Optimization of the Synthesis and Conjugation of the Methyl Rhamnan Tip of Pseudomonas aeruginosa A-Band Polysaccharide and Immunogenicity Evaluation for the Continued Development of a Potential Glycoconjugate Vaccine

Pseudomonas aeruginosa is an antimicrobial-resistant bacterium that has no vaccine approved for human use. Additionally, it has been identified by the World Health Organization as a priority pathogen for novel vaccines and therapeutic development. We previously developed a synthetic mimic of the A-band polysaccharide tip that showed promise in terms of immunogenicity for use as a glycoconjugate vaccine. In this current manuscript, we improve upon the previous work to continue the development of this glycoconjugate vaccine. Herein, we report a higher-yielding synthesis of mimics containing a handle and a spacer that improved conjugation efficiency, resulting in better carbohydrate-to-protein ratios and also good immunogenicity of these conjugates in mice and rabbits. The data suggested that perhaps only a tetrasaccharide was required to induce an immune response capable of recognizing whole cells of P. aeruginosa.

Pseudomonas aeruginosa (Pa) is a pathogen that has become of considerable concern due to its resistance to most commonly used antibiotics, including carbapenems. 1 This antimicrobial resistance (AMR) has prompted the World Health Organization and others to state that priorities include preventing infections by developing novel vaccines against AMR bacteria such as Pa due to the high mortality risk and the economic cost of these infections. 2,3Antibiotic-resistant Pa is often respiratoracquired and affects especially patients afflicted with cystic fibrosis, causing pneumonia and also a range of acute infections that can lead to sepsis. 4There is currently no vaccine on the market against Pa; however, a range of vaccines are being developed. 5,6−12 The A-band polysaccharide of Pa exhibits a methylated Drhamnose pentasaccharide tip at its nonreducing end. 11In the study by Cairns et al., we have conjugated the natural antigen extracted from the A-band polysaccharide to a carrier protein and immunized mice and rabbits with this conjugate. 11This antigen is recognized by a mAb (1B1) generated from the natural pentasaccharide unit. 11As mAb 1B1 exhibits opsonophagocytic killing activity, the pentasaccharide tip is an interesting target antigen to further pursue as a vaccine. 11s the extraction of the natural antigen was low-yielding from the bacterial biomass, an alternative and potentially more efficient approach to generate this antigen for a glycoconjugate vaccine was synthesis. 12The synthesis of the oligosaccharide mimics was achieved with an overall yield of 3.6, 1.7, and 1% for the trisaccharide, tetrasaccharide, and pentasaccharide, respectively. 12The synthetically generated mimics were confirmed as inhibitors via inhibition ELISA.All three mimics inhibited the binding of mAb 1B1 to its target LPS, suggesting that all three could be potentially used as an antigen. 11This could be significant for future commercialization of a glycoconjugate vaccine as the yield to synthesize a pentasaccharide is significantly lower than to synthesize a trisaccharide.All three mimics were conjugated to human serum albumin (HSA) via direct reductive amination of the anomeric aldehyde and led to 6 oligosaccharide units per HSA. 12 The resulting conjugates elicited good IgM and IgGspecific responses in mice. 12espite the encouraging results obtained from this original work, we strived to improve our conjugates to increase their immunogenicity.At first, we aimed to achieve the conjugation of the mimics to cross-reactive material 197 (CRM 197 ), as opposed to HSA, as CRM 197 is widely used in the clinic and is generally regarded as a safe and effective carrier protein. 13irect conjugation of a saccharide to a protein has the advantage of reducing the number of synthetic steps required and not introducing potential additional linker molecules that could elicit an immune response.However, factors such as steric hindrance, distance between the epitope and the carrier, and reactivity of the anomeric aldehyde for direct reductive amination can impact the effectivity of the conjugation negatively. 14,15A wide range of handles and spacers have been described in the past; aminoalkyl glycosides, in particular, have been widely used, attached at the anomeric position of a range of carbohydrates, as handles in the context of glycoconjugate vaccines. 16,17We thus envisioned that adding a handle (Figure 1) to the reducing end of the mimics might allow us to use different linking strategies to achieve improved conjugation to CRM 197 and with a higher carbohydrate-toprotein ratio than in our previous work (6 carbohydrates per HSA protein).Such strategies include activating CRM 197 to its aminooxy form, for example. 18,19This potentially higher ratio might also increase the immunogenicity of the conjugate.
