Vaccines | Free Full-Text | Development and Evaluation of an Immunoinformatics-Based Multi-Peptide Vaccine against Acinetobacter baumannii Infection
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1. Introduction
The prevalence of multi-drug-resistance (MDR) bacterial strains has increased yearly and has become a burden on healthcare systems globally, significantly increasing costs and mortality [1,2]. A significant contributor to this crisis is Acinetobacter baumannii (Ab), a nonmotile Gram-negative bacterium. It is one of the most successful opportunistic nosocomial pathogens due to its MDR phenotype and ability to avoid desiccation while thriving in healthcare settings under selective pressure [3]. Typically, Ab healthcare-associated infections present as ventilator-associated pneumonia, catheter-related bloodstream/urinary infections, and wound infections in military personnel [4,5,6]. The rise in cases over recent decades results from increased usage of the intensive care unit (ICU), increased length of hospital stays, and overuse of antibacterial therapy [7,8]. Recent isolates have exhibited extreme drug resistance (XDR), such as carbapenem-resistant Ab (CRAB) strains, prompting the World Health Organization (WHO) to assign CRAB the highest critical priority ranking for the identification of novel drug therapies to combat this pathogen [9]. Such designations highlight the need for the development of alternative strategies to combat MDR Ab as current antimicrobials become less effective year after year. This emergency has prompted researchers to investigate the efficacy of immunotherapies, e.g., vaccination or monoclonal antibodies, as viable alternatives.
Many early and current immunotherapeutic developments against this pathogen have demonstrated the adaptive potential to protect against acute Ab infection. Studies utilizing whole-cell vaccines, such as UV-inactivated, formalin-inactivated, and live-attenuated bacteria, provide robust protection to vaccinated mice [10,11,12,13]. The robust immunogenicity and broadly protective nature of these vaccines is due to the plethora of antigens available to prime the adaptive immune response to Ab. Despite their protective capabilities, no preclinical evaluation of these candidates has begun. The hesitation to utilize such vaccines is mainly due to safety concerns. Thus, current Ab vaccine efforts are focused on developing safer protein subunit vaccines, and additional work in understanding Ab pathogenesis has shed light on virulence factors and novel vaccine targets. Subunit vaccines have afforded partial protection across multiple studies, showing limited capability in providing robust protection against Ab’s ability to adapt to selective pressures due in part to targeting a single antigenic protein in a rapidly changing pathogen [14,15,16].
With the emergence of bioinformatics, it is now possible to quickly scan entire bacterial genomes for potential vaccine candidates, a process referred to as reverse vaccinology [17]. The abundance of targets can be filtered and downselected based on sequence conservancy among different strains, antigenic prediction, solubility upon overexpression, and in silico immune system simulation. These tools accelerate vaccine target selection and design with immunoinformatic tools capable of predicting T- and B-cell epitopes of interest in each antigen [18]. These epitopes can be fused together with linkers hypothetically providing protection to multiple antigens in contrast to vaccines targeting a single antigenic protein. Fusing epitopes together is not a novel concept and has shown great success in T-cell-based vaccines due to the manner in which the immune system processes protein antigens and subsequent peptides [19]. However, in silico, predicted B-cell epitopes are linear and not conformational. Thus, it remains to be seen if such linear B-cell epitopes elicit antibodies capable of recognizing three-dimensional antigen conformations.
In the last few years, there has been a significant increase in the publication of immunoinformatic-based vaccines against various pathogens [20,21]. However, few have been assessed for their immunogenicity and protectivity in vivo. In this study, we designed and evaluated the immunogenicity and protective efficacy of an Acinetobacter Multi Epitope Vaccine (AMEV) consisting of predicted immunogenic peptide regions across five different Ab virulence factors.
2. Materials and Methods
2.1. Animals
All animal experiments were performed using 7–8-week-old C57BL/6 mice purchased from Jackson Laboratories (Bar Harbor, ME, USA). Mice were housed at the University of Texas at San Antonio in an AAALAC-accredited animal facility. All animal experiments were performed in accordance with Institutional Animal Care and Use Committee Protocol MU070.
