Intranasal Immunization of Mice with Multiepitope Chimeric Vaccine Candidate Based on Conserved Autotransporters SigA, Pic and Sap, Confers Protection against Shigella flexneri

Round 1

Reviewer 1 Report

General: This manuscript describes the investigation of an intranasal immunization for Shigella flexneri using a multiepitope chimeric vaccine. The manuscript is well written and builds upon the authors previous in silico work. The authors provide a logical progression of experiments describing protein expression and purification, immunization and antibody titers toward the protein using different combinations of adjuvant. The authors also show expression of key cytokines indicative of the immune response induced and demonstrate lymphocyte proliferation in each of their groups. Finally, survival in a challenge study provides evidence of an efficacious vaccine in mice.

 

The major concern with the manuscript is how the data are collected and presented, which make it quite difficult to provide a proper analysis of the manuscript. Most notably, it appears that the titers are determined by identifying the lowest dilution that gives a positive response above background. As described, this method lack statistical rigor and prevents appropriate statistical testing. Furthermore, the values appear to hit an upper limit due to the highest dilution tested. Reciprocal endpoint titers have previously been defined as 2 times the background wells or 2 standard deviations above background wells, which is determined using linear regression by determining the slope of the titration curve and dividing the slope by (2 x bkgd). A more rigorous statistically defined endpoint determination has been reported (Frey, Canzio, & Zurakowski, J Immunol Method 221, 35-41, 1998.).

 

Using these methods, the authors will need to show variance in their groups using bar charts and error bars or using the reviewer preferred method of dot plots of individual data points for each group with group mean denoted. Appropriate statistical tests of the data will then need to be performed. It is expected that this method of calculating titers will strengthen the data.

 

Minor corrections:

Line 22: “administrated” should be “administered”

Line 22: “...administered to mice alone through the intranasal (i.n.) route...”

Line 23: CFA is Complete Freund’s Adjuvant, looks like the C stands for commercial

Line 25: “titles” should be “titers”

Line 27: “titles” should be “titers”

Line 50: “...mouse models, or in silico...”

Line 163: Once more rigorous titer method is implemented, expand explanation.

Line 258: “Specifics” should be “Specific”

Line 298: missing period in “i.n.”

Line 299: “With respect to TNF-α...”

Line 319: “...found in mice immunized by i.n. route using rMESF alone,...”

Line 371: does this refer to increasing cells or increasing cytokines? It reads like CD8+ T cell cytokines are helper, but CD4+ T cells are helper cells. Please rewrite for clarity

Line 410: delete “elevated” or “increased”, this is redundant

Line 422: “subsequence” should be “subsequent”

Author Response

Reviewer 1

General: This manuscript describes the investigation of an intranasal immunization for Shigella flexneri using a multiepitope chimeric vaccine. The manuscript is well written and builds upon the authors previous in silico work. The authors provide a logical progression of experiments describing protein expression and purification, immunization and antibody titers toward the protein using different combinations of adjuvant. The authors also show expression of key cytokines indicative of the immune response induced and demonstrate lymphocyte proliferation in each of their groups. Finally, survival in a challenge study provides evidence of an efficacious vaccine in mice. cutoff

The major concern with the manuscript is how the data are collected and presented, which make it quite difficult to provide a proper analysis of the manuscript. Most notably, it appears that the titers are determined by identifying the lowest dilution that gives a positive response above background. As described, this method lack statistical rigor and prevents appropriate statistical testing. Furthermore, the values appear to hit an upper limit due to the highest dilution tested. Reciprocal endpoint titers have previously been defined as 2 times the background wells or 2 standard deviations above background wells, which is determined using linear regression by determining the slope of the titration curve and dividing the slope by (2 x bkgd). A more rigorous statistically defined endpoint determination has been reported (Frey, Canzio, & Zurakowski, J Immunol Method 221, 35-41, 1998.).