Herein, we report the synthesis of the A-band polysaccharide tip mimics composed of 3-O-methyl-D-rhamnose repeats, including a trisaccharide, tetrasaccharide, and pentasaccharide with a handle at the reducing end.We implemented the usage of a heterobifunctional spacer composed of an aldehyde protected with a hemiacetal and an activated N-hydroxysuccinimide ester, as described by Pozsgay. 20We also report improvements to synthetic yields and a reduction in the number of steps for early intermediates.Additionally, an improved degree of conjugation of the generated mimics to CRM 197 after the derivatization of CRM 197 to its aminooxy form is reported.Finally, we evaluate the immune response to the CRM 197 mimics conjugates in mice and rabbits, establish immunogenicity, and potentially identify the minimum length of oligosaccharide that effectively mimics the natural antigen.We also evaluated whether rabbit and mouse sera bind to whole killed cells from a range of strains, including clinical isolates.

■ RESULTS AND DISCUSSION
To add a handle at the reducing end of the pentasaccharide tip mimics, we had to restock intermediate S1.Previously, the 3-O-methyl-D-rhamnose mimics we had generated had been synthesized using 1-O-benzyl intermediates (Scheme 1); however, late-stage deprotection of the anomeric benzyl group with HCl in acetonitrile proved challenging with a 65% yield, resulting in a low overall yield for the synthesis of S1.
It was envisioned that instead, a p-tolyl thioglycoside could be first generated with the p-tolyl group as an anomeric protecting group throughout the synthesis as well as a glycosyl donor during late-stage glycosylation as previously implemented by others. 21,22o start, acetylated D-mannose obtained as described previously 12 was converted to thioglycoside S2 in two steps with excellent yields by treatment with thiocresol under acidic conditions followed by acetate deprotection using Zempleń deacetylation.C6 deoxygenation was achieved, as previously reported, via the Appel reaction (S3), followed by reduction with palladium hydroxide to obtain D-rhamnose thioglycoside S4 in moderate yields (Scheme S1).Next, 2,3-O-acetonide protection (S5) and O-4 benzylation lead to acetonide S6 in 90% yield over two steps.
After acetonide deprotection (S7), regioselective O-3 methylation was achieved in high yields via the formation of a tin acetal and selective methylation using methyl iodide and cesium fluoride, giving 3-O-methyl thioglycoside S8.The presence of methyl at 3 was confirmed via heteronuclear multiple bonds coupling of the O-Me 1 H singlet at 3.51 ppm and C3 at 82.0 ppm.Lastly, the thioglycosyl donor S1 was isolated in 89% yield after O-2 acetylation.The p-tolyl 1-thio group remained intact throughout the synthesis, allowing access to key intermediate S1 with 50% greater efficiency and three steps shorter compared to our previous synthesis.
With S1 in hand, N-boc-protected monomer, 2, was formed via glycosidic linkage with the glycosyl donor, N-bocethanolamine (Scheme 2).This was achieved in 84% yield by activating donor S1 with NIS and TfOH (HRMS m/z calcd for C 23 H 35 NO 8 H [M + H] + , 454.2435; found, 454.2435).Glycosyl acceptor 3 was formed in excellent yields by addition to a stirred solution of activated Pearlman's catalyst in methanol under hydrogen gas.Iterative 1,4 glycosylations between 4-O deprotected handles (5 di, 5 tri, 5 tetra) and building block S1 generated oligosaccharide handles (4 di, 4 tri, 4 tetra, and 4 penta).4-O-Benzyl deprotections lead to near quantitative yields.Glycosylation yields were higher than what we previously observed with the benzyl glycoside as an acceptor, with all yields being above 64%, while we previously observed yields below 69%.The α configuration was exclusively observed consequent with prior work and as verified by the J H1−C1 coupling constants. 12ligosaccharides 5 tri, 5 tetra, and 5 penta were globally deprotected using sodium methoxide followed by TFA for Boc deprotection, yielding the free amino handles 6 tri, 6 tetra, and 6 penta (ESI-MS: m/z calcd for C 37 H 67 O 21 NH [M + H] + , 862.4278; found, 862.4265) in quantitative yields.Without further purification, these free handles were then conjugated to an activated N-hydroxysuccinimide ester spacer containing a protected aldehyde.HPLC purification using C-18 produced pure antigens 1 tri, 1 tetra, and 1 penta in respectively 1.2, 1.4, and 1.6% yield for evaluation in colorimetric inhibition ELISA assays and protein conjugation.
