Augmentation of vaccine-induced humoral and cellular immunity by a physical radiofrequency adjuvant

Protein/subunit vaccines often require external adjuvants to induce protective immunity. Due to the safety concern of chemical adjuvants, physical adjuvants were recently explored to boost vaccination. Physical adjuvants use physical energies rather than chemicals to stimulate tissue stress and endogenous danger signal release to boost vaccination. Here we present the safety and potency of non-invasive radiofrequency treatment to boost intradermal vaccination in murine models. We show non-invasive radiofrequency can increase protein antigen-induced humoral and cellular immune responses with adjuvant effects comparable to widely used chemical adjuvants. Radiofrequency adjuvant can also safely boost pandemic 2009 H1N1 influenza vaccination with adjuvant effects comparable to MF59-like AddaVax adjuvant. We find radiofrequency adjuvant induces heat shock protein 70 (HSP70) release and activates MyD88 to mediate the adjuvant effects. Physical radiofrequency can potentially be a safe and potent adjuvant to augment protein/subunit vaccine-induced humoral and cellular immune responses.


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, 3, 4, 5, 6: there is no description how many times these particular experiments were repeated to confirm reproducibility. 5. Figure 3: The authors describe "Log-rank test was used to compare differences in E.", but which method was used to correct for multiple comparisons between groups? 6. In Figure 6: The authors describe that "Student's t-test was used to compare differences between groups". In this experiment, there are three genotypes crossed with two treatments. Please clarify how the authors perform the statistical analysis. In Figure 6C: there are 3 test groups. Which method was used to correct for multiple comparisons between groups? 7. In Material and Method section, Page 14, Line 417: the authors state that "This device has three energy settings (low, medium, high) and high energy setting was used in this study". Please provide more specific pieces of information including frequency and actual power density used in this study. It is helpful to include detailed methodology how the authors determined these parameters on the mouse skin. In addition, technical details how the authors established and reproducibly perform the application help other investigators reproduce the results. 8. In page 12, line 342: the authors state that "To our knowledge, RFA represents the first physical adjuvant that simultaneously augments both arms of adaptive immunity with adjuvant potency". However, nanosecond pulsed laser is reported to augment both CD4 and CD8 T cell responses when it was combined with the intramuscular administration of a model vaccine (Chen X, PLOS ONE, 2010). Moreover, the same nanosecond pulsed laser alone was sufficient to enhance anti-tumor immunity when combined with dendritic cell vaccine (Chen X, Clin Cancer Res, 2012).