Using these methods, the authors will need to show variance in their groups using bar charts and error bars or using the reviewer preferred method of dot plots of individual data points for each group with group mean denoted. Appropriate statistical tests of the data will then need to be performed. It is expected that this method of calculating titers will strengthen the data.

We would like to thank your comments and we have incorporated your suggestions. Cut-off analyzes were calculated as described by Frey et al. (1998), this is noted on lines 162 and 164. Then we use these new cut-off values ​​to rebuild the graphs. Finally, the statistical analysis was carried out, it should be noted that some error bars are not visible due to similarities in the results between replications.

Minor corrections:                                                            

-Line 22: “administrated” should be “administered”. OK, was replaced

-Line 22: “...administered to mice alone through the intranasal (i.n.) route...”. OK, was replaced  “...administered to mice alone through the intranasal (i.n.) route...” 

-Line 23: CFA is Complete Freund’s Adjuvant, looks like the C stands for commercial. Its, ok. Sorry, was replaced by “Complete Freund’s Adjuvant” 

-Line 25: “titles” should be “titers”. Ok, was replaced by “titers”

-Line 27: “titles” should be “titers”. Ok, was replaced by “titers”

-Line 50: “...mice models, or in silico...” Its, OK, was changed by “...mouse models, or in silico...”

-Line 163: Once more rigorous titer method is implemented, expand explanation.

In lines 163 we add: “The antibody titers were expressed as mean ± standard deviation (SD) of log₁₀ of the last inverse serum dilution before cut-off” was added the following information: “The antibody titers were expressed as mean ± standard deviation (SD) of log₁₀ of the last reciprocal serum dilution before cut-off. The cut-off values were defined according to the method described by Frei et al. (1998)”.

-Line 258: “Specifics” should be “Specific”. Ok, was changed to “specific”.

-Line 298 missing period in “i.n.” Its ok, period was added to “i.n.” in line 301

-Line 299: “With respect to TNF-α...”. Its, OK, in line 302, “Respect to TNF-α..” was replaced by “With respect to TNF-α...”

Line 319: “...found in mice immunized by i.n. route using rMESF alone,...”. Its OK, in line 322, was replaced by “...found in mice immunized by i.n. route using rMESF alone...”

-Line 371: does this refer to increasing cells or increasing cytokines? It reads like CD8+ T cell cytokines are helper, but CD4+ T cells are helper cells. Please rewrite for clarity.

This sentences is refer to T cells activity and cytokines secreted by these cells, specifically CD4 T cells, however it is confuse, and therefore the phrase in line 371-372 “proinflammatory mediators from innate immune cells and increasing levels of T CD8+ and CD4+ helper cytokines that induce the production of antibodies [27,30]” was replaced In lines 371-373 by “proinflammatory mediators from innate immune cells and increasing activity of CD8+ T cells-mediated cytotoxicity and cytokines secreted by CD4+ T cells inducing the macrophages activation and the production of antibodies [27,30].”

-Line 410: delete “elevated” or “increased”, this is redundant. Its ok, now in line 411 the word “elevated” was deleted.

-Line 422: “subsequence” should be “subsequent”. Ok, now in line 423 the word “subsequence” was replaced by “subsequent”

 

Author Response File: Author Response.docx

Reviewer 2 Report

This reviewer commends the author’s for their study titled, “Intranasal immunization of mice with multiepitope chimeric vaccine candidate based on conserved  autotransporters SigA, Pic and Sap, confers protection   against Shigella flexneri.”

This reviewer is more familiar with clinical case management and epidemiological investigation and management of Shigella dysentery outbreaks than basic science research with vaccine development against Shigella flexneri.

However, I have the following questions to the authors about the designed vaccine candidate:

 Regarding the physicochemical and other. properties of this vaccine candidate, what is the:

1. molecular weight,
2. amino acid composition,
3. instability index,
4. half-life,
5. aliphatic index,
6. theoretical pI and
7. GRAVY (Grand average of hydropathicity )
8. What are the immunological properties, including antigenicity and allergenicity of the vaccine candidate?
9. Does the vaccine candidate provide partial or full protection?
10. How long will the protection last? Days? Weeks? Months? Years?
11. How big of a dose and how many doses are required in order to protect vaccine recipients?

