Analysis of vaccine-induced immune responses according to the immunization sequences of mRNA and protein vaccines

doi:10.21203/rs.3.rs-2361251/v1

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Analysis of vaccine-induced immune responses according to the immunization sequences of mRNA and protein vaccines

https://doi.org/10.21203/rs.3.rs-2361251/v1

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In response to the COVID-19 pandemic, different types of vaccines, such as inactive, live-attenuated, messenger RNA, and protein subunit, have been developed against SARS-CoV2. This circumstance has unintentionally led to heterologous prime-boost vaccination against a single virus in a large human population. Here, we aimed to analyze whether the immunization order of vaccine types influences the efficacy of heterologous prime-boost vaccination, especially mRNA and protein-based vaccines. We developed a new mRNA vaccine expressing hemagglutinin (HA) of influenza using the 3′UTR and 5′UTR of muscle cells (mRNA-HA) and tested its efficacy by heterologous immunization with an HA protein vaccine (protein-HA). The results demonstrated higher IgG2a levels and hemagglutination inhibition titers in mRNA-HA priming/protein-HA boosting (R-P) regimen than that induced by reverse immunization (protein-HA priming/mRNA-HA boosting, P-R). After the virus challenge, the R-P group showed lower virus titers and less inflammation in the lungs than the P-R group. Transcriptome analysis revealed that heterologous prime-boost groups had differentially activated immune response pathways, according to the order of immunization. In summary, our results demonstrate that the sequence of vaccination is critical to sculpt immune responses. This study provides the potential of a heterologous vaccination strategy using mRNA and protein vaccine platforms against viral infection.

Biological sciences/Immunology/Infectious diseases

Biological sciences/Immunology/Vaccines/RNA vaccines

mRNA vaccine

protein vaccine

heterologous prime-boost vaccination

Due to the rapid spread of SARS-CoV-2, the development of vaccines to reduce the morbidity and mortality associated with coronavirus disease (COVID-19) has spurred in several countries. The RNA vaccine platform is a next-generation platform consisting of messenger RNA (mRNA) that encodes the target antigen. In 1990, Wolff et al. ¹ first conceived the concept of an mRNA vaccine by demonstrating that in vitro-transcribed mRNA can be sufficiently expressed in mouse skeletal muscle cells through direct injection. However, its application was limited by the instability and ineffective in vivo delivery and the high innate immunogenicity of mRNA, which hinders the translation of antigens ^{2, 3}. Nonetheless, technological advances in lipid nanoparticles (LNPs) and the introduction of nucleoside modification by pseudouridine have shown the potential to overcome these limitations ^{4, 5}. mRNA does not integrate into the host genome, eliminating the need for insertional mutagenesis ⁶; it can be manufactured in a cell-free manner, allowing rapid, scalable, and cost-effective production ⁷.

Owing to these advantages, two COVID-19 mRNA vaccines (Moderna mRNA-1273 and Pfizer/BioNTech BNT162b2) have been authorized by the US Food and Drug Administration (FDA) for emergency use in under a year. Therefore, these early licensed mRNA vaccines have been given priority in many countries. Around the same time or later, various COVID-19 vaccines, including inactivated and viral vector vaccines, have been approved and globally administered. Furthermore, FDA has recently approved a traditional protein subunit type vaccine, Novavax, for emergency use ⁸. Because of the availability of these vaccines, people worldwide have unintentionally received a heterologous prime-boost vaccination schedule, i.e., the administration of different types of vaccines to achieve immunization against a single virus. It has been reported that heterologous prime-boost strategies induce more robust T-cell responses and higher neutralizing antibody titers ⁹. Increasing clinical results indicate that heterologous vaccination strategies such as priming with viral vectored vaccines followed by boosting with mRNA vaccines or priming with inactivated vaccines followed by boosting with mRNA vaccines against COVID-19 provided enhanced immune responses compared to homologous vaccination strategies ¹⁰. However, the immunogenicity and efficacy of heterologous priming-boosting using mRNA and protein vaccines have not yet been reported. It is also unknown how vaccine efficacy is affected by the sequence of immunization.

The influenza virus is one of the most important zoonotic viruses that has been estimated to cause ~ 3–5 million cases of severe illness and ~ 290,000–650,000 deaths every year globally ¹¹. Although current influenza vaccines are regarded as effective protection tools against disease, their effectiveness will be greatly reduced if novel pandemic strains emerge or fail to predict the vaccine strain ¹². Therefore, the need for developing an influenza mRNA vaccine that can be rapidly produced has been highlighted to respond to the emerging influenza pandemic. Currently, a few monovalent (Sanofi MRT-5400, MRT-5401; Pfizer PF-07252220) or quadrivalent (Moderna mRNA-1010) influenza mRNA vaccines encoding hemagglutinin (HA) from seasonal influenza strains are undergoing clinical testing, and several others are in the preclinical phase ¹³. Nevertheless, deducing an appropriate vaccination strategy along with increased available options is essential for preventing future pandemics.