Inhibition ELISA Assay.To ensure that the necessary conformation to mimic the natural antigen had been retained in the synthetic oligosaccharides with the handle and spacers, 1 tri, 1 tetra, and 1 penta were evaluated in an inhibition ELISA against mAb 1B1 that was raised to the natural antigen. 12n this colorimetric inhibition ELISA, we observed a reduction in absorbance for all of the saccharides, indicating that all bind to mAb 1B1 (Figure S1), with 1 penta blocking mAb 1B1 at a greater extent than 1 tetra than in turn 1 tri.
These results therefore corroborate our prior data on prepared oligosaccharides (without handle and spacer). 11,12he addition of the handle and spacer in these oligosaccharides does not alter the conformation of the oligosaccharide nor affect the oligosaccharide from binding and blocking mAb 1B1.Thus, the synthesized oligosaccharides with a handle and spacer effectively mimic the epitope recognized by mAb 1B1 on Pa.
Conjugation of Oligosaccharide to Activated CRM 197 and Activated BSA to Prepare Glycoconjugates.Lysines

ACS Infectious Diseases
on CRM 197 and BSA were first modified to their bromoacetic amide derivatives.Then, a reaction with (aminooxy)-1propanethiol led to aminooxy-activated CRM 197 /BSA.The conjugates were prepared from 1 tri, 1 tetra, and 1 penta after deprotection of the hemiacetal to the aldehyde using acetic acid and formation of the stable oxime after conjugation to the aminooxy-activated CRM 197 /BSA (Scheme 3).The carbohydrate/protein ratio, as determined by MALDI-MS analyses, revealed a satisfactory ratio between 5 and 6 for BSA and a good ratio between 8 and 10 for CRM 197 (Table S1).Even though the results obtained with BSA were similar to our prior work (albeit with HSA), conjugation to CRM 197 was improved with 8−10 saccharides as opposed to 6 (for HSA).
Screening of Derived Mice Sera vs BSA Conjugates and LPS.To determine whether the new conjugates were immunogenic, the mice were immunized under a prime and two boosts schedule.We evaluated whether the BSA− oligosaccharide conjugates and Pa wt LPS were recognized by individual mice sera and compared the responses to the preimmune sera via ELISA.
All mice produced a good IgM response, albeit a moderate IgG response both specific to the carbohydrates and Pa wt LPS (Figures S2−S11).Since mice that received immunizations with the CRM-1 tetra and 1 penta conjugates showed an improved IgG response to Pa wt LPS in ELISA relative to mice that received the CRM-1 tri, it may be suggested from the mice data that the minimum length of oligosaccharide required to effectively mimic the natural antigen is a tetrasaccharide Screening of Derived Rabbit Sera vs BSA Conjugates and LPS.With the encouraging mice results described above, we elected to evaluate the capacity of the conjugates to elicit a specific immune response in rabbits.As for mice, we evaluated whether the BSA−oligosaccharide conjugates and Pa wt LPS were recognized by individual rabbit sera.All rabbits produced a good immune response to the conjugates, as illustrated by their recognition of the BSA−oligosaccharide conjugates and Pa wt LPS.Differently from mice that exhibited a modest response, all rabbits produced a strong response with end-point titers in the 1:10,000 range that were capable of recognizing three LPS structures (Pa wt, Pa wzy5457, and Pa wzy5458 LPS) (Figures 2 and S12).
