A novel vaccine strategy using quick and easy conversion of bacterial pathogens to unnatural amino acid-auxotrophic suicide derivatives

ABSTRACT We propose a novel strategy for quick and easy preparation of suicide live vaccine candidates against bacterial pathogens. This method requires only the transformation of one or more plasmids carrying genes encoding for two types of biological devices, an unnatural amino acid (uAA) incorporation system and toxin-antitoxin systems in which translation of the antitoxins requires the uAA incorporation. Escherichia coli BL21-AI laboratory strains carrying the plasmids were viable in the presence of the uAA, whereas the free toxins killed these strains after the removal of the uAA. The survival time after uAA removal could be controlled by the choice of the uAA incorporation system and toxin-antitoxin systems. Multilayered toxin-antitoxin systems suppressed escape frequency to less than 1 escape per 109 generations in the best case. This conditional suicide system also worked in Salmonella enterica and E. coli clinical isolates. The S. enterica vaccine strains were attenuated with a >105 fold lethal dose. Serum IgG response and protection against the parental pathogenic strain were confirmed. In addition, the live E. coli vaccine strain was significantly more immunogenic and provided greater protection than a formalin-inactivated vaccine. The live E. coli vaccine was not detected after inoculation, presumably because the uAA is not present in the host animals or the natural environment. These results suggest that this strategy provides a novel way to rapidly produce safe and highly immunogenic live bacterial vaccine candidates. IMPORTANCE Live vaccines are the oldest vaccines with a history of more than 200 years. Due to their strong immunogenicity, live vaccines are still an important category of vaccines today. However, the development of live vaccines has been challenging due to the difficulties in achieving a balance between safety and immunogenicity. In recent decades, the frequent emergence of various new and old pathogens at risk of causing pandemics has highlighted the need for rapid vaccine development processes. We have pioneered the use of uAAs to control gene expression and to conditionally kill host bacteria as a biological containment system. This report proposes a quick and easy conversion of bacterial pathogens into live vaccine candidates using this containment system. The balance between safety and immunogenicity can be modulated by the selection of the genetic devices used. Moreover, the uAA-auxotrophy can prevent the vaccine from infecting other individuals or establishing the environment.

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Reviewer #1 (Comments for the Author): General: Authors demonstrated great efforts in designing suicide live vaccine candidates, which adapt both uAA incorporation system and toxin-antitoxin system.The employment of multi-layer toxin-antitoxin systems is an interesting approach results in low escape frequency.The novelty of this approach is unknown.The major concern is regarding the inconsistent administration route, dosage concentration and frequency of the bacteria and vaccine strains used throughout the study.The sample size is small.Despite the live vaccines tested here confer partial protection and IgG production, it is not conclusive to be translated for clinical use.More works can be done to profile the immune responses induced by the vaccine candidates, such as measurement of cytokines, phenotyping of immune cells using flow cytometry, or RNA sequencing etc.The results may serve as preliminary findings, which authors should propose future plan on improving these strains.Authors should be more moderate in using description words, avoid using "most", "extremely", "excellent", etc.

Importance:
• Suggest to emphasize on the unique advantages of using the proposed conditional killing system in live bacterial vaccine.

Introduction:
• Suggest to briefly compare the approach in this study with other conditional suicide systems.

Result:
• Did authors confirm the expression of the integrated plasmids and proteins encoded?
• How many independent experiments were conducted to generate the escape frequency?
• "To further reduce the escape frequency, we attempted to transform plasmid C-1 into χ3306 (ZK-k) and χ3306 (ZK-kd), but no transformants were obtained."Any further improvement to solve this problem?Would these strains still be practical to use if their escape frequencies do not meet NIH requirement?
• Authors should justify the use of BALB/c mouse strain in this study.Why the infection was done via intravenous administration?
• "χ3306 (ZK-kd) induced significantly more IgG than χ3306 (ZK-k)."Authors should be more precise in describing the result, by including the value, significance, fold-change.This comment applies to all results.
• "Mice were intraperitoneally vaccinated with a double-dose administration at 1 × 105 CFU/mouse of χ3306 (ZK-k) or χ3306 (ZK-kd)."Justify the dosage use.Discussion: • Instead of using "quick and easy preparation", suggest to use more precise, for example, the vaccine candidate can be prepared in how many days etc.
• Suggest to include literature or evidence from previous studies to support the paragraph mentioned "The selection of the uAA incorporation system affected escape frequency." • Authors should discuss why the vaccine candidates only confer partial protection?
• The limitation of study was not discussed.

