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.

Shigella spp. invade the colonic epithelium and cause bacillary dysentery in humans. Individuals living in areas that lack access to clean water and sanitation are the most affected. Even though infection can be treated with antibiotics, Shigella antimicrobial drug resistance complicates clinical management. Despite decades of effort, there are no licensed vaccines to prevent shigellosis. The highly conserved invasion plasmid antigens (Ipa), which are components of the Shigella type III secretion system, participate in bacterial epithelial cell invasion and have been pursued as vaccine targets. However, expression and purification of these proteins in conventional cell-based systems have been challenging due to solubility issues and extremely low recovery yields. These difficulties have impeded manufacturing and clinical advancement. In this study, we describe a new method to express Ipa proteins using the Xpress^+TM cell-free protein synthesis (CFPS) platform. Both IpaB and the C-terminal domain of IpaH1.4 (IpaH-CTD) were efficiently produced with this technology at yields > 200 mg/L. Furthermore, the expression was linearly scaled in a bioreactor under controlled conditions, and proteins were successfully purified using multimode column chromatography to > 95% purity as determined by SDS-PAGE. Biophysical characterization of the cell-free synthetized IpaB and IpaH-CTD using SEC-MALS analysis showed well-defined oligomeric states of the proteins in solution. Functional analysis revealed similar immunoreactivity as compared to antigens purified from E. coli. These results demonstrate the efficiency of CFPS for Shigella protein production; the practicality and scalability of this method will facilitate production of antigens for Shigella vaccine development and immunological analysis. Key points: • First report of Shigella IpaB and IpaH produced at high purity and yield using CFPS • CFPS-IpaB and IpaH perform similarly to E. coli–produced proteins in immunoassays • CFPS-IpaB and IpaH react with Shigella-specific human antibodies and are immunogenic in mice. Graphical

Group B Streptococcus (Streptococcus agalactiae or GBS) is the leading infectious cause of neonatal mortality, causing roughly 150,000 infant deaths and stillbirths annually across the globe. Approximately 20% of pregnant women are asymptomatically colonized by GBS, which is a major risk factor for severe fetal and neonatal infections as well as preterm birth, low birth weight, and neurodevelopmental abnormalities. Current clinical interventions for GBS infection are limited to antibiotics, and no vaccine is available. We previously described VAX-A1 as a highly effective conjugate vaccine against group A Streptococcus that is formulated with three antigens, SpyAD, streptolysin O, and C5a peptidase (ScpA). ScpA is a surface-expressed, well-characterized GAS virulence factor that shares nearly identical sequences with the lesser studied GBS homolog ScpB. Here, we show that GBS C5a peptidase ScpB cleaves human complement factor C5a and contributes to disease severity in the murine models of pneumonia and sepsis. Furthermore, antibodies elicited by GAS C5a peptidase bind to GBS in an ScpB-dependent manner, and VAX-A1 immunization protects mice against lethal GBS heterologous challenge. These findings support the contribution of ScpB to GBS virulence and underscore the importance of choosing vaccine antigens; a universal GAS vaccine such as VAX-A1 whose formulation includes GAS C5a peptidase may have additional benefits through some measure of cross-protection against GBS infections.

Periodontal diseases are chronic inflammatory diseases of the periodontium that result in progressive destruction of the soft and hard tissues supporting the teeth, and it is the most common cause of tooth loss among adults. In the US alone, over 100 million individuals are estimated to have periodontal disease. Subgingival bacteria initiate and sustain inflammation, and, although several bacteria have been associated with periodontitis,Porphyromonas gingivalishas emerged as the key etiological organism significantly contributing to the disease. Currently, intensive clinical maintenance strategies are deployed to mitigate the further progression of disease in afflicted individuals; however, these treatments often fail to stop disease progression, and, as such, the development of an effective vaccine for periodontal disease is highly desirable. We generated a conjugate vaccine, comprising of the purified capsular polysaccharide ofP. gingivalisconjugated to eCRM®, a proprietary and enhanced version of the CRM197 carrier protein with predetermined conjugation sites (Pg-CV). Mice immunized with alum adjuvanted Pg-CV developed robust serum levels of whole organism-specific IgG in comparison to animals immunized with unconjugated capsular polysaccharide alone. Using the murine oral bone loss model, we observed that mice immunized with the capsule-conjugate vaccine were significantly protected from the effects ofP. gingivalis-elicited oral bone loss. Employing a preclinical model of infection-elicited oral bone loss, our data support that a conjugate vaccine incorporating capsular polysaccharide antigen is effective in reducing the main clinical endpoint of periodontal disease—oral bone destruction. Further development of aP. gingivaliscapsule-based conjugate vaccine for preventing periodontal diseases is supported.

Chlamydia trachomatis (Ct) is the most common cause of bacterial sexually transmitted infections (STIs) worldwide. Ct infections are often asymptomatic in women, leading to severe reproductive tract sequelae. Development of a vaccine against Chlamydia is crucial. The Chlamydia major outer membrane protein (MOMP) is a prime vaccine antigen candidate, and it can elicit both neutralizing antibodies and protective CD4+ T cell responses. We have previously designed chimeric antigens composed of immunogenic variable regions (VDs) and conserved regions (CDs) of MOMP from Chlamydia muridarum (Cm) expressed into a carrier protein (PorB), and we have shown that these were protective in a mouse model of Cm respiratory infection. Here, we generated corresponding constructs based on MOMP from Ct serovar F. Preliminary structure analysis of the three antigens, PorB/VD1-3, PorB/VD1-4 and PorB/VD1-2-4, showed that they retained structure features consistent with those of PorB. The antigens induced robust humoral and cellular responses in mice with different genetic backgrounds. The antibodies were cross-reactive against Ct, but only anti-PorB/VD1-4 and anti-PorB/VD1-2-4 IgG antibodies were neutralizing, likely due to the antigen specificity. The cellular responses included proliferation in vitro and production of IFN-γ by splenocytes following Ct re-stimulation. Our results support further investigation of the PorB/VD antigens as potential protective candidates for a Chlamydia subunit vaccine.

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.