Physical radiofrequency adjuvant enhances immune responses to influenza H5N1 vaccination

Abstract

Pre-pandemic influenza H5N1 vaccine has relatively low immunogenicity and often requires high antigen amounts and two immunizations to induce protective immunity. Incorporation of vaccine adjuvants is promising to stretch vaccine doses during pandemic outbreaks. This study presents a physical radiofrequency (RF) adjuvant (RFA) to conveniently and effectively increase the immunogenicity and efficacy of H5N1 vaccine without modification of vaccine preparation. Physical RFA is based on a brief RF treatment of the skin to induce thermal stress to enhance intradermal vaccine-induced immune responses with minimal local or systemic adverse reactions. We found that physical RFA could significantly increase H5N1 vaccine-induced hemagglutination inhibition antibody titers in murine models. Intradermal H5N1 vaccine in the presence of RFA but not vaccine alone significantly lowered lung viral titers, reduced body weight loss, and improved survival rates after lethal viral challenges. The improved protection in the presence of RFA was correlated with enhanced humoral and cellular immune responses to H5N1 vaccination in both male and female mice, indicating no gender difference of RFA effects in murine models. Our data support further development of the physical RFA to conveniently enhance the efficacy of H5N1 vaccine.

Introduction

Influenza is a highly contagious respiratory illness caused by influenza viruses. Influenza viral genome is composed of 8 single-stranded RNA segments encoding at least 10 proteins (1). Influenza viruses can be divided into 4 types (A, B, C, and D) (2). Human influenza is often caused by influenza type A and B viruses (2). Influenza type A viruses can be further divided into subtypes based on surface expression of hemagglutinin (HA) and neuraminidase (NA). There are total 18 HA and 11 NA subtypes that give rise to possible 198 influenza A subtypes (1). Influenza A viruses can infect a diverse range of hosts, including pigs, birds, and ducks (3). Influenza A H1N1 and H3N2 viruses mainly cause annual influenza epidemics in humans (1). Influenza B viruses contain two lineages (B/Yamagata and B/Victoria) and can cause human diseases (1). Influenza viruses infect respiratory tracts and lung tissues and can cause mild to severe diseases or even deaths, especially in patients with comorbidities (4). According to a recent CDC study, an estimated 3% to 11% of the US population gets sick from influenza each year (5). According to WHO, influenza causes about 3–5 million cases of severe illnesses and about 290,000–650,000 deaths each year worldwide.

Besides seasonal influenza, pandemic influenza also poses a threat to global public health. Four major influenza pandemics have occurred in the last 100 years or so: the 1918 H1N1 pandemic, the 1957 H2N2 pandemic, the 1968 H3N2 pandemic, and the 2009 H1N1 pandemic (6). The 1918 H1N1 pandemic claimed about 50 million lives, while the 1957 H2N2 pandemic and the 1968 H3N2 pandemic each claimed about one million lives (6, 7). The most recent 2009 H1N1 pandemic claimed about 284,000 lives (8). Influenza viruses that cause pandemics are often generated by reassortment of two or more influenza A viruses (7, 9). In fact, the virus that caused the 2009 H1N1 influenza pandemic resulted from triple-reassortment of classical swine H1N1 virus, avian-like Eurasian H1N1 virus, and triple reassortant H3N2 virus (10). Reassortments can generate completely new viruses, to which humans lack pre-existing immunity, and cause rapid transmission and severe diseases (6, 7, 9, 11).

