Chih-Hsiang Leng, Ih-Jen Su, Pele Chong
National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes (NHRI), Taiwan
A core platform technology for the efficient production of recombinant lipoprotein composed of lipoepitope-tagged peptide as the enhancer of antigenicity for novel subunit vaccines was established by National Health Research Institutes (NHRI), Taiwan.
This platform technology is intellectually protected, and has the following advantages: (1) high expression of recombinant lipoproteins at a level of 50 to 100 mg/L in patented E. coli strains; (2) precisely defined lipoprotein synthesis initiated from a patented leader sequence; (3) validated optimal cultivation conditions for robust lipoprotein production, and (4) verified novel downstream bioprocess for high-yield lipoprotein purification. The lipid moiety of recombinant lipoproteins is identical to that of bacterial lipoproteins, which is recognized as a potent immune potentiator for both innate and adaptive immune systems (Figure 1). We have unambiguously demonstrated the feasibility and safety of this platform technology with various targets, such as a meningococcal group B vaccine (MGBvac), a dengue vaccine (Denvac) and an HPV-based therapeutic vaccine (HPVvac).
High yield of lipidated immunogens with a built-in adjuvant activity
The recombinant subunit vaccine holds tremendous potential as an inexpensive, safe and efficacious vaccine for human use. A number of recombinant subunit vaccine candidates were designed in the post genomic era, but few have advanced to clinical trials because of their poor intrinsic immunogenicity failing in eliciting a strong cytotoxic T-lymphocyte (CTL) response. To overcome this hurdle, the research team at NHRI has developed a platform technology for high yield of recombinant lipoproteins . This technology offers a synergistic combination of immunogen with the adjuvant function into a single fusion peptide for the designated recombinant subunit vaccine. Further, we have identified a variant E. coli strain, C43 (DE3), that has a multi-layer internal membrane for the expression of homologous and heterlogous lipoproteins with high yield. As testament to this platform technology, a lipoprotein Ag473 based meningococcal group B vaccine candidate (MGBvac) has been developed and successfully manufactured at the cGMP facility of National Institute of Infectious Disease and Vaccinology (NIIDV), NHRI. The MGBvac has been approved for IND by the Taiwan Food and Drug Administration (TFDA), and a Phase I trial has been scheduled.
Identification of a leader sequence necessary for expressing an antigen of non-lipoprotein as a recombinant lipoprotein
We chose a dengue subunit vaccine as an example to explore the feasibility of recombinant lipoproteins as vaccines. Contrary to what we believed in the past that a signal peptide is sufficient to convert a non-lipidated protein into a lipoprotein, the fusion of domain III of the E protein (E3) of dengue virus with the lipopeptide leader failed to express the recombinant lipo-E3 (rlipo-E3) to a detectable quantity. The same is true for other peptides, such as the TraK precursor, the peptidoglycan-associated precursor, the acriflavine-resistance protein E precursor, the membrane-bound lytic murein transglycosylase D precursor and the flagellar L-ring protein precursor. These results suggested that more lipopeptide sequences may be needed to express the rlipo-E3 to a scalable quantity. Therefore, E3 was individually fused to three different truncated fragments of Ag473 varying in the N-terminal sequences. We were able to identify a leader sequence (peptide sequence D1) containing more amino acids than the original signal peptide, that could substantially enhance the expression level of recombinant lipidated E3 (rlipo-D1E3) . This major breakthrough changes the landscape of subunit vaccine development because it makes the synergistic combination of an immunogen with a lipid adjuvant possible.
Characterization of a recombinant lipoprotein
The study of post-translational modifications of lipoproteins is typically problematic due to the hydrophobic property of the Figure 2. Enhancement of neutralizing antibody titers in animals immunized with rlipo-D1E3 without an adjuvant. Groups of BALB/c mice (n=5) were primed either with rlipo-D1E3 (20 µg) alone or with rE3 (20 µg) formulated in PBS or AlPO4. Two weeks later, all animals were boosted with the same immunogens used for the priming. Serum samples were collected two weeks after the last immunization. The ability of serum pools from different immunization groups to neutralize dengue virus-2 in vitro was assessed. The neutralizing antibody titer was determined as the fold of dilution of pooled immune sera that resulted in a 40% reduction in the number of foci compared to that obtained from the negative control group.fatty acid moieties, and it needs a certain amount of lipoprotein to go through the whole identification and analysis process. Thanks to our breakthrough in achieving a high lipoprotein yield, we could analyze the lipid identity and structure of a recombinant lipoprotein using the multi-stage mass spectrometry (MSn). Multiple-stage fragmentation analyses of the lipopeptides using both the positive and negative ion modes enabled us to identify the putative structure of the N-acyl-S-diacylglycerylcysteine as an amide bonding to palmitic acid via the N-terminal cysteine. Moreover, another unsaturated fatty acid of either hexadecenoic acid, or cyclopropaneoctanoic acid, or oleic acid or nonadecenoic acid of diacylglycerol residue in ester bonding has been identified . To our knowledge, this is the first time that the lipid structure of a Braun’s lipoprotein is elucidated at the molecular level. It is important to know that the recombinant lipid moiety contains an unsaturated bond which is different from that of a synthetic tripalmitoylated lipopeptide.
