High-Throughput Protein Engineering Improves the Antigenicity and Stability of Soluble HIV-1 Envelope Glycoprotein SOSIP Trimers.

Soluble envelope glycoprotein (Env) trimers (SOSIP.664 gp140) are attractive HIV-1 vaccine candidates, with structures that mimic the native membrane-bound Env spike (gp160). Since engineering trimers can be limited by the difficulty of rationally predicting beneficial mutations, here we used a more comprehensive mutagenesis approach with the goal of identifying trimer variants with improved antigenic and stability properties. We created 341 cysteine pairs at predicted points of stabilization throughout gp140, 149 proline residue substitutions at every residue of the gp41 ectodomain, and 362 space-filling residue substitutions at every hydrophobic and aromatic residue in gp140. The parental protein target, the clade B strain B41 SOSIP.664 gp140, does not bind the broadly neutralizing antibody PGT151 and so was used here to identify improved variants that also provide insight into the structural basis for Env antigenicity. Each of the 852 mutants was expressed in human cells and screened for antigenicity using four different monoclonal antibodies (MAbs), including PGT151. We identified 29 trimer variants with antigenic improvements derived from each of the three mutagenesis strategies. We selected four variants (Q203F, T538F, I548F, and M629P) for more comprehensive biochemical, structural, and antigenicity analyses. The T538F substitution had the most beneficial effect overall, including restoration of the PGT151 epitope. The improved B41 SOSIP.664 trimer variants identified here may be useful for vaccine and structural studies.IMPORTANCE Soluble Env trimers have become attractive HIV-1 vaccine candidates, but the prototype designs are capable of further improvement through protein engineering. Using a high-throughput screening technology (shotgun mutagenesis) to create and evaluate 852 variants, we were able to identify sequence changes that were beneficial to the antigenicity and stability of soluble trimers based on the clade B B41 env gene. The strategies described here may be useful for identifying a wider range of antigenically and structurally improved soluble trimers based on multiple genotypes for use in programs intended to create a broadly protective HIV-1 vaccine.

Exploring the Effect of Conjugation Site and Chemistry on the Immunogenicity of an anti-Group B Streptococcus Glycoconjugate Vaccine Based on GBS67 Pilus Protein and Type V Polysaccharide.

We have recently described a method for tyrosine-ligation of complex glycans that was proven efficient for the site selective coupling of GBS capsular polysaccharides (PSs). Herein, we explored the effect of conjugation of type V polysaccharide onto predetermined lysine or tyrosine residues of the GBS67 pilus protein with the dual role of T-cell carrier for the PS and antigen. For the preparation of a conjugate at predetermined lysine residues of the protein, we investigated a two-step procedure based on microbial Transglutaminase (mTGase) catalyzed insertion of a tag bearing an azide for following copper-free strain-promoted azide-alkyne [3 + 2] cycloaddition (SPAAC) with the polysaccharide. Two glycoconjugates were obtained by tyrosine-ligation through the known SPAAC and a novel thiol-maleimide addition based approach. Controls were prepared by random conjugation of PSV to GBS67 and CRM197, a carrier protein present in many commercial vaccines. Immunological evaluation in mice showed that all the site-directed constructs were able to induce good levels of anti-polysaccharide and anti-protein antibodies inducing osponophagocytic killing of strains expressing individually PSV or GBS67. GBS67 randomly conjugated to PSV showed carrier properties similar to CRM197. Among the tested site-directed conjugates, tyrosine-directed ligation and thiol-malemide addition was elected as the best combination to ensure production of anti-polysaccharide and anti-protein functional antibodies (in vitro opsonophagocytic killing titers) comparable to the controls made by random conjugation, while avoiding anti-linker antibodies. Our findings demonstrate that (i) mTGase based conjugation at lysine residues is an alternative approach for the synthesis of large capsular polysaccharide-protein conjugates; (ii) GBS67 can be used with the dual role of antigen and carrier for PSV; and (iii) thiol-maleimide addition in combination with tyrosine-ligation ensures the production of anti-polysaccharide and anti-protein functional antibodies while maintaining low levels of anti-linker antibodies. Site-specific conjugation methods aid in defining conjugation site and chemistry in carbohydrate-protein conjugates.

Tyrosine-directed conjugation of large glycans to proteins via copper-free click chemistry.