Highly pathogenic avian influenza (HPAI) H5N1 poses a risk of causing future pandemics (12). HPAI H5N1 mainly infects birds and can cause sporadic transmissions to humans who are in close contact with infected birds (13). HPAI H5N1 can cause severe diseases in humans and has a high mortality rate (~60%) (13). Since its first infection of humans in 1997, H5N1 has infected more than 700 people in 15 countries in Asia, Africa, the Pacific, Europe, and Near East (14). Mathematical modeling suggested the value of even low-efficacy targeted vaccination in the early phase of a pandemic in reducing disease transmission (15). As of such, pre-pandemic H5N1 vaccines have been stockpiled in several countries including the United States. Three types of pre-pandemic H5N1 vaccines (non-adjuvanted, AS03-adjuvanted, and MF59-adjuvanted) have been prepared the U.S. government (16). Non-adjuvanted H5N1 vaccine was approved in 2007, which contained 90 μg HA and required two immunizations to elicit protective immunity (17, 18). AS03-adjuvanted H5N1 vaccine was approved in 2013 to reduce vaccine antigen use (3.75 μg HA) (19). AS03-adjuvanted H5N1 vaccine requires mixing of H5N1 antigen and AS03 adjuvant prior to administration and requires two immunizations to induce protective immunity (19). MF59-adjuvanted H5N1 vaccine was approved in 2020, which contained 7.5 μg HA and also required two immunizations to induce protective immunity (20).

AS03 and MF59 are squalene nanoemulsion-based adjuvants (21). Both adjuvants need to be manufactured in GMP facilities and mixed with H5N1 vaccines before packaging or immunization. Furthermore, AS03 adjuvant needs to be stored together with H5N1 vaccine in the refrigerator, which significantly increases the cold-chain storage volume. Also, the impact of physiochemical properties of nanoemulsions on vaccine-induced immune responses and adverse reactions has not been extensively studied. Recently, we developed a physical radiofrequency (RF) adjuvant (RFA) based on a brief non-invasive RF treatment of a small area of the skin to generate thermal stress with the potential release of damage-associated molecular patterns (DAMPs) to enhance vaccine-induced immune responses (22). We found RFA could induce transient low-level local inflammation and significantly enhance model antigen ovalbumin (OVA) and influenza pandemic 2009 H1N1 (pdm09) vaccine-induced humoral and cellular immune responses (22). We further found RFA showed a similar or better adjuvant effect than MF59-like AddaVax adjuvant to boost pdm09 vaccination depending on vaccine doses (22). Recently, we found RFA could also significantly enhance recombinant nucleoprotein (NP)-induced cellular immune responses and confer significant protection against lethal viral challenges in murine models (23). Physical RFA adjuvant has the advantage that it does not need to be mixed with vaccines and it can be stored at room temperature and conveniently used to boost vaccination. This study explored whether the physical RFA could significantly enhance H5N1 vaccine immunogenicity and protective efficacy in murine models.

Materials and Methods

Animals

Male and female C57BL/6 mice (6–8 weeks old) were obtained from Jackson Laboratories. Animals were housed in the facility of the University of Rhode Island (URI) and anesthetized for hair removal, RF treatment, and immunization. In most of the experiments, hair on the lateral back skin was removed by shaving followed by topical application of hair removal lotion (Nair) one day before experiment. Immunized mice were shipped to the animal facility at Georgia State University (GSU) for challenge studies. All animal procedures were approved by the URI (AN1516-004) and GSU (A21004) Institutional Animal Care and Use Committee (IACUC) and carried out with the Guide for the Care and Use of Laboratory Animals of the NIH.

RF treatment

A handheld Dot Matrix RF Anti-aging Face Lift Device (Norlanya Technology Co., Hong Kong, China) equipped with 12 × 12 array of a microelectrode in 2 × 2 cm² was used to treat mouse skin for 1–2 min without causing visible or histological skin damages as indicated in our previous studies (22). Before treatment, a thin layer of ultrasound coupling medium was applied on the skin surface and RF device was firmly pressed to enable treatment tips a tight contact with the skin. For sham treatment, an ultrasound coupling medium was applied and the RF device was firmly pressed except the device was not activated.

