On-demand biomanufacturing of protective conjugate vaccines

Abstract

Conjugate vaccines are among the most effective methods for preventing bacterial infections. However, existing manufacturing approaches limit access to conjugate vaccines due to centralized production and cold chain distribution requirements. To address these limitations, we developed a modular technology for in vitro conjugate vaccine expression (iVAX) in portable, freeze-dried lysates from detoxified, nonpathogenic Escherichia coli. Upon rehydration, iVAX reactions synthesize clinically relevant doses of conjugate vaccines against diverse bacterial pathogens in 1 hour. We show that iVAX-synthesized vaccines against Francisella tularensis subsp. tularensis (type A) strain Schu S4 protected mice from lethal intranasal F. tularensis challenge. The iVAX platform promises to accelerate development of new conjugate vaccines with increased access through refrigeration-independent distribution and portable production.

Fig. 1. iVAX platform enables on-demand and portable production of antibacterial vaccines.

The iVAX platform provides a rapid means to develop and distribute conjugate vaccines against bacterial pathogens. Expression of pathogen-specific polysaccharide antigens (e.g., CPS and O-PS) and a bacterial oligosaccharyltransferase enzyme in nonpathogenic E. coli with detoxified lipid A yields low-endotoxin lysates containing all of the machinery required for synthesis of conjugate vaccines. Reactions catalyzed by iVAX lysates can be used to produce conjugates containing licensed carrier proteins and can be freeze-dried without loss of activity for refrigeration-free transportation and storage. Freeze-dried reactions can be activated at the point of care via simple rehydration and used to reproducibly synthesize immunologically active conjugate vaccines in ~1 hour.

Fig. 2. In vitro synthesis of licensed conjugate vaccine carrier proteins.

(A) All four carrier proteins used in approved conjugate vaccines were synthesized solubly in vitro, as measured via ¹⁴C-leucine incorporation. These include H. influenzae PD, the N. meningitidis porin protein (PorA), and genetically detoxified variants of the C. diphtheriae toxin (CRM197) and the C. tetani toxin (TT). Additional immunostimulatory carriers, including E. coli MBP and the fragment C (TTc) and light chain (TTlight) domains of TT, were also synthesized solubly. Values represent means and error bars represent SDs of biological replicates (n = 3). (B) Full-length product was observed for all proteins tested via Western blot. Different exposures are indicated with solid lines. Molecular weight ladder is shown at left. αHis, anti–hexa-histidine antibody.

Fig. 3. Reproducible glycosylation of proteins with FtO-PS in iVAX.

(A) iVAX lysates were prepared from cells expressing CjPglB and the biosynthetic pathway encoding FtO-PS. (B) Glycosylation of sfGFP^217-DQNAT with FtO-PS was only observed when CjPglB, FtO-PS, and the preferred DQNAT glycosylation sequence (sequon) were present in the reaction (lane 3). When plasmid DNA was omitted, sfGFP^217-DQNAT synthesis was not observed. (C) Biological replicates of iVAX reactions producing sfGFP^217-DQNAT using the same lot (left) or different lots (right) of iVAX lysates demonstrated reproducibility of reactions and lysate preparation. Top panels show signal from probing with αHis to detect the carrier protein, middle panels show signal from probing with commercial anti–FtO-PS antibody (αFtO-PS), and bottom panels show αHis and αFtO-PS signals merged. Images are representative of at least three biological replicates. Dashed lines indicate that samples are from nonadjacent lanes of the same blot with the same exposure. Molecular weight ladders are shown at the left of each image.

Fig. 4. On-demand production of conjugate vaccines against F. tularensis using iVAX.

(A) iVAX reactions were prepared from lysates containing CjPglB and FtO-PS and primed with plasmid encoding immunostimulatory carriers, including those used in licensed vaccines. (B) We observed on-demand synthesis of anti-F. tularensis conjugate vaccines for all carrier proteins tested. Conjugates were purified using Ni-NTA agarose from 1-ml iVAX reactions lasting ~1 hour. Top panels show signal from probing with αHis to detect the carrier protein, middle panels show signal from probing with αFtO-PS, and bottom panels show αHis and αFtO-PS signals merged. Images are representative of at least three biological replicates. Dashed lines indicate that samples are from nonadjacent lanes of the same blot with the same exposure. Molecular weight ladders are shown at the left of each image.

Fig. 5. Detoxified, lyophilized iVAX reactions produce conjugate vaccines.