Screening of Derived Mice and Rabbit Sera vs Killed Whole Cells.To verify whether postimmune mice and rabbit sera would recognize a range of strains, which is important to evaluate the strain coverage ability of the conjugates, whole-cell ELISA was performed on a range of Pa-killed cells (Table S2), including a wt strain, strains with mutations in their genes thought to be related to the A-band methyl rhamnan, serotype strains most commonly encountered in a clinical setting, and a small group of clinical isolates.
Postimmune mice sera pooled by the conjugate that they received showed recognition of approximately half the cells they were tested against (Figure S13).The CRM-1 tri conjugate showed a weaker response when compared to the clear-cut response provoked by the CRM-1 tetra and CRM-1 penta conjugate, illustrating that the methyl rhamnan tip epitope is visible in the context of the whole cells and that the synthetic conjugate was able to generate an immune response capable of recognizing this epitope elaborated on whole cells.
When postimmune rabbit sera were evaluated (Table S2 and Figures 3 and S14), a majority of the range of cells, including against a range of clinical isolates in our collection, were recognized, illustrating that the methyl rhamnan tip epitope is visible in the context of whole cells and that the conjugates (1 tri, 1 tetra, and 1 penta) were able to generate an immune response capable of recognizing this epitope, also illustrating the conservation of this epitope and the ability of oligosaccharide-based conjugates to be capable of generating the required immune response to afford recognition.
Consistent with the earlier data with LPS and BSA conjugates, whole-cell ELISA corroborated the requirement in mice for a tetrasaccharide as the minimum length of oligosaccharide necessary to facilitate an appropriate response.However, rabbit-derived sera did not exhibit this same minimum length requirement, as all conjugates were able to elicit a similar cross-reactive response.

■ CONCLUSIONS
Herein, we reported the synthesis of three targets: 3-O-methyl rhamnan trisaccharide, tetrasaccharide, and pentasaccharide, with an aminoethyl handle at the reducing end to create a glycoconjugate vaccine containing a mimic of the A-band polysaccharide tip of Pa.This improved synthesis demonstrated an increase in overall yield compared to our previous route (1.6% vs 1.0% overall yields for the pentasaccharide, for example) and with an added handle and spacer.Additionally, the addition of an amino handle at the reducing end of the sugar allowed for improved conjugation to CRM 197 , as exemplified by conjugates with carbohydrate loading of approximately 10 residues obtained for each oligosaccharide.
The results obtained with the immune sera from mice and rabbits indicate that CRM 197 conjugates of at least the synthetic 1 tetra and 1 penta representative of the methyl rhamnan A-band tip epitope are capable of provoking a specific immune response that recognizes the natural antigen elaborated on Pa wt LPS and whole cells representing the most commonly encountered serotypes in a clinical setting, and from a small collection of clinical isolates, illustrating their potential as viable alternatives to the isolated antigens as vaccine immunogens.Continued development will include performing opsonophagocytic assays with the polyclonal mice and rabbit sera and subsequently animal models.
There remains a shortfall of vaccines to combat the significant AMR threat of Pa, and this work has highlighted that these relatively simple oligosaccharides provide a potential synthetic route to an antigen for consideration as an alternative to antibiotics to combat this challenge.

■ METHODS
Reactants and reagents were purchased from MilliporeSigma, Oakwood Chemicals, or Fisher Scientific and used without further purification.NMR spectra were measured on either a Varian ( 1 H 500 MHz, 13 C 125 MHz), Bruker ( 1 H 600 MHz, 13 C 150 MHz), or Jeol ( 1 H 400 MHz, 13 C 100 MHz) spectrometer.Spectra were calibrated with the solvent's residual signals (CDCl 3 , 7.26 ppm for 1 H and 77.1 ppm for 13 C; CD 3 OD, 3.31 ppm for 1 H and 49.0 ppm for 13 C).NMR signals were assigned using standard two-dimensional NMR experiments (HSQC, COSY, HMBC).Purification occurred on CombiFlash RF and Biotage system with Silicycle silica columns.Mass-spectrometry data was recorded and analyzed using a single quadrupole detector 2 from Waters, and highresolution mass-spectrometry data were recorded and analyzed on a Waters Ultima using the LC/MS Calibrant Mix from  S2 for full details of strains).