We have demonstrated that the insertion of alkyne-containing bifunctional linkers into the tyrosine residues of the carrier protein, followed by the copper mediated azide-alkyne [3 + 2] cycloaddition of carbohydrates, is a robust approach for the preparation of glycoconjugates with defined glycans, carrier, and connectivity. Conjugation of Group B Streptococcus (GBS) capsular polysaccharides to streptococcal pilus protein could extend the vaccine coverage to a variety of strains. Application of our protocol to these large charged polysaccharides occurred at low yields. Herein we developed a tyrosine-directed conjugation approach based on the copper-free click chemistry of sugars modified with cyclooctynes, which enables efficient condensation of synthetic carbohydrates. Most importantly, this strategy was demonstrated to be more effective than the corresponding copper catalyzed reaction for the insertion of GBS onto the tyrosine residues of GBS pilus proteins, previously selected as vaccine antigens through the so-called reverse vaccinology. Integrity of protein epitopes in the modified proteins was ascertained by competitive ELISA, and conjugation of polysaccharide to protein was confirmed by SDS page electrophoresis and immunoblot assays. The amount of conjugated polysaccharide was estimated by high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). The described technology is particularly suitable for proteins used with the dual role of vaccine antigen and carrier for the carbohydrate haptens.

Investigating the immunodominance of carbohydrate antigens in a bivalent unimolecular glycoconjugate vaccine against serogroup A and C meningococcal disease.

Multicomponent constructs, obtained by coupling different glycans to the carrier protein, have been proposed as a way to co-deliver multiple surface carbohydrates targeting different strains of one pathogen and reduce the number of biomolecules in the formulation of multivalent vaccines. To assess the feasibility of this approach for anti-microbial vaccines and investigate the potential immunodominance of one carbohydrate antigen over the others in these constructs, we designed a bivalent unimolecular vaccine against serogroup A (MenA) and C (MenC) meningococci, with the two different oligomers conjugated to same molecule of carrier protein (CRM197). The immune response elicited in mice by the bivalent MenAC construct was compared with the ones induced by the monovalent MenA and MenC vaccines and their combinations. After the second dose, the bivalent construct induced good levels of anti-MenA and anti-MenC antibodies with respect to the controls. However, the murine sera from the MenAC construct exhibited good anti-MenC bactericidal activity, and very low anti-MenA functionality when compared to the monovalent controls. This result was explained with the diverse relative avidities against MenA and MenC polysaccharides, which were measured in the generated sera. The immunodominant effect of the MenC antigen was fully overcome following the third immunization, when sera endowed with higher avidity and excellent bactericidal activity against both MenA and MenC expressing strains were elicited. Construction of multicomponent glycoconjugate vaccines against microbial pathogens is a feasible approach, but particular attention should be devoted to study and overcome possible occurrence of immune interference among the carbohydrates.

Synthesis and immunological evaluation of protein conjugates of Neisseria meningitidis X capsular polysaccharide fragments.

A vaccine to prevent infections from the emerging Neisseria meningitidis X (MenX) is becoming an urgent issue. Recently MenX capsular polysaccharide (CPS) fragments conjugated to CRM197 as carrier protein have been confirmed at preclinical stage as promising candidates for vaccine development. However, more insights about the minimal epitope required for the immunological activity of MenX CPS are needed. We report herein the chemical conjugation of fully synthetic MenX CPS oligomers (monomer, dimer, and trimer) to CRM197. Moreover, improvements in some crucial steps leading to the synthesis of MenX CPS fragments are described. Following immunization with the obtained neoglycoconjugates, the conjugated trimer was demonstrated as the minimal fragment possessing immunogenic activity, even though significantly lower than a pentadecamer obtained from the native polymer and conjugated to the same protein. This finding suggests that oligomers longer than three repeating units are possibly needed to mimic the activity of the native polysaccharide.

Anti-Group B Streptococcus Glycan-Conjugate Vaccines Using Pilus Protein GBS80 As Carrier and Antigen: Comparing Lysine and Tyrosine-directed Conjugation.

