Immunogenicity of an AAV-Based COVID-19 Vaccine in Murine Models of Obesity and Aging

Round 1

Reviewer 1 Report

Line 170-171: "...ACE2 were seeded at 1.5X104 cells/well in poly-L-Lysine (0.01%) and coated in 96-well black plates..."; this line sounds like poly-L-Lysine was added to the cells. I believe they meant to say that they used 96-well black plates pre-coated with poly-L-Lysine.

Lines 176-177: This is not part of the manufacturer's protocol. Please clarify why plates were frozen at -80°C for 60 minutes (I assume to enhance lysis?).

Lines 182-184: Please clarify why 200-fold difference was chosen between virus stock and background. According to Crawford paper, they used >1000-fold difference for their neutralizing assays. This is probably all relative and dependent on readers used for detecting luminescence and target RLU for the assay.

Supplementary material: Please include neutralization curve data graphs used to generate EC50 values for samples.

Minor Formatting Corrections:

Consistency of values and their abbreviated units: throughout the document, the spacing between values and their associated abbreviate units are not consistent. Ex: lines 159-160 and line 175, you have 18µg, 9µg, 29µg, and 50µL with values attached to the abbreviated units, and other lines such as line 173 and line 174 (50 µL and 10 µg/mL) have a space between their values and units. Please double-check the article and make sure you stick with the same format for your values and units.

Superscript required for "power of" numbers: Lines 171, 214, and 221 need to have their "power of" #s in superscript.

Line 200: subheading needs to be in bold.

Line 219: kits needs an apostrophe (kit's)

 

Author Response

We thank reviewer 1 for the comments and suggestions. Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The study evaluates the immunogenicity of 2 AAV based vaccines against SARS-CoV-2 in wild type, aged, or obese B6 mice. The study is an important topic and of high interest currently. There are several things that could be improved upon to increase the relevance and rigor of the study.

Major:

Murine models of SARS-CoV-2 infection necessitate the use of hACE2 transgenic mice (or mouse adapted SARS-CoV-2), but this immunogenicity study is completed in B6 mice. It is possible that differential immune responses or level of chronic inflammation observed in obese or aged mice could occur between B6 and hACE2 transgenic mice. This could potentially explain the unexpected lack of impact of obesity to vaccine immunogenicity. Can the authors address this?

On a similar note, the paper notes that it is thought that chronic inflammation may dampen the immune responses induced by vaccines. Interestingly, the study did not find substantial differences between DIO mice and their lean counterparts in terms of vaccine immunogenicity. Did the study confirm the induction of chronic inflammation in this model? Or perhaps other hallmarks of this metabolic dysfunction such as elevated liver enzymes (ALT/AST)?

It is interesting that DIO results in enhanced T cell responses to peptides within the S2 subunit of spike. Why do the authors think this is? And why is this effect lost at higher doses of AC1 vaccine? This seems counterintuitive.

Was the potency of the T cell response induced in aged mice evaluated?

Is the efficacy of protection against SARS-CoV-2 infection compromised in aged or obese mice? This is a highly important question.

Were any immune responses at mucosal surfaces evaluated, or in the lungs perhaps? As the point of entry, this is likely of importance.

Minor:

Line 296 should say “sex” instead of “gender”

In figure 3b, were statistical comparisons not made or were they just non-significant?

A symbol key should be added to figure 1.

Author Response

Comments and Suggestions of Reviewer 2

We thank the reviewer for raising these interesting questions and comments. We have included the new section “Limitations of the study” in the manuscript to report some of the points made by Reviewer 2.

Murine models of SARS-CoV-2 infection necessitate the use of hACE2 transgenic mice (or mouse adapted SARS-CoV-2), but this immunogenicity study is completed in B6 mice. It is possible that differential immune responses or level of chronic inflammation observed in obese or aged mice could occur between B6 and hACE2 transgenic mice. This could potentially explain the unexpected lack of impact of obesity to vaccine immunogenicity. Can the authors address this?

We agree with the reviewer in this point, the immunogenicity and inflammation may vary between different mouse strains. Many vaccine efficacy studies performed in mice utilize the K18-hACE2 mouse model [1] that is permissive for SARS infection. This model has a C57BL/6 background and overexpresses the human hACE2 under the control of the K18 promoter. The expression of hACE2 is restricted to airway epithelia (not alveolar epithelia), as well as in epithelia of other internal organs including the liver, kidney, and gastrointestinal tract. Considering that the genetic background is the same and hACE2 is not expressed in immune cells or tissues, the immune response in both models is expected to be the same. Most of vaccine efficacy studies performed in this model characterize immunogenicity of vaccines in WT mice and perform challenge experiments in K18-hACE2, assuming that the immunogenicity will be comparable in both models [2-4]. Huang et al measured humoral immunity elicited by an COVID19 mRNA vaccine in C57BL/6 and K18-hACE2 mouse model and observed comparable binding and neutralizing antibody titers. We have not been able to find any data that suggests that hACE2 transgenic mice have different immunogenicity responses compared to wild type from vaccination. Therefore, we anticipate similar immune responses in both mouse strains [5].

