Escherichia coli σ70 promoters allow expression rate control at the cellular level in genome-integrated expression systems.

BACKGROUND: The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators (lacO) into the constitutive T5 and A1 promoter sequences. RESULTS: We showed that, in genome-integrated E. coli expression systems that used σ70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacIQ, on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level. CONCLUSIONS: Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

Pre-existing yellow fever immunity impairs and modulates the antibody response to tick-borne encephalitis vaccination.

Flaviviruses have an increasing global impact as arthropod-transmitted human pathogens, exemplified by Zika, dengue, yellow fever (YF), West Nile, Japanese encephalitis, and tick-borne encephalitis (TBE) viruses. Since all flaviviruses are antigenically related, they are prone to phenomena of immunological memory ('original antigenic sin'), which can modulate immune responses in the course of sequential infections and/or vaccinations. In our study, we analyzed the influence of pre-existing YF vaccine-derived immunity on the antibody response to TBE vaccination. By comparing samples from YF pre-vaccinated and flavivirus-naive individuals, we show that YF immunity not only caused a significant impairment of the neutralizing antibody response to TBE vaccination but also a reduction of the specific TBE virus neutralizing activities (NT/ELISA-titer ratios). Our results point to a possible negative effect of pre-existing cross-reactive immunity on the outcome of flavivirus vaccination that may also pertain to other combinations of sequential flavivirus infections and/or vaccinations.

Microbioreactor Cultivations of Fab-Producing Escherichia coli Reveal Genome-Integrated Systems as Suitable for Prospective Studies on Direct Fab Expression Effects.

Despite efforts to develop concepts for efficient antibody fragment (Fab) production in Escherichia coli (E. coli) and the high degree of similarity within this protein class, a generic platform technology is still not available. Indeed, feasible production of new Fab candidates remains challenging. In this study, a setup that enables direct characterization of host cell response to Fab expression by utilizing genome-integrated (GI) systems is established. Among the multitude of factors that influence Fab expression, the variable domain, the translocation mechanism, the host strain, as well as the copy number of the gene of interest (GOI) are varied. The resulting 32 production clones are characterized in carbon-limited microbioreactor cultivations with yields of 0-7.4 mg Fab per gram of cell dry mass. Antigen-binding region variations have the greatest effect on Fab yield. In most cases, the E. coli HMS174(DE3) strain performs better than the BL21(DE3) strain. Translocation mechanism variations mainly influence leader peptide cleavage efficiency. Plasmid-free systems, with a single copy of the GOI integrated into the chromosome, reach Fab yields in the range of 80-300% of plasmid-based counterparts. Consequently, the GI Fab production clones could greatly facilitate direct analyses of systems response to different impact factors under varying production conditions.

Implementation of a Fully Automated Microbial Cultivation Platform for Strain and Process Screening.

Advances in molecular biotechnology have resulted in the generation of numerous potential production strains. Because every strain can be screened under various process conditions, the number of potential cultivations is multiplied. Exploiting this potential without increasing the associated timelines requires a cultivation platform that offers increased throughput and flexibility to perform various bioprocess screening protocols. Currently, there is no commercially available fully automated cultivation platform that can operate multiple microbial fed-batch processes, including at-line sampling, deep freezer off-line sample storage, and complete data handling. To enable scalable high-throughput early-stage microbial bioprocess development, a commercially available microbioreactor system and a laboratory robot are combined to develop a fully automated cultivation platform. By making numerous modifications, as well as supplementation with custom-built hardware and software, fully automated milliliter-scale microbial fed-batch cultivation, sample handling, and data storage are realized. The initial results of cultivations with two different expression systems and three different process conditions are compared using 5 L scale benchmark cultivations, which provide identical rankings of expression systems and process conditions. Thus, fully automated high-throughput cultivation, including automated centralized data storage to significantly accelerate the identification of the optimal expression systems and process conditions, offers the potential for automated early-stage bioprocess development.

Immunization with Immune Complexes Modulates the Fine Specificity of Antibody Responses to a Flavivirus Antigen.

UNLABELLED: The antibody response to proteins may be modulated by the presence of preexisting antigen-specific antibodies and the formation of immune complexes (ICs). Effects such as a general increase or decrease of the response as well as epitope-specific phenomena have been described. In this study, we investigated influences of IC immunization on the fine specificity of antibody responses in a structurally well-defined system, using the envelope (E) protein of tick-borne encephalitis (TBE) virus as an immunogen. TBE virus occurs in Europe and Asia and-together with the yellow fever, dengue, West Nile, and Japanese encephalitis viruses-represents one of the major human-pathogenic flaviviruses. Mice were immunized with a dimeric soluble form of E (sE) alone or in complex with monoclonal antibodies specific for each of the three domains of E, and the antibody response induced by these ICs was compared to that seen after immunization with sE alone. Immunoassays using recombinant domains and domain combinations of TBE virus sE as well as the distantly related West Nile virus sE allowed the dissection and quantification of antibody subsets present in postimmunization sera, thus generating fine-specificity patterns of the polyclonal responses. There were substantially different responses with two of the ICs, and the differences could be mechanistically related to (i) epitope shielding and (ii) antibody-mediated structural changes leading to dissociation of the sE dimer. The phenomena described may also be relevant for polyclonal responses upon secondary infections and/or booster immunizations and may affect antibody responses in an individual-specific way. IMPORTANCE: Infections with flaviviruses such as yellow fever, dengue, Japanese encephalitis, West Nile, and tick-borne encephalitis (TBE) viruses pose substantial public health problems in different parts of the world. Antibodies to viral envelope protein E induced by natural infection or vaccination were shown to confer protection from disease. Such antibodies can target different epitopes in E protein, and the fine specificities of polyclonal responses can differ between individuals. We conducted a mouse immunization study with TBE E protein alone or complexed to monoclonal antibodies specific for each of the three protein domains. We demonstrated that phenomena such as epitope shielding and antibody-induced structural changes can profoundly influence the fine specificity of antibody responses to the same immunogen. The study thus provided important new information on the potential immunomodulatory role of preexisting antibodies in a flavivirus system that can be relevant for understanding individual-specific factors influencing antibody responses in sequential flavivirus infections and/or immunizations.

