Development of an effective vaccine against the leading human bacterial pathogen group A Streptococcus (GAS) is a public health priority. The species defining group A cell wall carbohydrate (GAC, Lancefield antigen) can be engineered to remove its immunodominant N-acetylglucosamine (GlcNAc) side chain, implicated in provoking autoimmune cross-reactivity in rheumatic heart disease, leaving its polyrhamnose core (GACPR). Here we generate a novel protein conjugate of the GACPR and test the utility of this conjugate antigen in active immunization. Instead of conjugation to a standard carrier protein, we selected SpyAD, a highly conserved GAS surface protein containing both B-cell and T-cell epitopes relevant to the bacterium that itself shows promise as a vaccine antigen. SpyAD was synthesized using the XpressTM cell-free protein expression system, incorporating a non-natural amino acid to which GACPR was conjugated by site-specific click chemistry to yield high molecular mass SpyAD-GACPR conjugates and avoid disruption of important T-cell and B-cell immunological epitopes. The conjugated SpyAD-GACPR elicited antibodies that bound the surface of multiple GAS strains of diverse M types and promoted opsonophagocytic killing by human neutrophils. Active immunization of mice with a multivalent vaccine consisting of SpyAD-GACPR, together with candidate vaccine antigens streptolysin O and C5a peptidase, protected against GAS challenge in a systemic infection model and localized skin infection model, without evidence of cross reactivity to human heart or brain tissue epitopes. This general approach may allow GAC to be safely and effectively included in future GAS subunit vaccine formulations with the goal of broad protection without autoreactivity.
Surface-expressed
bacterial polysaccharides
are important vaccine antigens but must be conjugated to a carrier
protein for efficient antigen presentation and development of strong
memory B cell and antibody responses, especially in young children.
The commonly used protein carriers include tetanus toxoid (TT), diphtheria
toxoid (DT), and its derivative CRM197, but carrier-induced epitopic
suppression and bystander interference may limit the expanded use
of the same carriers in the pediatric immunization schedule. Recent
efforts to develop a vaccine against the major human pathogen group
A
Streptococcus
(GAS) have sought to combine two
promising vaccine antigens—the universally conserved group
A cell wall carbohydrate (GAC) with the secreted toxin antigen streptolysin
O (SLO) as a protein carrier; however, standard reductive amination
procedures appeared to destroy function epitopes of the protein, markedly
diminishing functional antibody responses. Here, we couple a cell-free
protein synthesis (CFPS) platform, allowing the incorporation of non-natural
amino acids into a C-terminally truncated SLO toxoid for the precise
conjugation to the polyrhamnose backbone of GAC. The combined immunogen
generated functional antibodies against both conserved GAS virulence
factors and provided protection against systemic GAS challenges. CFPS
may represent a scalable method for generating pathogen-specific carrier
proteins for multivalent subunit vaccine development.
Diarrhea caused by
Shigella
species results in long-term disability and mortality globally, disproportionally affecting younger children living in poor countries. Although it is treatable by antibiotics, the rapid and widespread emergence of resistant strains and the highly contagious nature of the disease compel the development of preventive tools.
Strain-promoted azide–alkyne
cycloaddition (SPAAC)
reactions
like click chemistry have the potential to be highly scalable, robust,
and cost-effective methods for generating small- and large-molecule
conjugates for a variety of applications. However, despite method
improvements, the rates of copper-based click chemistry reactions
continue to be much faster than the rates of copper-free click chemistry
reactions, which makes broader deployment of click chemistry challenging
from a safety and compatibility standpoint. In this study, we used
a zwitterionic detergent, namely, lauryldimethylamine
N
-oxide (LDAO), in a copper-free click chemistry reaction to investigate
its impact on the generation of conjugate vaccines (CVs). For this,
we utilized an Xpress cell-free protein synthesis (CFPS) platform
to generate a proprietary variant of CRM197 (eCRM) containing non-native
amino acids (nnAA) with azide-containing side chains as a carrier
protein for conjugation to several clinically relevant dibenzocyclooctyne
(DBCO)-derivatized
S. pneumoniae
serotypes
(types 3, 5, 18C, and 19A). For conjugation, we performed copper-free
click chemistry in the presence and absence of LDAO. Our results show
that the addition of LDAO significantly enhanced the reaction kinetics
to generate larger conjugates, which were similarly immunogenic and
equally stable to conjugates generated without LDAO. Most importantly,
the addition of LDAO substantially improved the efficiency of the
conjugation process. Thus, our results for the first time show that
the addition of a zwitterionic surfactant to a copper-free click chemistry
reaction can significantly accelerate the reaction kinetics along
with improving the efficiency of the conjugation process.
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