2.2. Bacteria
Acinetobacter baumannii clinical isolate 79 (Ci79) obtained from the San Antonio Military Medical Center (SAMMC; Fort Sam Houston, San Antonio, TX, USA) was provided by Dr. James Jorgensen (University of Texas Health Science Center at San Antonio, San Antonio, TX, USA) [22,23]. Bacteria were streak-plated on Luria-Bertani (LB) agar plates supplemented with ampicillin (100 µg/mL) from a frozen stock. An overnight culture was prepared from a single colony, incubated overnight at 37 °C, subcultured the following day to an OD600nm = 0.03 in fresh LB broth, and grown for 3.5 h until mid-log phase. Subcultures were centrifuged at 5000 rpm for 5 min and bacteria pellets were resuspended and washed in phosphate-buffered saline (PBS). Following the first PBS wash, bacteria were diluted to an OD600nm = 0.5 (≈2 × 108 CFUs/mL). This inoculum was either further diluted for the intraperitoneal (i. p.) challenge or centrifuged and concentrated for the intranasal (i. n.) challenge. Bacterial inoculum CFU/mL was determined by serial dilution and plating.
2.3. rAMEV2 Cloning and Purification
The rAMEV2 expression vector contained an Ab-leading protein sequence linked to 5 identified peptides via a rigid EAAAK amino acid linker. Peptides were joined by the short amino acid linkers, GPGPG or KK, and the resulting construct contained a 6× His tag at the C-terminus for protein purification. The E. coli codon-optimized nucleotide sequence for rAMEV2 was synthesized by GenScript (Piscataway, NJ, USA). The pET23a- AMEV2 vector was transformed into BL21(DE3) competent E. coli. rAMEV2 expression was induced in the presence of 1 mM IPTG and purified under denaturing conditions using a HisPur cobalt spin column (Thermo Fisher, Waltham, MA, USA) according to manufacturer’s guidelines. The denatured recombinant protein was refolded by serial dialysis against PBS in the presence of 2-fold diluted urea (4 to 1 M). Following final dialysis against PBS-purified rAMEV2 protein was aliquoted and stored at −80 °C until used. The purified rAMEV2 protein sequence was confirmed by standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) from trypsin-digested fragments.
2.4. AMEV2 Vaccination and A. baumannii Challenge
C57BL/6 (7–8-weeks-old) mice were randomly divided into Mock (PBS + Adjuvant) or AMEV2 (rAMEV2 + Adjuvant) groups. rAMEV2 (10 µg/mouse) or PBS alone (100 µL/mouse) was formulated with AddaS03 adjuvant (InvivoGen, San Diego, CA, USA) at a 1:1 (v/v) ratio and injected subcutaneously on day 0. All mice were boosted with the same dose on days 14 and 28 and rested for four weeks before evaluating protective efficacy. Briefly, Ab Ci79 inocula containing 2 × 109 CFUs/mL were prepared for intranasal challenge. Mice were anesthetized by isoflurane inhalation, and 50 µL bacteria (108 CFU/mouse) was administered dropwise to the nares. Animals were monitored daily for morbidity (weight loss) and mortality for 30 days following the bacterial challenge.
2.5. Organ Bacterial Burden Measurement
Lungs, spleens, kidneys, and blood were collected from PBS mock, PBS + AddaS03 mock, and rAMEV2 + AddaS03 vaccinated mice (n = 5 per group per time point) at 24 and 48 h post intranasal challenge with Ab Ci79 (108 CFU/mouse) in 2 mL PBS. Tissues were homogenized, serially diluted, and plated on LB agar supplemented with ampicillin (100 µg/mL) to enumerate bacterial burdens.