Nevertheless, if the K18-hACE2 model was to be found not suitable for future efficacy studies, other alternative models may be used. The prototypic SARS-CoV-2 Wuhan strain has been adapted to engage mouse ACE2 and it is able to infect C57BL/6 mice [6], which was also tested in obese and aged mice showing increased pathogenicity. Additionally, the variants of concern (VOC) that have the N502Y mutation can infect C57BL/6 wild type mice and therefore the efficacy of protection from some VOCs may also be studied in C57BL/6 mice [7]. The caveat of some of these models is that the severity of infection is lower than in the K18-hACE2 model [4], although the K18-hACE2 model develops encephalitis symptoms associated to neuroinvasion of SARS-CoV-2 that do not recapitulate disease in human.

Regarding the absence of impact of obesity in the immunogenicity elicited by our vaccine candidates in the C57BL/6 model, Kim et al test the efficacy of an influenza vaccine in the C57BL/6 DIO vaccine and they report a decrease in humoral immune responses and protection from influenza challenge [8]. Therefore, the C57BL/6 DIO model is susceptible to lower vaccine efficacy, but based on the data reported in this paper, we believe that this impact in efficacy is vaccine dependent.

On a similar note, the paper notes that it is thought that chronic inflammation may dampen the immune responses induced by vaccines. Interestingly, the study did not find substantial differences between DIO mice and their lean counterparts in terms of vaccine immunogenicity. Did the study confirm the induction of chronic inflammation in this model? Or perhaps other hallmarks of this metabolic dysfunction such as elevated liver enzymes (ALT/AST)?

 

The diet-induced obese (DIO) mouse model has been extensively studied and it is known that mice fed with high-fat diet present several metabolic and inflammatory alterations [9-12]. As mentioned in our discussion, the upregulation of pro-inflammatory cytokines in obesity has been linked to chronic inflammation in human and mice [13]. Since the metabolic and inflammatory alterations were well described in published literature, we did not measure any inflammation or metabolic marker in our study, and we monitored the weights of the animals to ensure that the model was behaving as expected. We were not able to measure any serum markers in our study animals in the short time (10 days) provided for this revision. However, we would be willing to measure inflammatory and/or metabolic markers if it is required for publication of the manuscript.

It is interesting that DIO results in enhanced T cell responses to peptides within the S2 subunit of spike. Why do the authors think this is? And why is this effect lost at higher doses of AC1 vaccine? This seems counterintuitive.

We also find this observation interesting, however we do not have an explanation for it. We could hypothesize that this is an effect of the metabolic and proinflammatory disfunction of the obesity model, however, the lack of effect in high dose is counterintuitive, as the referee mentions.

Was the potency of the T cell response induced in aged mice evaluated?

We did not evaluate the T cell response because we were interested in studying the durability of responses (Figures 4E and 4F).

Is the efficacy of protection against SARS-CoV-2 infection compromised in aged or obese mice? This is a highly important question.

We agree with the reviewer that the efficacy of protection in aged and obese models is of high importance. Literature suggests that the severity of SARS-CoV-2 infection in some mouse models is affected by age and obesity [6]. However, we could not find any literature regarding efficacy of protection of COVID19 vaccines in aged and obese mice. Some human trials suggest that protection is compromised in vaccinated obese individuals [14]. Graham et al, demonstrated that aged (12-months-old) mice vaccinated with a live virus vaccine were protected from lethal SARS-CoV-1 infection, but the protection efficacy was not compared to young, vaccinated animals [15]. As mentioned above, some influenza literature suggests that both the antibody responses elicited by vaccine and the protection are compromised in obese animals [8]. However, we believe that the protection is vaccine modality dependent and should be studied for each vaccine candidate. Additionally, serum neutralizing antibodies are considered the best correlate of protection [16, 17]. Based on the data reported in this manuscript, obese mice developed same neutralizing responses as lean mice, and therefore, same protection efficacy is to be expected. On the other hand, 24-month-old mice showed decreased vaccine immunogenicity, and a decreased protection is also expected. Of course, protection needs to be evaluated in a SARS-CoV-2 challenge study to confirm this hypothesis.

Were any immune responses at mucosal surfaces evaluated, or in the lungs perhaps? As the point of entry, this is likely of importance.

We agree with the reviewer that the mucosal immunity will likely determine the protection from a SARS-CoV-2 infection. Unfortunately, we did not harvest samples from mucosal surfaces or lungs. However, serum neutralizing antibody levels are considered the best correlate of protection [16, 17]. We have previously published efficacy data of AC1 vaccine in a cynomolgus macaque SARS-CoV-2 challenge model, in which we show high neutralizing antibody titers in serum and near-sterilizing protection of upper and lower respiratory tracts [18]. This said, mucosal immunity might be impacted by obesity or aging, and it should be further explored, likely in combination with a protection readout.

Minor:

Line 296 should say “sex” instead of “gender”

Thanks, we have substituted “gender” by sex.

In figure 3b, were statistical comparisons not made or were they just non-significant?

Statistical comparisons made but not included in the initial submission, apologies for the oversight. We have now added the statistical analysis in figure 3.

A symbol key should be added to figure 1.

We have added a symbol key, sorry for the oversight.

 

References

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