Innovative bioprocesses for the large-scale production of lipoproteins
In order to optimize the growth condition of E. coli C43 (DE3) strain, and to obtain a completely lipidated recombinant lipoprotein, a series of studies on the compositions of culture media were conducted. The M9 medium was found to be superior to the M63 and other media for the high-level expression of recombinant lipoproteins in the C43 (DE3) strain. Other factors such as culture nitrogen and carbon sources, phosphate, ferrous ions, calcium, magnesium, and pH that might influence the expression of lipoprotein have been reported . For downstream process development, an ordered procedure of membrane chromatography, ultra-diafiltration and reverse-phase chromatography was determined to be optimal for purification. The above mentioned technologies were intellectually protected through the submission of patent application to the office of Patent Cooperation Treaty (PCT).
Comparison of the immune-potentiation activity of a recombinant lipoprotein to that of a synthetic lipopeptide
Bone marrow-derived dendritic cells (BM-DCs) were used as a model to investigate the biological activity of the recombinant lipoprotein. Based on cytokine and chemokine assays, we demonstrated that the recombinant lipoprotein could stimulate innate immune responses by acting as a TOLL-like-receptor (TLR) agonist . The lipoprotein-induced production of cytokines from BM-DCs was in fact mediated by TLR2. The recombinant lipoprotein induced the activation of spleen cells and BM-DCs in wild-type and TLR4-deficient mice, but not in TLR2 knockout mice . Because the recombinant lipoprotein contains an unsaturated lipid which is different from that of the commercially available synthetic lipopeptide adjuvant, it is of interest to study whether recombinant lipoprotein would induce cytokines distinct from those by a synthetic lipopeptide adjuvant. We found that the recombinant lipoprotein induced a higher level of IL-23, IL-27 and MIP-1a secretion than that of a synthetic lipopeptide adjuvant, it is of interest to study whether recombinant lipoprotein would induce cytokines distinct from those by a synthetic lipopeptide adjuvant. We found that the recombinant lipoprotein induced a higher level of IL-23, IL-27 and MIP-1a secretion than that of a synthetic lipopeptide. These results suggest that the recombinant lipoprotein and the synthetic lipopeptide activate BM-DCs through the same receptor (TLR2) and thus via an identical MAPK signaling pathway. However, they may differ in the level of induction of gene expression to regulate immune responses .
The dengue subunit vaccine candidate, rlipo-E3, induces strong neutralizing antibody and memory immune responses
To fully explore the lipoprotein technology, a novel dengue subunit vaccine was rationally designed for comparing in animals the immunogenicity between the recombinant E3 and the recombinant lipidated E3 (that is, rlipo-D1E3 mentioned above). rlipo-D1E3 (not rE3) was capable of activating antigen-presenting cells, and strongly enhancing cellular and humoral immune responses in mouse (Figure 2) [1,5], rabbit and macaque [unpublished data]. Furthermore, with a single-dose immunization without extra adjuvant, rlipo-D1E3 could elicit neutralizing antibodies against all four serotypes of dengue viruses, and maintained both long term cellular and humoral immune memory responses in mouse . A quick, anamnestic neutralizing antibody response upon the dengue virus challenge was observed at week 28 post-immunization. These results demonstrated the possibility of a future tetravalent dengue vaccine that requires only one-dose vaccination with rlipo-D1E3 .
Recombinant HPV E7 lipoprotein induces strong cellular protective immunity
Since a lipidated immunogen like rlipo-D1E3 could elicit strong immune responses, it is of interest to investigate whether lipoprotein technology could be further applied for the development of immunotherapeutic vaccines against cancers. The E7 oncoprotein of human papillomavirus (HPV) is an ideal target and has been used as the immunogen for immunotherapeutics against HPV-associated tumors. The biologically inactive E7 (E7m) mutant protein was fused to the D1 sequence and expressed in E. coli C43(DE3) as recombinant lipoprotein rlipo-E7m. In in vitro studies, we found that rlipo-E7m could induce the maturation of bone marrow-derived dendritic cells in mouse through the TLR2 signaling pathway, skewing the immune responses toward the Th1 pathway for E7-specific CTL responses . We further studied the ability of rlipo-E7m in providing the protection against a TC-1 tumor cell challenge to the host in an animal model. Mice prophylactically immunized with two consecutive 10-µg doses of rlipo-E7m were found to be free of TC-1 tumor outgrowth. Experiments done with another therapeutic immunization model showed that the tumor volume in mice receiving a single dose of rlipo-E7m was less than 0.01 cm3 on day 40, whereas the tumor volume in mice treated with rE7m and that in the control group was over 2 cm3, respectively. In addition, we demonstrated that the CD8+ T cells played a major role in the anti-tumor activity in animals administered with rlipo-E7m (Figure 3). These results demonstrated that rlipo-E7m could be a promising candidate for treating HPV-associated tumors .
We have demonstrated that fusing a tumor antigen with a bacterial lipid moiety rendered this antigen capable of stimulating the BM-DCs through TLR2 to elicit a Th1 immune response. As a result, tumor growth was remarkably inhibited via antigen-specific CTL responses. Importantly, these lipoimmunogens could elicit both humoral and cellular immune responses. As a prophylactic vaccine against the viral pathogen, the lipidated immunogen was able to elicit stronger virus-neutralizing antibody responses than those of non-lipidated antigen formulated with an alum adjuvant. As for a therapeutic cancer vaccine, the lipidated immunogen could generate tumor-protective immunity in a mouse tumor model. This platform technology may provide a novel approach for the development of potent, safe prophylactic and therapeutic vaccines. Our study demonstrated the merit of recombinant lipoprotein-based immunogens with a built-in immuno-potentiation activity for the development of novel vaccines.
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