2.6. Determination of Antibody and Isotype Levels by ELISA
Microtiter plates were coated with rAMEV2 protein (500 ng/well) or peptides (1 µM per well) overnight in sodium bicarbonate buffer (pH 9.5). Plates were then washed with PBS containing 0.05% Tween 20 (PBST) and blocked with PBS containing 10% (v/v) fetal bovine serum (blocking buffer) for 1 h at room temperature (23–25 °C). Following blocking, plates were washed with PBST and sera diluted with blocking buffer were added to the plates and incubated for 2 h at room temperature. Following three washes with PBST, plates were incubated for 1 h with goat anti-mouse total Ig, IgG1, and IgG2c conjugated to HRP (Southern Biotechnology Associates, Birmingham, AL, USA) diluted 1:4000 in blocking buffer. Plates were washed three times with PBST and TMB substrate reagent (BD Biosciences, San Diego, CA, USA) was added to each well for color development. Color development was stopped after 15 min with the addition of 2 M H2SO4. Absorbance at 450 and 570 nm was recorded for each well using a microplate reader (TECAN, Männedorf, Switzerland). Endpoint titers were determined as the highest dilution with an absorbance reading of 0.1 greater than the blank absorbance reading.
2.7. B-Cell ELISpot Assays
The frequency of antigen-specific antibody-secreting cells was evaluated using the B-cell ELISpot assay. Spleen and bone marrow were collected 1 week after the second vaccination. PVDF membrane ELISpot plates (Millipore Sigma, Burlington, MA, USA) were coated overnight with 1 µg per well of either hen egg lysozyme (HEL, an unrelated antigen negative control), goat anti-mouse Ig (Southern Biotechnology Associates, a positive control), or rAMEV2. PBS-only wells served as a negative control. Plates were washed with PBS and blocked with RPMI-1640 supplemented with 10% (v/v) fetal bovine serum, 100 U penicillin/mL, 100 µg streptomycin/mL, and 2 mM L-glutamine (R10). 2.5 × 105 Splenocytes and bone marrow cells were seeded into the wells and incubated for 6 h at 37 °C with 5% CO2. Following incubation, plates were washed with PBST before adding goat-anti-mouse-Ig-AP-conjugated secondary antibody (Southern Biotechnology Associates) diluted 1:5000 with 1% (w/v) bovine serum albumin (BSA) in PBS and incubated at 4 °C overnight. Plates were then washed with PBST and incubated with BCIP/NBT phosphatase substrate (SeraCare, Milford, MA, USA) for spot development. The reaction was terminated with water and dried before reading using an ImmunoSpot analyzer (CTL) as previously described [24].
2.8. T-Cell ELISpot Assays
T-cell reactivity was evaluated using IFNγ, IL-4, and IL-17 ELISpot assays as previously described with modification [25]. Spleens from AMEV2-vaccinated or adjuvant-only treated mock mice were isolated one-week post-second vaccination. Splenocytes were evaluated for in vitro recall with rAMEV2 (10 µg/mL) or UV-inactivated Ab (1 × 106 CFU/well). Media-only wells served as a negative control for background, HEL (10 µg/mL) served as an antigen-specificity control, and α-CD3 (clone: 145-2C11, 1 µg/mL) served as a positive control. PVDF membrane ELISpot plates (Millipore Sigma) were coated overnight with either IFNγ (clone: AN-18, 2 µg/mL), IL-4 (clone: 11B11, 4 µg/mL), or IL-17 (clone: 17F3, 2 µg/mL) capture antibodies. The following day, plates were washed with PBS and blocked with complete R10 media while spleen tissue was collected and prepared. 5 × 105 splenocytes were seeded in IFNγ and IL-4 wells, while 106 cells were seeded into the IL-17 wells. Antigens at indicated concentrations were added and incubated at 37 °C with 5% CO2. IFNγ and IL-17 plates were stimulated for 24 h, while IL-4 plates were left for 48 h. Following the indicated incubations, plates were washed with PBS before secondary antibodies were added. Biotinylated IFNγ (clone: R4-6A2, 0.5 µg/mL), IL-4 (clone: BVD6-24G2, 2 µg/mL), and IL-17 (clone: eBioTC11-8H4, 0.125 µg/mL) detection antibodies were added to the respective wells and incubated at 4 °C overnight. Plates were then washed with PBS before incubation with Streptavidin Alkaline Phosphatase conjugate (Invitrogen, Waltham, MA, USA) for 2 h at room temperature. Following an additional wash, BCIP/NBT phosphatase substrate (SeraCare) was added for spot development. Reactions were quenched with water, followed by drying and spots were counted using an ImmunoSpot analyzer (CTL).