Preparation of challenge virus and inactivated split vaccine

To prepare the challenge virus and inactivated split vaccine, we used reassortant H5N1 virus (rgH5N1) containing H5 HA with the polybasic cleavage site deleted and N1 NA derived from A/Vietnam/1203/2004 and the backbone from A/Puerto Rico/8/1937 (24). The rgH5N1 virus was amplified in embryonated chicken eggs and harvested from allantoic fluids of the eggs. We next prepared inactivated rgH5N1 split vaccine as described (25). Briefly, live rgH5N1 viruses were inactivated with formalin (1:4000, v/v) (26) and concentrated by ultracentrifugation (123,760 × g) for 1 hour at 4°C. Viruses were then resuspended in phosphate-buffered saline (PBS) and disrupted with 1% Triton X-100. The viral particles were dialyzed against PBS using 30K molecular weight cutoff dialysis cassettes (Thermo Fisher Scientific). The total protein concentration of rgH5N1 split vaccine was determined by DC (detergent compatible) protein assay kit (Bio-Rad). The functional activities and contents of inactivated rgH5N1 split vaccine were determined by using hemagglutination units against chicken red blood cells (RBC) and Bio-Rad protein assay kit. Both challenge virus and split vaccine from rgH5N1 were aliquoted and stored at −80°C until use.

Immunization and challenge of mice

C57BL/6 mice (n=7–9 per gender) were intradermally (ID) immunized with H5N1 split vaccine (0.2 μg HA) alone (ID), or in the presence of RFA (RFA/ID), or intramuscularly (IM) immunized with the same amount of H5N1 vaccine in the presence of AddaVax adjuvant (1:1 volume ratio, AddaVax/IM), or non-immunized (NI, mock). Immunization was repeated 3 weeks later. Blood samples were collected 9 weeks after boost to measure serum antibody responses. To determine the protective efficacy, mice were intranasally challenged with 3 × LD₅₀ of rgH5N1 10 weeks after boost. A few mice were randomly picked to measure lung viral titers 5 or 6 days post infection (dpi), while the remaining mice were monitored for body weight loss and survival daily for 14 days.

Hemagglutination inhibition (HAI) assay

Immune sera were subjected to receptor destroying enzymes (RDE, Sigma-Aldrich) treatment and heat inactivation (56°C, 30 min). Serum HAI titers were determined using 4 HA units of rgH5N1 virus and 0.5% chicken RBCs (Lampire Biological Laboratories) as previously described (27).

Enzyme-linked immunosorbent assay (ELISA)

Antibodies specific for the rgH5N1 in immune sera were determined as previously described (26). Briefly, 96-well plates were coated with inactivated rgH5N1 (2 μg/mL) and then incubated with diluted immune sera. Plates were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, IgG1, and IgG2a antibodies (SouthernBiotech, Birmingham, AL) to measure anti-H5N1 IgG and subtype IgG1 and IgG2c antibody levels. Plates were further incubated with tetramethylbenzidine (TMB) substrates (eBiosciences, San Diego, CA) and the optical absorbance at 450 nm was read in a microplate reader. To measure H5N1-specific IgG antibody levels in cell cultures, mediastinal lymph node (MLN) and spleen were harvested 5 or 6 days after challenge and single-cell suspensions were prepared for use in this study. 96-well plates were precoated with inactivated rgH5N1 (2 μg/mL) and then incubated with MLN cells or splenocytes at 10⁶ cells/well for one or five days. Cells were then removed, and bound IgG antibody levels were measured with HRP-conjugated goat anti-mouse IgG antibodies as above described. The concentration of IgG was calculated using standard IgG (Southern Biotech).

Lung viral titration

Lung extracts were prepared by mechanical grinding of lung tissues harvested 5 or 6 dpi in 1.5 mL RPMI 1640 without fetal bovine serum (FBS) followed by lung viral titer determination by infecting embryonated chicken eggs (Hy-Line North America, Elizabethtown, PA) as previously described (28). Viral titers were calculated and expressed as 50% egg infection dose (EID₅₀)/mL according to the method of Reed and Muench (29).