Variation of the specificity of the human antibody responses after tick-borne encephalitis virus infection and vaccination.

UNLABELLED: Tick-borne encephalitis (TBE) virus is an important human-pathogenic flavivirus endemic in large parts of Europe and Central and Eastern Asia. Neutralizing antibodies specific for the viral envelope protein E are believed to mediate long-lasting protection after natural infection and vaccination. To study the specificity and individual variation of human antibody responses, we developed immunoassays with recombinant antigens representing viral surface protein domains and domain combinations. These allowed us to dissect and quantify antibody populations of different fine specificities in sera of TBE patients and vaccinees. Postinfection and postvaccination sera both displayed strong individual variation of antibody titers as well as the relative proportions of antibodies to different domains of E, indicating that the immunodominance patterns observed were strongly influenced by individual-specific factors. The contributions of these antibody populations to virus neutralization were quantified by serum depletion analyses and revealed a significantly biased pattern. Antibodies to domain III, in contrast to what was found in mouse immunization studies with TBE and other flaviviruses, did not play any role in the human neutralizing antibody response, which was dominated by antibodies to domains I and II. Importantly, most of the neutralizing activity could be depleted from sera by a dimeric soluble form of the E protein, which is the building block of the icosahedral herringbone-like shell of flaviviruses, suggesting that antibodies to more complex quaternary epitopes involving residues from adjacent dimers play only a minor role in the total response to natural infection and vaccination in humans. IMPORTANCE: Tick-borne encephalitis (TBE) virus is a close relative of yellow fever, dengue, Japanese encephalitis, and West Nile viruses and distributed in large parts of Europe and Central and Eastern Asia. Antibodies to the viral envelope protein E prevent viral attachment and entry into cells and thus mediate virus neutralization and protection from disease. However, the fine specificity and individual variation of neutralizing antibody responses are currently not known. We have therefore developed new in vitro assays for dissecting the antibody populations present in blood serum and determining their contribution to virus neutralization. In our analysis of human postinfection and postvaccination sera, we found an extensive variation of the antibody populations present in sera, indicating substantial influences of individual-specific factors that control the specificity of the antibody response. Our study provides new insights into the immune response to an important human pathogen that is of relevance for the design of novel vaccines.

Aluminum hydroxide influences not only the extent but also the fine specificity and functional activity of antibody responses to tick-borne encephalitis virus in mice.

Aluminum hydroxide is the most widely used adjuvant in human vaccines and serves as a potent enhancer of antibody production. Its stimulatory effect strongly depends on the adsorption of the antigen to the adjuvant, which may influence antigen presentation and, as a consequence, the fine specificity of antibody responses. Such variations can have functional consequences and can modulate the effectiveness of humoral immunity. Therefore, we investigated the influence of aluminum hydroxide on the fine specificity of antibody responses in a model study in mice using an inactivated purified virus particle, the flavivirus tick-borne encephalitis (TBE) virus, as an immunogen. To dissect and quantify the specificities of polyclonal antibodies in postimmunization sera, we established a platform of immunoassays using recombinant forms of the major target of neutralizing antibodies (protein E) as well as individual domains of E (DIII and the combination of DI and DII [DI+DII]). Our analyses revealed a higher proportion of neutralizing than virion binding (as detected by enzyme-linked immunosorbent assay) antibodies after immunization with aluminum hydroxide. Furthermore, the induction of antibodies to DIII, a known target of potently neutralizing antibodies, as well as their contributions to virus neutralization were significantly greater in mice immunized with adjuvant and correlated with a higher avidity of these antibodies. Thus, our data provide evidence that aluminum hydroxide can lead to functionally relevant modulations of antibody fine specificities in addition to its known overall immune enhancement effect.

Dissection of antibody specificities induced by yellow fever vaccination.

The live attenuated yellow fever (YF) vaccine has an excellent record of efficacy and one dose provides long-lasting immunity, which in many cases may last a lifetime. Vaccination stimulates strong innate and adaptive immune responses, and neutralizing antibodies are considered to be the major effectors that correlate with protection from disease. Similar to other flaviviruses, such antibodies are primarily induced by the viral envelope protein E, which consists of three distinct domains (DI, II, and III) and is presented at the surface of mature flavivirions in an icosahedral arrangement. In general, the dominance and individual variation of antibodies to different domains of viral surface proteins and their impact on neutralizing activity are aspects of humoral immunity that are not well understood. To gain insight into these phenomena, we established a platform of immunoassays using recombinant proteins and protein domains that allowed us to dissect and quantify fine specificities of the polyclonal antibody response after YF vaccination in a panel of 51 vaccinees as well as determine their contribution to virus neutralization by serum depletion analyses. Our data revealed a high degree of individual variation in antibody specificities present in post-vaccination sera and differences in the contribution of different antibody subsets to virus neutralization. Irrespective of individual variation, a substantial proportion of neutralizing activity appeared to be due to antibodies directed to complex quaternary epitopes displayed on the virion surface only but not on monomeric E. On the other hand, DIII-specific antibodies (presumed to have the highest neutralizing activity) as well as broadly flavivirus cross-reactive antibodies were absent or present at very low titers. These data provide new information on the fine specificity as well as variability of antibody responses after YF vaccination that are consistent with a strong influence of individual-specific factors on immunodominance in humoral immune responses.