Materials and Methods:
• Any reason for using young age (3-week-old) animal?
Reviewer #3 (Comments for the Author): This paper describes a novel approach for generating live-attenuated vaccines for bacterial pathogens using genetic engineering.Live attenuated vaccines are desirable because they are often more immunogenic and provide greater protection than other methods of vaccination; however, major problems can arise if the pathogen used as a vaccine is not effectively attenuated.In this paper, the authors used toxin-antitoxin genes to generate E. coli (laboratory and pathogenic strains) and Salmonella enterica, that can only survive in media supplemented with an unnatural amino acid (uAA).These engineered bacteria can be grown in media containing a uAA, which facilitates stop codon readthrough, and expression of an antitoxin gene.The cultured cells can then be used to inoculate an animal as a vaccine.Because the uAA does not exist within the animal, the antitoxin gene is not expressed, and the microbes die shortly after injection.Importantly, the bacteria are still able to illicit an immune response inducing the animal to generate antibodies to the pathogen.Building on their previous work, herein, the authors refine this method of generating bacteria that are auxotrophic for a uAA and successfully demonstrate the use of this technology for immunizing laboratory mice against pathogenic strains of bacteria.This paper is well written and technically sound.The data are sufficient to support the conclusions made.I would rate the novelty of the work as moderate-uAAs have been used to generate live attenuated vaccines previously, as have toxin-antitoxin genes.Thus, this work is somewhat incremental.Moreover, it is limited to bacterial strains for which multiple expression plasmids are readily available.However, given the quality of the work and the pressing needs for rapid and effective methods for generating vaccines, I believe the work will be of interest to a diverse audience.I find no major issues that the authors must address at this time.

Reviewer Comment: General:
Authors demonstrated great efforts in designing suicide live vaccine candidates, which adapt both uAA incorporation system and toxin-antitoxin system.The employment of multi-layer toxin-antitoxin systems is an interesting approach results in low escape frequency.The novelty of this approach is unknown.The major concern is regarding the inconsistent administration route, dosage concentration and frequency of the bacteria and vaccine strains used throughout the study.The sample size is small.Despite the live vaccines tested here confer partial protection and IgG production, it is not conclusive to be translated for clinical use.More works can be done to profile the immune responses induced by the vaccine candidates, such as measurement of cytokines, phenotyping of immune cells using flow cytometry, or RNA sequencing etc.The results may serve as preliminary findings, which authors should propose future plan on improving these strains.Authors should be more moderate in using description words, avoid using "most", "extremely", "excellent", etc.

Importance:
 Suggest to emphasize on the unique advantages of using the proposed conditional killing system in live bacterial vaccine.

Introduction:
 Suggest to briefly compare the approach in this study with other conditional suicide systems.

Result:
 Did authors confirm the expression of the integrated plasmids and proteins encoded?
 How many independent experiments were conducted to generate the escape frequency?
 "To further reduce the escape frequency, we attempted to transform plasmid C-1 into χ3306 (ZK-k) and χ3306 (ZK-kd), but no transformants were obtained."Any further improvement to solve this problem?Would these strains still be practical to use if their escape frequencies do not meet NIH requirement?
 Authors should justify the use of BALB/c mouse strain in this study.Why the infection was done via intravenous administration?
 "χ3306 (ZK-kd) induced significantly more IgG than χ3306 (ZK-k)." Authors should be more precise in describing the result, by including the value, significance, fold-change.This comment applies to all results.
 "Mice were intraperitoneally vaccinated with a double-dose administration at 1 × 10 5 CFU/mouse of χ3306 (ZK-k) or χ3306 (ZK-kd)."Justify the dosage use.Discussion:  Instead of using "quick and easy preparation", suggest to use more precise, for example, the vaccine candidate can be prepared in how many days etc.
 Suggest to include literature or evidence from previous studies to support the paragraph mentioned "The selection of the uAA incorporation system affected escape frequency."  Authors should discuss why the vaccine candidates only confer partial protection?
 The limitation of study was not discussed.

Materials and Methods:
 Any reason for using young age (3-week-old) animal?
This paper describes a novel approach for generating live-attenuated vaccines for bacterial pathogens using genetic engineering.Live attenuated vaccines are desirable because they are often more immunogenic and provide greater protection than other methods of vaccination; however, major problems can arise if the pathogen used as a vaccine is not effectively attenuated.In this paper, the authors used toxin-antitoxin genes to generate E. coli (laboratory and pathogenic strains) and Salmonella enterica, that can only survive in media supplemented with an unnatural amino acid (uAA).These engineered bacteria can be grown in media containing a uAA, which facilitates stop codon readthrough, and expression of an antitoxin gene.The cultured cells can then be used to inoculate an animal as a vaccine.Because the uAA does not exist within the animal, the antitoxin gene is not expressed, and the microbes die shortly after injection.Importantly, the bacteria are still able to illicit an immune response inducing the animal to generate antibodies to the pathogen.Building on their previous work, herein, the authors refine this method of generating bacteria that are auxotrophic for a uAA and successfully demonstrate the use of this technology for immunizing laboratory mice against pathogenic strains of bacteria.
This paper is well written and technically sound.The data are sufficient to support the conclusions made.I would rate the novelty of the work as moderate-uAAs have been used to generate live attenuated vaccines previously, as have toxin-antitoxin genes.Thus, this work is somewhat incremental.Moreover, it is limited to bacterial strains for which multiple expression plasmids are readily available.However, given the quality of the work and the pressing needs for rapid and effective methods for generating vaccines, I believe the work will be of interest to a diverse audience.I find no major issues that the authors must address at this time.
Intravenous administration is one method of administering Salmonella to mice.It is more susceptible than oral infection, and intravenous administration was used to test the difference in susceptibility between the wild strain and the ZK-kd or ZK-d strains.
The citations for oral infection, peritoneal infection, and intravenous administration were added to the Methods section.