Flow cytometry

Lung tissues were homogenized and spun on 44% and 67% Percoll gradients (GE Healthcare Bio-Sciences, Pittsburgh, PA) at 727.5 × g for 15 min. The layers containing lymphocytes were harvested. For intracellular cytokine staining, purified lung lymphocytes were stimulated with inactivated rgH5N1virus at 4 μg/mL in the presence of Brefeldin A (20 μg/mL, BD Biosciences, San Jose, CA) for 5 hours at 37 °C as described (26, 28). In vitro stimulated cells were then stained with fluorescence dye-conjugated anti-CD3 (clone 17A2, BD), anti-CD4 (clone RM405, eBioscience), and anti-CD8 antibodies (clone 53–6.7, eBioscience), fixed and then permeabilized using BD Cytofix/CytopermTM Plus Kit (BD Biosciences, San Jose, CA). Cells were then stained with fluorescence dye-conjugated anti-IFN-γ (clone XMG1.2, BD) and anti-TNF-α (clone MP6-XT22, Biolegend) followed by flow cytometry analysis in BD LSR-II/Fortessa (BD, San Diego, CA). Percentage of cytokine-secreting CD4⁺ and CD8⁺ T cells were analyzed by FlowJo software (Tree Star Inc.).

Statistical analyses

Values were expressed as Mean ± SEM (standard error of the mean). Student’s t-test was used to compare differences between groups and one-way ANOVA with Tukey’s Multiple Comparison test was used to compare differences for more than 2 groups or otherwise specified. Log-rank (Mantel-Cox) test with Bonferroni’s correction was used to compare differences of survival between groups. P value was calculated by PRISM software (GraphPad, San Diego, CA) and considered significant if it was less than 0.05.

Results

RFA increases antibody responses

Non-adjuvanted H5N1 vaccine requires high antigen amounts and two immunizations to induce protective immunity. We recently developed a physical RFA capable of enhancing pdm09 vaccine-induced immune responses with the RFA potency comparable or superior to MF59-like AddaVax adjuvant depending on vaccine doses (22). In this study, we explored whether the RFA was effective to enhance the efficacy of an inactivated rgH5N1 split vaccine in murine models. Considering gender has been an important biological variable, we also explored whether RFA could similarly enhance the H5N1 vaccine efficacy in both genders of mice.

Male and female mice were prime/boost immunized and serum HAI titers were measured 9 weeks after boost (Fig.1). In female mice, ID H5N1 vaccine in the presence of RFA significantly increased serum HAI titers when compared to non-immunized (NI) group (Fig.2A). IM H5N1 vaccine in the presence of AddaVax increased serum HAI titers more significantly, while ID vaccine alone failed to significantly increase serum HAI titers (Fig.2A). Similar results were observed in male mice (Fig.2B). In both genders, serum HAI titer in NI and ID groups was below 10, while those in RFA/ID group were above 32 and that in AddaVax/IM group was above 256. Serum HAI titers in male mice were comparable or slightly higher than those in female mice in all groups.

Fig.1.

Schematic illustration of experimental procedures and timelines

Fig.2. RFA enhances H5N1 vaccine-induced HAI titers.

C57BL/6 mice of both genders were subjected to ID immunization of H5N1 vaccine (0.2 ug HA) alone or in the presence of RFA, or IM immunization of the same amount of H5N1 vaccine in the presence of AddaVax adjuvant or left NI. Immunization was repeated 3 weeks later. Serum HAI titers were measured 9 weeks after boost. A. Serum HAI titers of female mice. B. Serum HAI titers in male mice. n=4 in NI and AddaVax/IM and n=6 in ID and RFA/ID. Kruskal-Wallis one-way ANOVA with Dunnett’s multiple comparison test was used to compare differences between NI and other groups. *, p<0.05; ***, p<0.001.