Agilent as the internal standard.TLC was eluted using hexane/ ethyl acetate or MeOH/CH 2 Cl 2 as solvents and visualized with charring after dipping in a 5% H 2 SO 4 solution in absolute ethanol.
Conjugation of 1 tri, 1 tetra, and 1 penta to Activated CRM and Activated BSA.BSA and CRM 197 were activated as described prior 18 and within the Supporting Information.Then, 1 tri, 1 tetra, and 1 penta (3 mg/mL) were dissolved in 50% acetic acid.After 7 h at 37 °C, the reaction mixture was cooled and then lyophilized, which led to the deprotection of the acetal to the aldehyde for further conjugation with the aminooxy-proteins.Deprotected 1 tri, 1 tetra, or 1 penta (3 mg) were then separately dissolved in sodium phosphate buffer (50 μL, 200 mM, pH 6) and separately added to the aminooxy-proteins (CRM 197 -oxy, respectively 2.1, 1.7 and 1.5 mg).The amount of aminooxy-protein used was adjusted in each case to keep an approximate 8× molar excess of oligosaccharide per aminooxy on the protein.The product was isolated after 18 h at RT using an Amicon Ultra-10 30K MWCO filter against PBS (3×) and stored at 4 °C.The carbohydrate/protein ratio was determined by MALDI-MS (Table S1).
Immunization of Mice and Rabbits with Conjugates.Female BALB/c mice, 6 to 8 weeks old, were immunized intraperitoneally (3×).Each mouse received the same amount of conjugate, as well as SIGMA adjuvant, and PBS buffer at each time point.The mice were primed on day 0 and received boosters on days 21 and 42, and blood samples were taken on day 0, day 35, and day 56.Each mouse in group MRha3V received 3 μg of the trisaccharide conjugate and 28 μg of CRM, along with 50% v/v SIGMA adjuvant, and PBS buffer totaling 100 μL, administered intraperitoneally.Each mouse in group MRha4V received 3 μg of the tetrasaccharide conjugate and 25.5 μg of CRM, along with 50% v/v SIGMA adjuvant, and PBS buffer totaling 100 μL, administered intraperitoneally.Finally, each mouse in group MRha5V received 3 μg of the pentasaccharide and 23 μg of CRM, along with 50% v/v SIGMA adjuvant, and PBS buffer totaling 100 μL, administered intraperitoneally.Blood samples were obtained by the submandibular vein collection method to yield approximately 100 μL of serum after blood separation.
Female New England white rabbits, 1.5−2.0kg, were immunized three times subcutaneously at two separate sites for each immunization.Each rabbit received the same amount of oligosaccharide conjugate, as well as SIGMA adjuvant, and PBS buffer at each time point.The rabbits were primed on day 0 and received boosters on days 21 and 49, and blood samples were taken on day 0, day 35, and day 70.Each rabbit in group RRha3V received 20 μg of the trisaccharide conjugate and 188 μg of CRM 197 , along with 50% v/v SIGMA adjuvant, and PBS buffer totaling 500 μL, administered subcutaneously with 250 μL in each of 2 sites.Each rabbit in group RRha4V received 20 μg of the tetrasaccharide conjugate and 170 μg of CRM 197 , along with 50% v/v SIGMA adjuvant, and PBS buffer totaling 500 μL, administered subcutaneously with 250 μL in each of 2 sites.Each rabbit in group RRha5V received 20 μg of the pentasaccharide and 151.5 μg of CRM 197 , along with 50% v/v SIGMA adjuvant, and PBS buffer totaling 500 μL, administered subcutaneously with 250 μL in each of 2 sites.Blood samples were obtained by marginal ear vein collection method to yield approximately 500 μL of serum after blood separation.
ELISA.ELISA experiments with mice and rabbit sera against BSA conjugates, LPS, and whole cells were performed as described previously. 11ASSOCIATED CONTENT * sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsinfecdis.4c00049.
Additional experimental procedures, experimental data, NMR spectra, Maldi spectra, inhibition ELISA results, mice ELISA results, rabbit ELISA results from BSA conjugates, screening of bacterial clinical cell isolates with mice, and rabbit pre and post immune sera.(PDF)