Why the CFU dosage is different for each vaccination?
The first immunization was set to 1 × 10 3 CFU/mouse to avoid the onset of septic shock due to LPS.

"χ3306 (ZK-kd) induced significantly more IgG than χ3306 (ZK-k)."
Authors should be more precise in describing the result, by including the value, significance, foldchange.This comment applies to all results.
The following text has been modified to the manuscript.

Page 10, line 17-28:
After fourth-dose vaccination, single-dose administration at 1 × 10 3 CFU/mouse following triple-dose administration at 1 × 10 4 CFU/mouse for χ3306 (ZK-k) or χ3306 (ZK-kd), the immunized mice were challenged with intraperitoneal injection of the wildtype χ3306 at 1 × 10 CFU/mouse (Figure 4B).We evaluated the induction of anti-S.enterica lipopolysaccharide (LPS) IgG after the intraperitoneal administration with χ3306 (ZK-k) and χ3306 (ZK-kd) (Figure 4C).As shown in Figures 4B and 4C, χ3306 (ZKkd) immunized mice showed significantly higher anti-LPS IgG compared to controls at more than 28 days after immunization (p=0.007).On the other hand, anti-LPS antibody levels in χ3306 (ZK-k) immunized mice did not differ significantly from controls even 56 days after immunization, but a tendency to increase induction of the anti-LPS IgG was observed.
Page 10, line 35 -page 11, line 4: Mice were intravenously vaccinated with triple-dose administration at 1 × 10 5 CFU/mouse of χ3306 (ZK-k) or χ3306 (ZK-kd).When total IgG titers were assayed, production was observed after two inoculations (Figure 4F).χ3306 (ZK-kd) and χ3306 (ZK-k) induced significantly more anti-LPS IgG than control at day 40 postimmunization (p=0.017 and p=0.0005, respectively).In Figure 4, the relevant panels are indicated by distinct colors.Figure 4A is a safety test of vaccine candidates.Figures 4B-D and E-G show protection studies using intraperitoneal injection and oral infection models, respectively.As the reviewer points out, the order of the panels is not regular, but we believe the present layout is reasonable.

Discussion:
Instead of using "quick and easy preparation", suggest to use more precise, for example, the vaccine candidate can be prepared in how many days etc.
To indicate the specific details of the "quick and easy preparation", we have made an additional note (page 14, line 6-7).
Suggest to include literature or evidence from previous studies to support the paragraph mentioned "The selection of the uAA incorporation system affected escape frequency." Following the reviewer's suggestion, we have made an additional note and cited additional references (page 14, line 9-14.Ref. 50,51).

Authors should discuss why the vaccine candidates only confer partial protection?
Although the only partial protection in survival rate was shown as the reviewer pointed out, the significant difference between the survival curves of the wild-type and the vaccine candidates indicates a protective effect of vaccination.In addition, S. Typhimurium commonly transmits via oral route (58).In our experiment, the vaccination exerted a better protective effect for survival rate against oral challenge than intraperitoneal challenge (Fig. 4D and G).This result suggests that our vaccination system has a potential to be put practical use.
We have summarized above remarks in the Discussion section as: Page 17, line 8-11: "Notably, the vaccination exerted a better protective effect for survival rate against oral challenge than intraperitoneal challenge (Fig. 4D and G

Materials and Methods:
Any reason for using young age (3-week-old) animal?

•
Fig 4, panel arrangement can be improved, either from left to right or top to bottom.• Fig 4 A, why the survival day was short (max 6 days)?• Fig 4 C and F, the standard unit for IgG titers should be IU/ml?Significant values and statistical tests for this experiment are not mentioned.• Fig 5 C, histology stains are not clear.Suggest to show overview tissue stain, and high resolution of area that show bacterial aggregation.


Fig 4, panel arrangement can be improved, either from left to right or top to bottom. Fig 4 A, why the survival day was short (max 6 days)? Fig 4 C and F, the standard unit for IgG titers should be IU/ml?Significant values and statistical tests for this experiment are not mentioned. Fig 5 C, histology stains are not clear.Suggest to show overview tissue stain, and high resolution of area that show bacterial aggregation.

Fig 4 ,
Fig 4, panel arrangement can be improved, either from left to right or top to bottom.

Fig 4 A
Fig 4 A, why the survival day was short (max 6 days)?

Fig 4 C
Fig 4 C and F, the standard unit for IgG titers should be IU/ml?Significant values and statistical tests for this experiment are not mentioned.
www.who.int/news-room/fact-sheets/detail/salmonella-(non-typhoidal)The limitation of study was not discussed.The limitations of our research at this time have been discussed for each technology.In addition, we have summarized the technologies that are inadequately complete and added the direction of development required for translational research (page 18, line 6-14).