Besides HAI titers, we also measured anti-H5N1 IgG as well as subtype IgG1 and IgG2c antibody levels. As shown in Fig.3A, ID immunization of H5N1 vaccine alone failed to significantly increase anti-H5N1 IgG or subtype IgG1 or IgG2c antibody levels in female mice. ID immunization in the presence of RFA significantly increased serum anti-H5N1 IgG and subtype IgG1 but not IgG2c antibody levels in female mice (Fig.3A). IM immunization with AddaVax most significantly increased serum anti-H5N1 IgG and subtype IgG1 and IgG2c antibody levels in female mice (Fig.3A). Although serum anti-H5N1 IgG and subtype IgG1 and IgG2c antibodies in male mice showed a similar trend to that in female mice, RFA significantly increased serum IgG2c antibody levels in male mice (Fig.3B). The highest anti-H5N1 antibody levels were found in AddaVax/IM group followed by RFA/ID group, and then ID and NI groups in male mice (Fig.3B). Interestingly, ID immunization alone also significantly increased serum anti-H5N1 IgG and IgG1 antibody levels in male mice (Fig.3B).

Fig.3. RFA enhances H5N1 vaccine-induced IgG antibody responses.

Serum anti-H5N1 IgG and subtype IgG1 and IgG2c antibody levels were measured 9 weeks after boost. A. Serum IgG (left) and subtype IgG1 (middle) and IgG2c antibody levels (right) in female mice. B, Serum IgG (left) and subtype IgG1 (middle) and IgG2c antibody levels (right) in male mice. n=4 in NI and AddaVax/IM and n=6 in ID and RFA/ID. Two-way ANOVA with Dunnett’s multiple comparison test was used to compare absorbance reading differences between NI and other groups at each serum dilution. *, p<0.05; **, p<0.01; ***, p<0.001.

Reduced lung viral titers after challenge

The above studies indicated that RFA significantly increased H5N1 vaccine-induced antibody responses. Next, mice were intranasally challenged with a lethal dose of homologous viruses to evaluate the protection. A few mice were euthanized 5 or 6 dpi to quantify lung viral titers in embryonic eggs. As shown in Fig.4A, ID immunization alone failed to significantly reduce lung viral titers, while ID immunization in the presence of RFA significantly reduced lung viral titers and IM immunization with AddaVax more significantly reduced lung viral titers in female mice. Lung viral titers in male mice showed a similar trend to those in female mice with significantly lower lung viral titers observed in RFA/ID and AddaVax/IM groups (Fig.4B). Lung viral titers were more significantly reduced in AddaVax/IM group than those in RFA/ID group (Fig.4A–B).

Fig.4. RFA-adjuvanted H5N1 vaccine significantly reduces lung viral titer.

A. Female mice were challenged with rgH5N1 viruses (3 × LD₅₀) 10 weeks after boost. Lung viral titers were measured 6 dpi. B. Male mice were challenged with rgH5N1 viruses (3 × LD₅₀) 10 weeks after boost. Lung viral titers were measured 5 dpi. n=4. Kruskal-Wallis one-way ANOVA with uncorrected Dunnett’s test was used to compare differences between NI and other groups. *, p<0.05; **, p<0.01; ***, p<0.001.

Increased antibody responses in MLN and spleen after challenge

Antigen exposure may stimulate memory B cell differentiation into antibody-secreting plasma cells in secondary lymphoid organs following intranasal viral challenges. To this end, MLNs and spleen were harvested 6 dpi in female mice and 5 dpi in male mice. Single-cell suspensions were prepared and stimulated with pre-coated inactivated rgH5N1 for 1 or 5 days. Antibody levels were quantified and compared among groups. As shown in Fig.5A, anti-H5N1 IgG antibodies were produced at the highest levels by MLN cells in RFA/ID and AddaVax/IM groups followed by ID group in female mice. Anti-H5N1 antibody levels were comparable between RFA/ID and AddaVax/IM groups, which were significantly higher than those in ID group (Fig.5A). Interestingly, 5-day incubation did not further increase anti-H5N1 antibody levels (Fig.5A). A similar trend of anti-H5N1 antibody secretion was observed in male mice and 5-day incubation slightly increased antibody secretion (Fig.5B). Splenocytes showed the highest anti-H5N1 antibody secretion in AddaVax/IM group in both male and female mice (Fig.5C–D). Anti-H5N1 antibody levels secreted by splenocytes were significantly higher in RFA/ID group than those in ID group, which occurred only in female mice (Fig.5C–D). These results suggested that ID immunization in the presence of RFA and IM immunization with AddaVax induced comparable B cell memory responses in lung-draining MLNs, but the latter induced more potent systemic B cell memory responses in spleen.