 

Author Response

This reviewer commends the author’s for their study titled, “Intranasal immunization of mice with multiepitope chimeric vaccine candidate based on conserved autotransporters SigA, Pic and Sap, confers protection against Shigella flexneri.”

This reviewer is more familiar with clinical case management and epidemiological investigation and management of Shigella dysentery outbreaks than basic science research with vaccine development against Shigella flexneri.

However, I have the following questions to the authors about the designed vaccine candidate:

 Regarding the physicochemical and other. properties of this vaccine candidate, what is the:

1. molecular weight,
2. amino acid composition,
3. instability index,
4. half-life,
5. aliphatic index,
6. theoretical pI and
7. GRAVY (Grand average of hydropathicity )
8. What are the immunological properties, including antigenicity and allergenicity of the vaccine candidate?
9. Does the vaccine candidate provide partial or full protection?
10. How long will the protection last? Days? Weeks? Months? Years?
11. How big of a dose and how many doses are required in order to protect vaccine recipients?

 

 

Regarding your question about the physicochemical or other properties of the candidate vaccine (points 1 to 8), this was already disclosed in our previous publication (León et al., 2020, cited in line 103), but we will gladly summarize it for you. This vaccine candidate has a molecular weight of 84.47 kDa with a total of 829 aa (see below) and an isoelectric point of 5.01. The half-life of the multiepitope protein was 1 h in mammal reticulocytes (in vitro), 30 min in yeast (in vivo) and more than 10 h in E. coli (in vivo). The aliphatic index and the GRAVY value were 83.91 and -0.191 respectively. The instability index was 23.13 which classifies the multiepitope protein as stable. In this work antigenicity of the multiepitope vaccine was evaluated using VaxiJen v2.0 and ANTIGENpro servers for the three passenger domains of SigA, Pic and Sap autotransporters of S. flexneri. The most abundant epitopes were for B cells from which seven peptides were selected based on his score (over 1.7 in VaxiJen server). With respect to CTL epitopes the best IC50 value to MHC-I alleles with values between 0.09 and 201 and VaxiJen score between 1.0807 and 2.2930 were selected giving a total of six epitopes. Also three THL epitopes with a score from 0.8985 to 1.3404 were selected. Finally, three IFN-gamma inducing epitopes which showed positive scores between 0.17 and 0.61 were included.  The allergenicity of the vaccine, evaluated by AlgPred and AllergenFP servers, classified it as non-allergenic.

 

Regarding points 9 to 11, we can say that: In this study the vaccine candidate elicits protection levels ranging from 20% to 100% with high protection levels for rMESF alone through intranasal route, where mice survived 30 days after the last immunization. This route induces high levels of systemic and secretory IgA, indicating a positive effect in the protection against Shigella (Figure 2D and Figure. 3). Future experiments are required to know if this vaccinal approach will confer long-term immunity.

Protection against Shigella challenge was achieved using a dose of 25 µg/mouse and two booster doses (on the 14th and 28th days) administered by intranasal route (material and methods, section 2.5, line 141-142).

 

 

Point 2. Amino acid composition is presented here:

