Unique aspects of Lawsonia intracellularis cultivation (in vitro growth) conditions specified in US5885823
L. intracellularis is a microaerophilic (i.e., requiring reduced oxygen concentration), obligate intracellular (grows and replicates only inside the host cell), gram-negative, non-spore-forming, curved or “S”-shaped rod-like (i.e., bacillus) bacteria sometimes equipped with a single polar flagellum (i.e., tail)1,2. In the seminal work of Lawson and colleagues, the conditions allowing the infection and cultivation of this hard-to-culture microorganism were established for the first time. In particular, it was demonstrated that the monolayers of immortalized IEC-18 rat small intestinal epithelial (host) cells can be infected with L. intracellularis-containing filtrates derived from infected pigs2. First, 10–30-fold diluted filtrates were added to IEC-18 monolayers growing in classical two-dimensional (2D) plastic-adherent cell culture. Second, the IEC-18 monolayers and bacteria mixtures were mildly centrifuged (~2,000 g) to promote the precipitation of bacteria onto the host cells and the atmosphere within a vessel (anaerobic jar) containing host cells, bacteria, and liquid growth medium was changed to 8.0% O2 (oxygen), 7.0% CO2 (carbon dioxide), and 85% H2 (hydrogen). Third, the cultures of IEC-18 cells and bacteria were incubated under the atmosphere specified above for 3 hours (h) to promote infection. It was demonstrated both in vitro and in vivo that L. intracellularis enters the host cells in an endocytic vacuole, but rapidly (within less than 3h) escapes and replicates in the cytoplasm3. Fourth, 3h post-infection (p.i.), the medium was replaced and antibiotics added, whereas the atmosphere was set to 8.0% O2, 8.8% CO2, and 83.2% H2 and left unchanged until the end of culturing.
Lawson and colleagues reported: “The immunostaining of cells exposed to infection provides strong evidence for the multiplication of the bacteria; organisms are localized to groups of cells within the monolayer, and infection does not spread to adjacent cells once the monolayer has reached maturity2.” Consequently, the ability of L. intracellularis to grow in the IEC-18 monolayers, i.e., confluent cell culture, where cells contact each other, strongly suggests that this intracellular pathogen replicates efficiently in cells that are not actively dividing. Another important observation made by Lawson et al. was the fact that much of the spread of infection in IEC-18 monolayers was due to the division of initially infected cells. Thus, two important conclusions can be drawn from the above evidence. First, L. intracellularis growth is not contact-inhibited by adjacent cells within a monolayer two-dimensional (2D) cell culture. Second, due to predominant cell division-based spread of infection the efficiency of initial infection will determine the ultimate overall yield, i.e., bacteria total amount, of L. intracellularis as a result of culturing.
The analysis of US5885823 patent (Boehringer Ingelheim Vetmedica Inc.) reveals that the infection conditions used were essentially equivalent to those used by Lawson et al.2, whereas the culture conditions used after the initial infection, i.e., those used 3h p.i., are the ones that were patented. Thus, the initial infection conditions established by Lawson and colleagues can be used directly and preferably in the optimized form (as specified in the Solution) without infringing on the US5885823 patent.
Knittel and Roof describe the culture conditions they used in US5885823 patent in the following way: “Prior to the instant invention, it was generally believed that cells must be attached to a surface in order to be infected by L. intracellularis. The cell suspensions of the instant invention are unique and contradict this theory...” However, as deduced in the above paragraphs, the initial infection largely determines the yield of the pathogen and the infection conditions used in the patent were essentially taken from the study by Lawson et al.2. It follows that the patent statements highlighted above are not supported by evidence. Thus, the major novelty of the US5885823 patent is the ability to grow the host cells in a suspension culture after their infection in adherent-state by using previously established conditions not covered by the patent. In particular, the growth in suspension was achieved by mechanically scraping of infected host cell monolayers and transferring them into suspension culture with reduced oxygen concentration, essentially as specified in the claim number 4 of the patent. It was found that scraped-off monolayers consisting of infected HEp-2 epithelial carcinoma cells could grow in suspension culture, whereas IEC-18 and McCoy cells required micro-carriers (gelatin, agarose, collagen, acrylamide, silica beads, etc.) for suspension growth.
Overall, the US5885823 patent specifies that it is possible to transfer, i.e., by mechanically scraping, L. intracellularis-infected host cells, e.g., HEp-2, from plastic cell culture dishes into suspension culture and grow these cells for a prolonged time (months) to obtain an attenuated strain that can be used as a vaccine. In addition, the US5885823 specifies that during L. intracellularis growth in suspension culture, the oxygen concentration should be externally regulated and should be below 18%, 8% or 3%.
It is known, that L. intracellularis requires reduced oxygen concentration for efficient growth. For example, Lawson et al. reported that 8% oxygen and 7% carbon dioxide were essential for this pathogen growth2. Knittel et al. reported that a gas mixture of 8% oxygen and 10% carbon dioxide was necessary to support growth, whereas other combinations were attempted but were not successful4. Furthermore, one of the intentions of US5885823 patent was to patent essentially all various oxygen concentrations less than 18%. This was done largely to prevent competition and also due to the fact that the optimal oxygen concentration required for L. intracellularis growth has never been exactly determined. Finally, according to US5885823 patent, it is required to culture the infected host cells for 150–250 days to obtain a live attenuated L. intracellularis strain suitable for use as a vaccine. This is a fairly long time period.
In this Solution, NextGenRnD describes the design of novel experimental system allowing large-scale growth of L. intracellularis-infected cells at external oxygen concentrations above 18% using unique approach compatible with the bioreactor suspension culture requirements. Moreover, the oxygen concentration is regulated autonomously (intrinsically) in the system described by NextGenRnD. In particular, when the oxygen concentration becomes optimal for L. intracellularis replication–the replication of bacteria is triggered automatically. Using our approach may lead to a much faster mutation accumulation, which for example might lead to increased rate of bacteria replication and as a result more rapid attenuation. As a consequence, faster attenuation might decrease the vaccine development time considerably. Our Solution will catalyze the creation of novel L. intracellularis attenuated live vaccine capable of outcompeting the Enterisol® Ileitis live vaccine (Boehringer Ingelheim Vetmedica Inc.).