Fig.5. Antibody responses in cultured MLN cells and splenocytes after challenge.

MLNs and spleen were harvested 6 dpi in female mice and 5 dpi in male mice. Single-cell suspensions of MLNs and spleen were prepared and stimulated with pre-coated inactivated rgH5N1 for 1 or 5 days. Antibody levels against H5N1 were quantified by ELISA. A. Antibody levels secreted by MLN cells of female mice. B. Antibody levels secreted by MLN cells of male mice. C. Antibody levels secreted by splenocytes of female mice. D. Antibody levels secreted by splenocytes of male mice. n=4. Two-way ANOVA with Dunnett’s multiple comparison test was used to compare differences between ID and other groups. *, p<0.05; **, p<0.01; ***, p<0.001.

Increased cellular immune responses in lung after challenge

Cellular immune responses in lung tissues were also evaluated 6 dpi in female mice and 5 dpi in male mice. Due to the crucial roles of IFN-γ and TNF-α in antiviral immunity (30), we also measured the number of IFN-γ and TNF-α-secreting CD4⁺ or CD8⁺ T cells. As shown in Fig.6A, ID immunization in the presence of RFA but not ID immunization alone significantly increased IFN-γ⁺CD8⁺ and TNF-α⁺CD8⁺ T cells in lung tissues of female mice. Interestingly, IM immunization with AddaVax failed to increase IFN-γ⁺CD8⁺ or TNF-α⁺CD8⁺ T cells in lung tissues of female mice (Fig.6A). ID immunization in the presence of RFA also induced the highest levels of IFN-γ⁺CD4⁺ and TNF-α⁺CD4⁺ T cells in lung tissues of female mice, while IM immunization with AddaVax did not increase IFN-γ⁺CD4⁺ or TNF-α⁺CD4⁺ T cells (Fig.6B). As observed in female mice, ID immunization in the presence of RFA also induced the highest levels of IFN-γ⁺CD8⁺ and TNF-α⁺CD8⁺ T cells (Fig.6C) as well as IFN-γ⁺CD4⁺ and TNF-α⁺CD4⁺ T cells (Fig.6D) in lung of male mice. Similarly, IM immunization with AddaVax adjuvant did not elicit significant IFN-γ⁺CD8⁺, TNF-α⁺CD8⁺, IFN-γ⁺CD4⁺, or TNF-α⁺CD4⁺ T cells in lung of male mice (Fig.6C–D).

Fig.6. Cellular immune responses in lung tissues after challenge.

Lung was harvested 6 dpi in female mice and 5 dpi in male mice. Lymphocytes were then isolated and stimulated with inactivated rgH5N1 for 5 hours followed by intracellular cytokine staining and flow cytometry analysis. A. IFN-γ⁺CD8⁺ T cells and TNF-α⁺CD8⁺ T cells in lung tissues of female mice. B. IFN-γ⁺CD4⁺ T cells and TNF-α⁺CD4⁺ T cells in lung tissues of female mice. C. IFN-γ⁺CD8⁺ T cells and TNF-α⁺CD8⁺ T cells in lung tissues of male mice. D. IFN-γ⁺CD4⁺ T cells and TNF-α⁺CD4⁺ T cells in the lung tissues of male mice. n=4. Two-way ANOVA with Tukey’s multiple comparison test was used to compare differences between groups. *, p<0.05; **, p<0.01; ***, p<0.001.