EAAAKMAAKDVKFGNDARVKMLRGVNVLADAVKVTLGPKGRNVVLDKSFGAPTITKDGVSVAREIELEDKFENMGAQMVKEVASKANDAAGDGTTTATVLAQSIITEGLKAVAAGMNPMDLKRGIDKAVAAAVEELKALSVPCSDSKAIAQVGTISANSDETVGKLIAEAMDKVGKEGVITVEDGTGLQDELDVVEGMQFDRGYLSPYFINKPETGAVELESPFILLADKKISNIREMLPVLEAVAKAGKPLLIIAEDVEGEALATLVVNTMRGIVKVAAVKAPGFGDRRKAMLQDIATLTGGTVISEEIGMELEKATLEDLGQAKRVVINKDTTTIIDGVGEEAAIQGRVAQIRQQIEEATSDYDREKLQERVAKLAGGVAVIKVGAATEVEMKEKKARVEDALHATRAAVEEGVVAGGGVALIRVASKIADLKGQNEDQNVGIKVALRAMEAPLRQIVLNCGEEPSVVANTVKGGDGNYGYNAATEEYGNMIDMGILDPTKVTRSALQYAASVAGLMITTECMVTDLPKSDAPDLGAAGGMGGMGGMGGMMEAAAKQKVGNNNSGGSGSGDLRDTNGEGSGSFVDKNDGTGGSGSEKATGSDNSGSGSRDGGGQSLNGSGSSGTRSDGKAGSGSEKSGSGTLTGSGSDGFKAEADKAAAAKAAAYAHASGDIHASAASTVAAYGALVTSTAATVTGTNHEYGAEALERAGLSIGRDANVKGHAAYNNLKIGNGTVILHEYGAEALERAGNPAQGQALNIPLAAYAGKNGKITLSGTHEYGAEALERAGIRDGGGQSLNGLAAYGSVEKSGSGTLTHEYGAEALERAG

 

 

 

Author Response File: Author Response.docx

Reviewer 3 Report

Angel A. Oñate and colleagues report on the development of a multi-epitope vaccine against shigellosis. The vaccine construct is based on sequences encoding antigenic epitopes from HSP GroEL, SigA, Pic and Sap autotransporters of Shigella flexneri. The authors examined the immunogenicity of the vaccine in the mouse model of shigellosis by administering the vaccine with or without different adjuvants and different routes of administration. This is a major weakness of the manuscript as the experiments are not well designed and controlled to directly compare the effect of the four different immunization routes on vaccine-induced immune responses, or the compare the effect of the alum versus complete Freund’s adjuvant on vaccine-induced immune responses. Section 3.2 describes the antibody responses without providing a summary of the vaccination regimens. The x-axis was not defined for Figure 2. It is presumed there are 4 time points, but it is unclear in the figure when the booster immunizations were provided. Figure 2 should be reformatted so that each group is defined by the group number as well as the combination of vaccine and adjuvant. It does not appear that the booster immunization increased the antibody responses to the vaccine.  Immunization of mice with the vaccine regimens resulted in protective immunity marked by reduced bacterial burden. However, additional evidence that the vaccine construct induces physiologically relevant antibodies is to examine the antigenicity of the purified immunogen with naïve or convalescent sera obtained from mice infected with Shigella flexneri. Although the introduction indicated induction of broad immunity against multiple serotypes of Shigella sp was a requirement for a shigellosis vaccine, the current study was limited by not examining the breadth of immunity induced by the most potent vaccine regimen.

Author Response

Reviewer 3

 

Thank you for your comments, which have obviously helped improve our work.

 

Angel A. Oñate and colleagues report on the development of a multi-epitope vaccine against shigellosis. The vaccine construct is based on sequences encoding antigenic epitopes from HSP GroEL, SigA, Pic and Sap autotransporters of Shigella flexneri. The authors examined the immunogenicity of the vaccine in the mouse model of shigellosis by administering the vaccine with or without different adjuvants and different routes of administration.

 

This is a major weakness of the manuscript as the experiments are not well designed and controlled to directly compare the effect of the four different immunization routes on vaccine-induced immune responses, or the compare the effect of the alum versus complete Freund’s adjuvant on vaccine-induced immune responses.

