against A. baumannii, while its toxicity might be fatal. That’s why, further studies should be directed towards the evaluation of a new method that depends on developing the detoxification of the OMC/OMV vaccine by gentle lipolytic methods, or the production of natural self-conjugated vaccines between the detoxified LPS and the OMPs, as previously described by the author’s research on Klebsiella pneumoniae. This later one has the advantage of eliciting a longtime thymus dependent cellular immune response against the conjugate individual components, as well as being of welldefined standard ingredient in terms of quality and quantity. Acknowledgments [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] The authors would like to thank Mr. Aly Salim the Iraqi scholar at Alexandria University for his share in writing this review. Simultaneously Miss. Yara Hussein and Miss. Enas Mostafa for the support they offered to retrieve the necessary articles. 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