Gram-positive Streptococcus agalactiae or group B Streptococcus (GBS) is a leading cause of invasive infections in pregnant women, newborns, and elderly people. Vaccination of pregnant women represents the best strategy for prevention of neonatal disease, and GBS polysaccharide-based conjugate vaccines are currently under clinical testing. The potential of GBS pilus proteins selected by genome-based reverse vaccinology as protective antigens for anti-streptococcal vaccines has also been demonstrated. Dressing pilus proteins with surface glycan antigens could be an attractive approach to extend vaccine coverage. We have recently developed an efficient method for tyrosine-directed ligation of large glycans to proteins via copper-free azide-alkyne [3 + 2] cycloaddition. This method enables targeting of predetermined sites of the protein, ensuring that protein epitopes are preserved prior to glycan coupling and a higher consistency in glycoconjugate batches. Herein, we compared conjugates of the GBS type II polysaccharide (PSII) and the GBS80 pilus protein obtained by classic lysine random conjugation and by the recently developed tyrosine-directed ligation. PSII conjugated to CRM197, a carrier protein used for vaccines in the market, was used as a control. We found that the constructs made from PSII and GBS80 were able to elicit murine antibodies recognizing individually the glycan and protein epitopes on the bacterial surface. The generated antibodies were efficacious in mediating opsonophagocytic killing of strains expressing exclusively PSII or GBS80 proteins. The two glycoconjugates were also effective in protecting newborn mice against GBS infection following vaccination of the dams. Altogether, these results demonstrated that polysaccharide-conjugated GBS80 pilus protein functions as a carrier comparably to CRM197, while maintaining its properties of protective protein antigen. Glycoconjugation and reverse vaccinology can, therefore, be combined to design vaccines with broad coverage. This approach opens a path to a new generation of vaccines. Tyrosine-ligation allows creation of more homogeneous vaccines, correlation of the immune response to defined connectivity points, and fine-tuning of the conjugation site in glycan-protein conjugates.

Recombinant Clostridium difficile toxin fragments as carrier protein for PSII surface polysaccharide preserve their neutralizing activity.

Clostridium difficile is a Gram-positive bacterium and is the most commonly diagnosed cause of hospital-associated and antimicrobial-associated diarrhea. Despite the emergence of epidemic C. difficile strains having led to an increase in the incidence of the disease, a vaccine against this pathogen is not currently available. C. difficile strains produce two main toxins (TcdA and TcdB) and express three highly complex cell-surface polysaccharides (PSI, PSII and PSIII). PSII is the more abundantly expressed by most C. difficile ribotypes offering the opportunity of the development of a carbohydrate-based vaccine. In this paper, we evaluate the efficacy, in naive mice model, of PSII glycoconjugates where recombinant toxins A and B fragments (TcdA_B2 and TcdB_GT respectively) have been used as carriers. Both glycoconjugates elicited IgG titers anti-PSII although only the TcdB_GT conjugate induced a response comparable to that obtained with CRM197. Moreover, TcdA_B2 and TcdB_GT conjugated to PSII retained the ability to elicit IgG with neutralizing activity against the respective toxins. These results are a crucial proof of concept for the development of glycoconjugate vaccines against C. difficile infection (CDI) that combine different C. difficile antigens to potentially prevent bacterial colonization of the gut and neutralize toxin activity.

Synthetically defined glycoprotein vaccines: current status and future directions.

Primary examples in vaccine design have shown good levels of carbohydrate-specific antibody generation when raised using extracted or fully synthetic capsular polysaccharide glycans covalently coupled to a protein carrier. Herein, we cover recent clinical developments of carbohydrate-based vaccines and describe how novel cutting-edge methodology for the total synthesis of oligosaccharides and for the precise placement of carbohydrates at pre-determined sites within a protein may be used to further improve the safety and efficacy of glycovaccines.

Immunoactivity of protein conjugates of carba analogues from Neisseria meningitidis a capsular polysaccharide.

Neisseria meningitidis type A (MenA) is a Gram-negative encapsulated bacterium that is a major cause of epidemic meningitis, especially in the sub-Saharan region of Africa. The development and manufacture of a liquid glycoconjugate vaccine against MenA are hampered by the poor hydrolytic stability of its capsular polysaccharide (CPS), consisting of (1→6)-linked 2-acetamido-2-deoxy-α-d-mannopyranosyl phosphate repeating units. The replacement of the ring oxygen with a methylene group to generate a carbocyclic analogue leads to enhancement of its chemical stability. Herein, we report conjugation of carbocyclic analogue monomer, dimer, and trimer to the protein carrier CRM197. After immunization in mice, only the conjugated trimer was able to induce specific anti-MenA polysaccharide IgG antibodies with in vitro bactericidal activity, although to a lesser extent than pentadecamer and hexamer oligomers obtained from mild acid hydrolysis of the native polysaccharide conjugated to the same protein carrier. This study represents the first proof-of-concept that hydrolytically stable structural analogues of saccharide antigens can be used for the development of efficacious antimicrobial preventative therapies. Conjugates with longer carbocyclic oligomers and/or precise acetylation patterns could further increase the induced immune response to a level comparable with those of commercially available anti-meningococcal glycoconjugate vaccines.