 

The article was reviewed and as you suggested were made modifications in the design and analyses of the experiments to improve this study. Nevertheless, these modifications were also suggested by other reviewer, who indicated that a statistical comparison of the different routes is a mistake, which was considered by our group. Therefore, the statistical comparations of the different administration routes were eliminated (lines 266, 267, 269, 270, among others), changing the results presentation (Figures 2 to 7) and description of them in different sections of the manuscript. Also, these modifications included the elimination of comparative analyzes between alum versus Freund's complete adjuvant effect on vaccine-induced immune responses.

 

 

Section 3.2 describes the antibody responses without providing a summary of the vaccination regimens.

 

With respect to the immunization regimen, it was clarified in material and methods section 2.6, lines 141-142: “Subsequently two booster doses were given to all experimental mice groups on the 14^th and 28^th days after the first immunization”.

 

 

The x-axis was not defined for Figure 2. It is presumed there are 4 time points, but it is unclear in the figure when the booster immunizations were provided. Figure 2 should be reformatted so that each group is defined by the group number as well as the combination of vaccine and adjuvant.

 

Figure 2 and 3 were reformatted according to reviewer suggestions, i.e., the x-axis labeling (T0, T1, T2 and T3 in Figure 2, and T2 and T3 in Figure 3) was changed as days. Information about boost immunization was added in Figure 2 legend.

 

It does not appear that the booster immunization increased the antibody responses to the vaccine. 

 

With respect to the immunization regimen, it was clarified in material and methods section 2.6, lines 141-142: “Subsequently two booster doses were given to all experimental mice groups on the 14^th and 28^th days after the first immunization”.

 

 

Immunization of mice with the vaccine regimens resulted in protective immunity marked by reduced bacterial burden. However, additional evidence that the vaccine construct induces physiologically relevant antibodies is to examine the antigenicity of the purified immunogen with naïve or convalescent sera obtained from mice infected with Shigella flexneri. Although the introduction indicated induction of broad immunity against multiple serotypes of Shigella sp was a requirement for a shigellosis vaccine, the current study was limited by not examining the breadth of immunity induced by the most potent vaccine regimen.

 

Suggestions about testing if mouse serum will be capable of neutralize or develop a microbicidal activity through complement activation and the immunity conferred by this vaccine against other Shigella serotypes or E. coli, could be performed in future research by this group for another publication.

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

All concerns have been addressed and the paper is improved.

Author Response

We appreciate your previous comments, this allows us to substantially improve the article. 

Reviewer 2 Report

 Re: Manuscript titled:  “Intranasal immunization of mice with multiepitope chimeric vaccine candidate based on conserved autotransporters SigA, Pic and Sap, confers protection against Shigella flexneri.”    

Regarding the physicochemical and other. properties of this vaccine candidate, what is the:

1. molecular weight,
2. amino acid composition,
3. instability index,
4. half-life,
5. aliphatic index,
6. theoretical pI and
7. GRAVY (Grand average of hydropathicity )
8. What are the immunological properties, including antigenicity and allergenicity of the vaccine candidate?
9. Does the vaccine candidate provide partial or full protection?
10. How long will the protection last? Days? Weeks? Months? Years?
11. How big of a dose and how many doses are required in order to protect vaccine recipients?

The authors’ have responded that the first 8 questions out of 11 were addressed in a previous publication, and reproduced the summary in their response.  This begs the question of how much of the previous publication is recycled in the current manuscript?  

Author Response

Regarding your question, you will forgive us, but we believe that there is a small error. This article nowhere describes what was addressed in the generalities of this comments. This was previously published and all of these were used as the basis for testing this vaccine in a murine model. Nonetheless, the current manuscript evaluated the immunogenicity and the protective efficacy of the quimeric multiepitope protein named rMESF in mice against lethal infection with S. flexnieri. rMESF quimeric protein was obtained from the cloning and expression of the in silico candidate vaccine developed previously.  

Both the published paper (León et al., 2020) and the current manuscript are closely related due that fact that both work are part of a project that attempts the design and evaluation of a vaccine candidate against Shigella flexnieri infection. 

 

León et al., 2020. doi.org/1o.1016/j.molimm.2020.02.008