A point-by-point response to reviewers
First of all, we would like to thank reviewers for their valuable comments and suggestions. We have conducted new experiments and included new data in this revised manuscript, including (1) comparison of RFA with AddaVax to boost pandemic 2009 H1N1 influenza (pdm09) vaccination ( Fig. 8), (2) evidence of heat shock protein 70 (HSP70) induction following RFA treatment (  S5), (4) effects of RFA on DC subsets in skin and draining lymph nodes ( Fig. 2 & 3). Besides the requested experiments, we also conducted adoptive transfer of OT-I T cells to confirm activation of OVA-specific CD8+ T cells following OVA immunization in the presence of RFA (Fig. 6). We also thoroughly addressed other minor concerns raised by reviewers either in the following point-by-point response or in the body of the manuscript whenever appropriate. These suggestions and modifications have improved the manuscript significantly.
Reviewer #1 (Remarks to the Author): 1) Very interesting, may solve the issue of adjuvant induced autoimmunity (Refer to it in your discussion). Response: We have discussed the less likelihood of RFA to induce autoimmune/inflammatory syndromes induced by adjuvant (ASIA) in the revised manuscript (P16, 1 st paragraph).
2) Do the authors have a comparison of titers of antibodies against a peptides compared by HF to a regular adjuvant after 1st injection or a second. Response: We compared RFA and MF59-like AddaVax adjuvant to boost pdm09 vaccineinduced HAI titer and protection against lethal viral challenges. We found RFA was at least comparable to AddaVax to boost pdm09 vaccination in murine models (Fig. 8).
Reviewer #2 (Remarks to the Author): The authors reported for the first time use of radio frequency (RF) as a vaccine adjuvant (RFA), although a very similar observation has been reported in a patent application (WO2009144567) HIGH-ENERGY PULSED ELECTRIC FIELD VACCINE ADJUVANTS by a Russian group. Compared to the previously explored laser adjuvant, RFA alone significantly augmented T-cell response, while lasers needed to be combined with other therapeutics such as microneedles (Wang J, PNAS, 2015;Wang J, Nat Commun, 2014). This study suggests that the mechanism(s) of action of RFA may be similar to those of lasers; recruitment and activation of antigen presenting cells (APCs) by local and limited inflammation. Moreover, heat proved to play a significant role, which is consistent with a recent report (Ye Y, Sci Immunol, 2017). This report, however, failed to identify molecular cues, especially pathway(s) activated by RFA and/or subsequent heat generation to ultimately activate MyD88 and augment immune responses including CD8+T cell response. In additional, more pieces of information on technical details of the RFA treatment, reproducibility of each experiment, and statistical strategy should be provided. Response: We thank this reviewer to provide closely related literature to our work. We have incorporated related literature in the discussion portion of the revised manuscript. To address the lack of molecular cues, we identified HSP70 as one endogenous danger signal stimulated by RFA, which likely mediated the observed RFA effects. In addition, we also provided more technical details about the RFA treatment, reproducibility of each experiments, and more information about the statistical analysis. Major 1. In page 10, line 289: the authors state that "It has been reported that the majority of endogenous danger signals or damage-associated molecular patterns (DAMPs) can bind to tolllike receptors (TLRs)". Indeed, heat generation has been reported to increase the expression of heat shock protein, ROS generation, recruitment of immune cells, and blood and lymphatic flow, resulting in activation of innate and adaptive immune responses. However, none of these signals was examined in this study. As a result, mechanism(s) of action of the RFA remain unclear. In addition, in page 12, line 342: the authors state "To our knowledge, RFA represents the first physical adjuvant that simultaneously augments both arms of adaptive immunity with adjuvant potency". In order to confirm this conclusion, it is critical to specify pathway(s) involved in augmentation of both humoral and cell-mediated immunity. Response: We thank the reviewer to raise this question. We identified HSP70 as one endogenous danger signal induced after RFA treatment (Fig. 9A & 9B). The available literature on the potent adjuvant effects of HSP70 is well in line with the observed RFA effects, hinting HSP70 may be the key endogenous danger signal mediating RFA effects (P16, 3 rd paragraph). Figure 2 to distinguish migratory DCs, lymphoid tissueresident DCs and macrophages in skin-draining lymph nodes or skin are not adequate to draw any conclusions. Response: We agree dendritic cells (DCs) cannot be simply gated based on CD11c, especial when different DC subsets exist in skin and draining lymph nodes. To address this, we stained skin and lymph node cells with antibodies against CD11c, MHC II, CD11b, Langerin, CD103, CD8α, and CD80 in order to identify and explore RFA effects on different DC subsets in the revised manuscript (Fig. 2 & 3).