2.9. Statistical Analysis
GraphPad Prism 10.0 was used to determine statistical significance tests. Differences between mock and AMEV2 vaccinated groups were assessed using the Student’s t test, multiple Mann–Whitney tests, and one-way or two-way analysis of variance (ANOVA). Survival rates were analyzed with the Log-rank Mantel-Cox test. Differences were considered statistically significant when p ≤ 0.05.
4. Discussion
Here, we describe a novel multi-peptide Acinetobacter baumannii vaccine designed using an immunoinformatic approach. Despite efforts over the past two decades to develop vaccines against this pathogen, none have advanced to clinical evaluation. Current efforts to develop subunit vaccines against Ab include whole recombinant proteins, immunogenic antigen peptides, and multi-peptides composed of short discontinuous B-cell and T-cell epitopes [40,41,42,43]. Our focus was to keep immunological sequences intact to improve T-cell-dependent antibody response, taking advantage of the chimeric nature of the construct’s potential for multivalent protection to counteract potential vaccine escape. This multivalent construct consisted of peptide segments from five virulence factors involved in Ab pathogenesis: NlpE, NucAB, TonB, ZnuD, and Omp38. The leader protein for this multi-peptide construct is Ab TrxA, an additional virulence factor well characterized by our laboratory and functions as a solubility enhancer [31,44,45]. NlpE is a copper-resistance protein involved in biofilm formation and bacterial adhesion in many MDR Ab strains [46]. Of the five virulence factors, NlpE is the only virulence factor that has not been evaluated previously as a vaccine candidate, although copper resistance gene knockouts have demonstrated attenuated virulence in a murine pneumonia model [47]. NucAB is an outer membrane nuclease and has been predicted in silico to be a desirable target due to its location. It is also highly conserved among Ab isolates [48]. Garg and coworkers have demonstrated the protective efficacy of recombinant NucAB with 20% survival following active immunization and 40% with passive immunization against pulmonary Ab infection. TonB is a general siderophore receptor important for iron acquisition and growth in the host [49]. A well-characterized siderophore receptor, BauA, rendered mice actively vaccinated using its recombinant form partial protection and complete protection following passive vaccination [50]. Like TonB, ZnuD is a zinc piracy receptor involved in resistance to host nutritional immunity [51]. A vaccine composed of ZnuD surface loops on a hybrid antigen afforded mice complete protection in an Ab bacteremia model [52]. Lastly, Omp38 belongs to the outer membrane protein A (OmpA) family, which has been thoroughly characterized as a therapeutic target against Ab infection [53]. It is capable of inducing apoptosis in epithelial cells and inhibits the host complement system [54,55]. Additionally, one of the first subunit vaccines against Ab targeted OmpA and demonstrated 50 and 90% protection following active and passive vaccination, respectively [56]. AMEV’s multivalent potential includes inhibition of biofilm formation, bacterial adhesion, nutritional immunity, and host immunomodulation. These virulence factors have shown promise as vaccine candidates individually and contain protective epitopes against Ab infection.