RFA-adjuvanted H5N1 vaccine reduces body weight loss after challenge

As shown in Fig.7A, ID immunization alone failed to provide significant protection against body weight loss in female mice, while ID immunization in the presence of RFA or IM immunization with AddaVax induced significant protection against body weight loss (Fig.7A). Mice in the ID group lost more than 20% body weight, while mice in the RFA/ID group lost approximately 9% body weight and mice in AddaVax/IM group experienced almost no body weight loss (Fig.7A). These results were consistent with the serum IgG antibody and HAI titer data. A similar trend of protection was observed in male mice with the most significant protection observed in AddaVax/IM group followed by RFA/ID group and then ID group (Fig.7B).

Fig.7. RFA enhances H5N1 vaccine-induced protection against body weight loss.

A. Female mice were challenged with rgH5N1 (3 × LD₅₀) 10 weeks after boost. Body weight was monitored daily for 14 days. n=4. B. Male mice were challenged with rgH5N1 (3 × LD₅₀) 10 weeks after boost. Body weight was monitored daily for 14 days. n=3 for NI, n=4 for ID and RFA/ID, n=5 for AddaVax/IM.

RFA-adjuvanted H5N1 vaccine induces 100% survival rates after challenge

Survival of the viral challenged mice was also monitored. As shown in Fig.8A, all female mice in NI group died in 8 days and only 20% female mice survived in ID group. In contrast, all female mice survived in RFA/ID and AddaVax/IM groups. ID immunization alone failed to significantly increase survival of female mice, while ID immunization in the presence of RFA or IM immunization with AddaVax significantly increased survival of female mice (Fig.8A). ID immunization in the presence of RFA and IM immunization with AddaVax also induced significantly higher survival rates than ID immunization alone in female mice (Fig.8A). Survival of male mice showed a similar trend to female mice except that ID immunization alone also significantly increased survival of mice (Fig.8B), which was in good agreement to serum IgG antibody levels of male mice (Fig. 3B).

Fig.8. RFA enhances H5N1 vaccine-induced protection against lethality.

A. Survival of female mice after lethal viral challenges was monitored daily for 14 days. n=4. B. Survival of male mice after lethal vial challenges was monitored daily for 14 days. n=3 for NI, n=4 for ID and RFA/ID, and n=5 for AddaVax/IM. Log-rank test with Bonferroni’s correction was used to compare differences of survival between groups. *, p<0.05; **, P < 0.01; ***, P < 0.001.

Discussion

Our data support the effectiveness of the physical RFA to boost poorly immunogenic H5N1 vaccination. RFA significantly increased ID H5N1 vaccine-induced antibody titers and protection in murine models, while ID vaccine alone failed to induce significant antibody titers or confer significant protection. Different from our previous finding that RFA was comparable or superior to AddaVax adjuvant to boost pdm09 vaccination (22), weaker RFA effects were observed in this study in boosting H5N1 vaccination when compared to AddaVax, which was reflected by the lower serum antibody titers and higher lung viral titers and more body weight loss after challenge in RFA than AddaVax group. Such a discrepancy needs further exploration but may reflect the differential potency of RFA to boost different influenza vaccination. Despite slightly weaker adjuvant effects against H5N1 vaccination, ID immunization in the presence of RFA conferred 100% protection against lethal viral challenges, similar to IM immunization with AddaVax adjuvant.