Macrophages can express CD11c and MHC-II; contrary, dendritic cells can express F4/80. The prototypical markers used in
3. The authors state that there is no applicable side effect with the RFA, but, little evidence (ex, lack of systemic adverse effects) for this claim is presented in this study. Response: We added local and systemic safety data in the revised manuscript (Fig. S5). Minor 1. In page 3, line 34: "Alum adjuvant induces Th2-biased immune responses and enhances mainly humoral immune responses 2, 4" is not correct, as Alum induces Th1-Th2 mixed response in humans (Coffman RL, Sher A, Seder RA., Immunity, 2010) Response: Alum adjuvant induces Th1 and Th2-mixed immune responses, which doesn't contradict with the description that 'Alum adjuvant induces Th2-biased immune responses' since Alum adjuvant more strongly induce Th2 versus Th1 immune responses (Petrovsky et al., Immunology and cell biology, 2004;Sunita et al., Front Immunol. 2013;James et al., J Immunol, 1999).
2. Figure 1C-G: It is helpful to show the gating strategy to identify neutrophils, monocytes, macrophages, eosinophils, and skin mDCs. Response: We followed a published method for gating the different cell types. In brief, live cells were gated based on FSC and SSC profile and then plotted based on expression of CD11b and CD11c. For gating neutrophils, CD11b + CD11ccells were gated and then plotted based on Ly6C and Ly6G. Ly6C + Ly6G hi cells were gated and analyzed for F4/80 expression. F4/80cells were gated as neutrophils. For gating eosinophils, CD11b + CD11ccells were gated and then plotted based on Ly6C and Ly6G. Ly6C -Ly6G int cells were gated and analyzed for F4/80 expression. F4/80int cells were gated as eosinophils.
For gating mDCs, CD11b + CD11c + cells were gated and then plotted based on MHC II and F4/80. MHC II hi F4/80 low cells were gated as mDCs. For gating macrophages and inflammatory monocytes, CD11b + CD11ccells were gated and then analyzed for F4/80 expression. F4/80 hi cells were gated as macrophages. F4/80 int cells were gated and analyzed for Ly6C expression. Ly6C + cells were gated as inflammatory monocytes.
3. In page 7, line 176: Ovalbumin from Sigma (Grade V) is known to contain LPS. The results and conclusion using this OVA may have been compromised by the existence of LPS, which strongly activates the innate immune response. If the authors perform LPS clean-up, it should be noted. Response: Endotoxin levels of OVA were reduced with Detoxi-Gel Endotoxin Removing Column (20344, Thermo Fisher Scientific) from original ~60 EU/ml to less than 15 EU/ml determined by Pierce LAL Chromogenic Endotoxin Quantification Kit (88282, Thermo Fisher Scientific) for in vivo application (See Materials and Methods). Figure 2, 3, 4, 5, 6: there is no description how many times these particular experiments were repeated to confirm reproducibility. Response: Experiments in all figures were repeated at least once and representative results were shown. 5. Figure 3: The authors describe "Log-rank test was used to compare differences in E.", but which method was used to correct for multiple comparisons between groups? Response: Log-rank test with Bonferroni correction was used to compare differences in E.

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6. In Figure 6: The authors describe that "Student's t-test was used to compare differences between groups". In this experiment, there are three genotypes crossed with two treatments. Please clarify how the authors perform the statistical analysis. In Figure 6C: there are 3 test groups. Which method was used to correct for multiple comparisons between groups? Response: We reanalyzed the multiple-group data using One-way ANOVA with Tukey's multiple comparison test in the revised manuscript ( Fig. 9C & 9D, Fig. S7). 7. In Material and Method section, Page 14, Line 417: the authors state that "This device has three energy settings (low, medium, high) and high energy setting was used in this study". Please provide more specific pieces of information including frequency and actual power density used in this study. It is helpful to include detailed methodology how the authors determined these parameters on the mouse skin. In addition, technical details how the authors established and reproducibly perform the application help other investigators reproduce the results. Response: This device works at around 1MHz frequency based on the manufacturer. We selected high energy level and 1.5 minutes of treatment time in order to induce significant thermal stress without skin damage for exploration of vaccine adjuvant effects. More technical details have been described in Materials and Methods. 8. In page 12, line 342: the authors state that "To our knowledge, RFA represents the first physical adjuvant that simultaneously augments both arms of adaptive immunity with adjuvant potency". However, nanosecond pulsed laser is reported to augment both CD4 and CD8 T cell responses when it was combined with the intramuscular administration of a model vaccine (Chen X, PLOS ONE, 2010). Moreover, the same nanosecond pulsed laser alone was sufficient to enhance anti-tumor immunity when combined with dendritic cell vaccine (Chen X, Clin Cancer Res, 2012).