The protective epitopes within these virulence factors were determined in silico using the EigenBio epitope prediction software. Five peptides, between 40 and 60 amino acids in length, containing B- and T-cell epitopes from each virulence factor were selected. Screening each peptide against sera from mice that recovered from an Ab infection indicated four of the five peptides exhibited specificity for α-Ab antibodies. Additionally, antisera reacted strongly to the complete recombinant AMEV2 construct. In vivo, immunogenic evaluation of rAMEV2 demonstrated that four of the five predicted peptides were immunogenic and did not give rise to the formation of new junctional epitopes between the linked peptides. The Omp38 peptide demonstrated the highest reactivity to serum following Ab infection, indicating the likelihood of it being a protective epitope. It remains to be determined which of the five peptides is necessary for protection to optimize the AMEV construct. Overall, these in silico-selected peptides demonstrate the ability to remain immunogenic, generating effective antibodies when linked together in a multipeptide construct.
rAMEV2 subcutaneously administered with AddaS03 adjuvant generates a robust humoral response, which has been the primary focus of Ab vaccine development due to the acute nature of the murine infection model. This aligns with most Ab vaccines demonstrating the effectiveness of antibody-mediated protection with passive sera transfer and monoclonal antibody generation [11,13,14,15,56,57,58,59,60,61]. However, the impact of cell-mediated protection against Ab remains poorly understood, given the limited cell-mediated immunity data from whole-cell vaccines. Our laboratory has previously characterized a live-attenuated strain of thioredoxin deficient Ci79 for its cell-mediated response and observed a minor increase in IL-17 secretion but no significant elevation in IFNγ or IL-4 upon restimulation with Ab [60]. However, vaccination of mice with an Ab D-glutamate auxotrophic strain deficient in wall peptidoglycan synthesis by Cabral et al. demonstrated significant IL-4 and IL-17 responses [13]. An outer membrane vesicle vaccine demonstrated an increase in the Th2 subset (CD4+/IL-4+) of T-cells in vaccinated splenocytes, indicating the adaptive immune response to this pathogen being Th2 dominant [62]. Protein subunit vaccines, in combination with highly immunogenic adjuvants, have encouraged investigation of the relevance of cell-mediated immunity against this pathogen with various adjuvants. We have shown that rAMEV2-sensitized splenocytes are capable of secreting IFNγ and IL-4 in response to restimulation to the protein construct with significantly higher IL-4 rAMEV2 specific T-cell frequency indicative of a Th2 response. This Th2 memory response is also elicited upon restimulation with whole Ab bacteria. The very acute nature of these infections leaves little room for investigating a potentially effective T-cell response, but IL-4 secretion and a Th2 response could be required for modulation of the protective B-cell response. This T-cell-dependent antibody response is instrumental in enhancing B-cell activation and effective antibody production, as evidenced by AMEV2′s substantial IgG1 and IgG2c antibody titers.
Using this design, we have demonstrated partial protection by AMEV2 vaccine to an otherwise lethal intranasal dose of a virulent Ab strain. AMEV2 vaccination reduces bacterial burden and dissemination to other tissues, ultimately leading to abrogation of TLR4-mediated septic shock compared to mock vaccinated control mice [63,64]. The mechanisms of this seemingly antibody-mediated protection remain to be established, thus requiring further study. Partial protection afforded by the AMEV2 vaccine is in agreement with other protein subunit vaccines, but it still underperforms in comparison to complete protection achieved by whole-cell vaccination to this pathogen [10,11,12,13,58]. Discovering additional conserved protective peptides will undoubtedly expand the AMEV vaccine constructs’ effectiveness and reactivity against additional Ab strains. For example, in a pilot study, we observed the broad protective efficacy of the AMEV2 vaccine in a systemic challenge model against an alternative hypervirulent strain of Ab, AB5075. Mice (n = 10) fully vaccinated with rAMEV2 in conjunction with Titermax Gold adjuvant and challenged intraperitoneally achieved 80% survival over 30 days compared to the PBS mock control group, which succumbed to bacteremia after 24 h (Supplemental Figure S2). This strategy indicates that AMEV constructs can be further refined to achieve desirable vaccine candidate status as a viable immunotherapeutic alternative for combating MDR Ab infection.
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