(A) iVAX lysates were detoxified via deletion of lpxM and expression of F. tularensis LpxE in the source strain used for lysate production. (B) Growth rates of CLM24 WT and CLM24 ∆lpxM strains. No growth defects were observed across two clones of the knockout strain. Values represent means and error bars represent SDs of n = 4 replicates. ns, not significant. (C) The resulting lysates exhibited significantly reduced endotoxin activity, as measured by activation of human TLR4 in HEK-Blue hTLR4 reporter cells. **P = 0.003, as determined by two-tailed t test. Values represent means and error bars represent SDs of n = 3 replicates. (D) FtO-PS conjugate vaccines produced and purified from detoxified iVAX reactions contained 0.21 ± 0.3 EU/10-μg dose, as measured by human TLR4 activation. Dashed line represents endotoxin levels reported in commercial conjugate vaccines (<12 EU/dose). Value represents mean and error bars represent SD of n = 6 replicates. (E) iVAX reactions producing sfGFP^217-DQNAT were run immediately or following lyophilization and rehydration. (F) Glycosylation activity was preserved following lyophilization, demonstrating the potential of iVAX reactions for portable biosynthesis of conjugate vaccines. Top panel shows signal from probing with αHis to detect the carrier protein, middle panel shows signal from probing with αFtO-PS, and bottom panel shows αHis and αFtO-PS signals merged. Images are representative of at least three biological replicates. Molecular weight ladder is shown at the left of each image.

Fig. 6. iVAX-derived conjugates elicit FtLPS-specific antibodies and protect mice from lethal pathogen challenge.

(A) Freeze-dried iVAX reactions assembled using detoxified lysates were used to synthesize anti–F. tularensis conjugate vaccines for immunization studies. (B) Groups of BALB/c mice were immunized subcutaneously with PBS or 7.5 μg of purified, cell-free synthesized unmodified or FtO-PS–conjugated carrier proteins. FtO-PS–conjugated MBP^4xDQNAT prepared in living E. coli cells using PCGT was used as a positive control. Each group was composed of six mice except for the PBS control group, which was composed of five mice. Mice were boosted on days 21 and 42 with identical doses of antigen. FtLPS-specific IgG titers were measured by enzyme-linked immunosorbent assay (ELISA) in endpoint (day 70) serum of individual mice (black dots) with F. tularensis LPS immobilized as antigen. Mean titers of each group are also shown (red lines). iVAX-derived conjugates elicited significantly higher levels of FtLPS-specific IgG compared to all other groups (**P < 0.01, Tukey-Kramer post hoc test). (C) IgG1 and IgG2a subtype titers measured by ELISA from endpoint serum revealed that iVAX-derived conjugates boosted production of FtO-PS–specific IgG1 compared to all other groups tested (**P < 0.01, Tukey-Kramer post hoc test). These results indicate that iVAX conjugates elicited a T_H2-biased immune response typical of most conjugate vaccines. Values represent means and error bars represent SEs of FtLPS-specific IgGs detected by ELISA.

Fig. 7. iVAX-derived conjugates protect mice from lethal F. tularensis challenge.

(A) Groups of five BALB/c mice were immunized intraperitoneally (IP) with PBS or 10 μg of purified, cell-free synthesized unmodified or FtO-PS–conjugated carrier proteins. FtO-PS–conjugated carriers prepared in living E. coli cells using PCGT were used as positive controls. Mice were boosted on days 21 and 42 with identical doses of antigen. IN, intranasally. (B) On day 56, FtLPS-specific IgG titers were measured by ELISA in serum of individual mice immunized with PBS or anti–F. tularensis conjugate vaccines (FtO-PS-CV) (black dots) with F. tularensis LPS immobilized as antigen. Mean titers of each group are also shown (red lines). Only iVAX-derived conjugates elicited significantly higher levels of FtLPS-specific IgG compared to PBS immunized controls across all carrier proteins tested (**P < 0.01, Tukey-Kramer post hoc test; CV, conjugate vaccine). On day 66, mice were challenged intranasally with 6000 CFU (60 times the intranasal LD₅₀) F. tularensis subsp. holarctica LVS Rocky Mountain Laboratories and monitored for survival for an additional 25 days. Kaplan-Meier curves for immunizations with (C) MBP^4xDQNAT, (D) PD^4xDQNAT, and (E) EPA^DNNNS-DQNRT as the carrier protein are shown. iVAX-derived vaccines protected mice from lethal pathogen challenge as effectively as vaccines synthesized using the state-of-the-art PGCT approach. *P < 0.05; **P < 0.01, Fisher’s exact test.