Anti-H5N1 antibodies secreted from cultured splenocytes were well correlated with serum HAI titers and anti-H5N1 antibody responses with the highest production from the AddaVax/IM group followed by the RFA/ID and then ID group. Interestingly, similar levels of anti-H5N1 antibodies were secreted from MLN cells of mice in the RFA/ID and AddaVax/IM groups after 5-day incubation regardless of the gender of mice. Considering the mice were challenged 10 weeks after boost, antibodies in vitro released from cultured MLN cells were most likely produced from a mixture of plasma cells differentiated from memory B cells upon viral exposure and long-lived plasma cells induced by vaccination (31). Thus, ID immunization in the presence of RFA and IM immunization with AddaVax likely stimulated similar levels of vaccine-specific plasma cells in lung-draining MLNs despite weaker systemic humoral immune responses induced by the former. Investigation of cellular immune responses after challenge identified the highest levels of IFN-γ and TNF-α-secreting CD8⁺ and CD4⁺ T cells in lung tissues of mice in RFA/ID group followed by ID group regardless of genders, while IM immunization with AddaVax did not generate these cell types in either male or female mice. Considering viral infection alone in NI mice only slightly induced these cells in the lung tissue when compared to non-infected mice, significant induction of these cells in RFA/ID group and to a lesser extent in ID group might reflect the uniqueness of the immunization route and the ability of RFA to potentiate lung cellular immune responses after challenges, which require further investigation.

Gender has been recognized as a biological variable in vaccine-induced humoral immune responses (32). Females usually develop higher antibody responses and experience more adverse reactions following vaccination than males (32). It is plausible that adjuvant effects may vary with genders. In this study, we found that RFA similarly enhanced H5N1 vaccine-induced HAI titers and IgG antibody responses in male and female mice. RFA effects on antibody secrection from culture MLN cells and splenocytes and cellular immune responses of the lung after challenge, as well as vaccine-induced protection, such as lung viral titers, weight loss, and survival, exhibited a similar trend among the different groups between male and female mice. These results indicated that RFA effects were not affected by gender in murine models.

RFA has the below advantages to boost H5N1 vaccination. First, physical RFA is delivered by a device. Thus, it does not need to be packaged and shipped together with vaccines. Second, physical RFA does not need cold-chain storage and can be conveniently stored at room temperature without the need of refrigeration. This is critical for use in resource-poor territories. Thirdly, there is no requirement of changing vaccine formulations with the use of physical RFA, and thus currently stockpiled non-adjuvanted H5N1 vaccine can be conveniently used in conjunction with RFA to reach more people. Furthermore, physical RFA can be used repeatedly for cost-effective adjuvantation. Besides dose-sparing, the physical RFA may also be used to enhance H5N1 vaccine efficacy in the elderly or immunocompromised populations. More work is needed to explore whether RFA could induce cross-protective immune responses against other H5N1 strains. Besides RFA, laser adjuvants have been also explored to boost influenza vaccination with similar advantages to RFA. Laser adjuvants deliver non-invasive laser beams to increase motility of antigen-presenting cells (APCs) or micro-fractional laser beams to create microthermal damages to enhance ID vaccine-induced immune responses (33–37). Laser adjuvants demonstrated substantial adjuvant effects against seasonal influenza vaccination in preclinical animal models (33–37). A near-infrared laser adjuvant has advanced to a pilot clinical test and was found to promote cutaneous immune cell trafficking without skin damage (38). Recently, we also reported an ablative fractional laser technology capable of alerting innate immune systems for more immunogenic vaccination without needle injection (39). These promising works support the development of alternative physical adjuvants to boost vaccination due to the slow pace to develop safe and effective chemical adjuvants.

RFA is safe to use and RFA effects are achieved without induction of visible or histological skin damages (22). Our previous studies found RFA induced transient low-level local inflammation, such as cytokine and chemokine gene expression and immune cell recruitment, in contrast to chemical adjuvants that induced lasting and more intense local inflammation (22). These results suggest that RFA stimulates well-controlled local inflammation. RFA is also less likely to induce systemic adverse reactions due to its transient localized effects. The underlying mechanisms of RFA is still under investigation and may be related to thermal stress with the potential release of DAMPs to enhance vaccine-induced immune responses. The safety and potency support further exploration of RFA for human use.

Data Availability

The raw/processed data required to reproduce these findings are available from the corresponding author upon request.