In this study, our primary aim was to evaluate if the order of immunization of vaccine types affects the efficacy of a heterologous prime-boost vaccination strategy. To achieve this, we developed a novel mRNA platform expressing HA of the influenza virus using 3′ UTR and 5′ UTR of muscle cells (mRNA-HA). We tested its efficacy with a commercially available HA protein subunit vaccine (protein-HA) following homologous or heterologous immunization strategies to induce immune responses and protect from influenza infection. These results will provide insights into the rationale for the heterologous prime-boost strategy that was inevitably inoculated during the pandemic.

mRNA-HA priming and protein-HA boosting elicited balanced IgG1/IgG2a and high hemagglutination inhibition (HI) titers

We designed an mRNA platform that encodes the HA sequence of influenza strain A/Puerto Rico/8/1934 H1N1. The expression of HA protein in Vero cells was confirmed after transfection with mRNA-HA using western blotting (Fig. 1a). Next, we compared humoral responses induced by homologous or heterologous vaccination with those induced by mRNA-HA or protein-HA, following the strategy shown in Fig. 1b. The sera were diluted 50- to 819,200-fold to set an endpoint at which antibodies were no longer detected, and then IgG1 and IgG2a levels were measured at a serum dilution of 1:10,000 (Supplementary Fig. 1). As shown in Fig. 1c, mRNA-HA priming induced high levels of IgG2a, while protein-HA priming induced an IgG1-biased response. Balanced IgG1/IgG2a responses were observed in the heterologous mRNA-HA/protein-HA-immunized (R-P) and homologous mRNA-HA-immunized groups (R-R) groups (Fig. 1d). Because inducing neutralizing antibodies is a requirement for successful vaccine development, we checked the HI and microneutralization (MN) titers in the serum of each group. The R-P group showed higher levels of HI and MN titers than the P-R group, which were comparable to those of the homologous R-R group (Fig. 1e,f). The protein-HA homologous immunized group (P-P) showed the lowest HI and MN titers (Fig. 1e,f).

mRNA-HA priming and protein-HA boosting induced strong T-cell responses

Many studies have demonstrated that T-cells play a key role in protective immunity against influenza viruses ^{14, 15}. Therefore, we examined the T-cell responses induced by homologous or heterologous immunization with mRNA-HA and protein-HA. Mice were intramuscularly primed and boosted with LNP-formulated mRNA-HA (5 µg) or AddaVax™-formulated HA protein (1 µg) at 2-week intervals and then sacrificed 1 week after boosting (Fig. 2a). No statistical difference was observed in the enzyme-linked immunospot (ELISpot) activity of interferon-γ (IFN-γ) cytokine-producing cells in splenocytes between R-P and P-R groups according to the immunization sequence, whereas the activity was significantly stronger in these groups than that in the P-P group (Fig. 2b). A similar result was obtained using IFN-γ enzyme-linked immunosorbent assay (ELISA; Supplementary Fig. 1).

The frequency of antigen-specific IFN-γ producing cells in CD4⁺ T cells was higher in the R-P group than those in the P-P and P-R groups (Fig. 2c). Although it did not reach statistical significance, the frequency of antigen-specific tumor necrosis factor-α (TNF-α)-producing cells in CD4⁺ T cells was increased in the R-P group compared to those in the P-P and P-R groups (Fig. 2c; Supplementary Fig. 1). The frequencies of IFN-γ or TNF-α producing cells in CD8⁺ T cells were significantly increased only in the R-R group, and the frequency of interleukin-2 (IL-2)-producing cells in CD4⁺ T cells was increased in P-R and R-P groups compared to that in the control group (Fig. 2c). In addition, more CD4⁺ and CD8⁺ T cells were detected in the spleen tissues of the P-R and R-P groups than those in the P-P group (Fig. 2d).

Heterologous prime-boost regime enabled the timely activation of distinct immune response

To investigate the underlying mechanism of the heterologous prime-boost regime, we conducted RNA-seq of the splenic tissue samples of mice undergoing different vaccination regimes 7 days after boosting. Figure 3 shows the result of transcriptome analysis. In principal component analysis, different gene expression patterns were observed in the four vaccinated groups compared to the negative control Fig. 3a). As shown in Fig. 3b and c, the gene expression patterns differed among the P-P and R-R groups but not between the P-R and R-P groups. Furthermore, the comparison of the gene expression patterns according to the prime-boost strategies revealed more similar gene expression patterns depending on the type of vaccine used for priming (Fig. 3d). Gene Ontology pathway enrichment analysis (See Supplementary methods) using the Immune System Process database revealed increased mast cell and neutrophil degranulation pathways in the P-P group compared to that in the negative control group (Fig. 3e). The P-R group also showed an increase in mast cell and neutrophil degranulation pathways compared to the control group. Moreover, helper T cell diapedesis, cytotoxic T cell differentiation pathways, and stimulatory C-type lectin receptor signaling pathways were also increased in the P-R group than those in the control group (Fig. 3f). The R-P group showed enriched pathways similar to those in the P-R group; however, the regulation of Th2 differentiation and CD8⁺ T-cell activation pathways were increased in the R-P group compared to the P-R group (Fig. 3g). The R-R group showed increased innate immune response signaling, regulation of the dendritic cell pathway, and negative regulation of the T-cell cytokine production pathway, unlike other groups (Fig. 3h). Similar to the R-P group, the R-R group also showed increased regulation of the Th2 and cytotoxic T-cell differentiation pathways. Furthermore, the differentially expressed genes (DEG) analysis revealed that Bcl6, a transcription factor for follicular helper T-cells, and Hmgb1 were significantly increased compared to the negative control group only in the mRNA-primed groups (Fig. 3i). V-set immunoregulatory receptor and interferon regulatory factor 1 (Irf1) were specifically increased in the R-R group. Figure 3j shows the pathways and genes related to Hmgb1 among the enriched pathways associated with genes that were significantly increased in the mRNA-primed group when the gene expression of the two heterologous immunized groups was compared.

Evaluation of the protective efficacy of the heterologous priming-boosting regimen and analysis of immune responses after influenza infection

Next, we compared the protective efficacy of heterologous and homologous prime-boost regimes. Mice were subjected to priming and boosting as described previously (Fig. 1b) and challenged with PR8 virus 2 weeks after boosting (Fig. 4a). The prime-boost regimen with different vaccination sequences did not significantly affect body weight, clinical score, or survival rate (Fig. 4b–d). Although the induced HI titer in the P-R group was lower than that in the R-P group, it was sufficient to protect against the virus challenge. In H&E-stained spleen tissue, no difference in the size of the spleen or the ratio of white pulp was observed between the immunized groups. Vacuolation, indicative of spleen damage, occurred least in the R-P group (Supplementary Fig. 2).

Next, we assessed histopathological changes in all lung samples from the mice challenge study. One week post-challenge, all lung tissues from the virus-inoculated control group (Nil) showed severe histopathological changes characterized by bronchiolitis, inflammatory cell infiltration in the parenchyma, and epithelial hyperplasia (Fig. 5a). In contrast, mild to moderate (P-R) and minimal to mild (R-P) changes were observed in lung tissues collected from immunized mice. Moreover, virus titers in the lungs and BALF collected 1 week after the virus challenge were significantly reduced in the R-P group compared to that in the P-R group (Fig. 5b). Similar changes were observed in the lungs of mice stained with influenza A virus NP-specific antibody (Fig. 5c).

To further analyze the vaccine effectiveness after the virus challenge, we assessed the HA-specific IgG1, IgG2a, and IgA levels. IgG1 level was similar, but the IgG2a level was higher in the P-R group than that in the R-P group (Fig. 6a). No significant difference was observed between the two groups with respect to serum HA-specific IgA levels (Supplementary Fig. 3). The HI titer tended to increase in the R-P group compared to that in the P-R group (p > 0.05; Fig. 6b). HA-specific T-cells were significantly increased in the heterologously immunized groups compared to those in the control group. However, no statistical difference was observed in the frequency of HA tetramer-specific CD8⁺ T-cells between the heterologous immune groups according to the immunization sequence (Fig. 6c). Interestingly, different patterns of CD4⁺ and CD8⁺ T-cell responses were observed between the two groups (Fig. 6d). The percentages of IFN-γ, TNF-α, and IL-2 producing CD4⁺T cells were higher in the R-P group than those in the P-R group. In contrast, the percentages of IFN-γ, TNF-α, and IL-2 producing CD8⁺ T cells were higher in the P-R group than those in the R-P group (Fig. 6d, e). ELISpot activity of IFN-γ cytokine-producing cells in splenocytes of the heterologous prime-boosted groups was higher than that in those of the control group (Supplementary Fig. 3). In addition, the --- of proliferating effector CD4⁺ and CD8⁺ and central CD8⁺ T cells in the lungs after the virus challenge were the lowest in the R-P group compared to those in the control and P-R groups. This result indicated that the R-P group protected the lungs from virus infection (Supplementary Fig. 4).

Several studies have demonstrated that a heterologous prime-boost vaccination strategy is more effective than a homologous prime-boost strategy ^{10, 16}. For instance, a heterologous vaccination strategy with COVID-19 mRNA and viral vector vaccines has been shown to induce higher levels of spike-specific neutralizing antibodies and T-cells than a homologous vaccination strategy with only viral vector vaccines ⁹. A recent study has demonstrated that a booster shot of the COVID-19 mRNA vaccine after two doses of inactivated vaccine significantly increased immune responses to SARS-CoV-2 and has been speculated to provide better protection against severe COVID-19 than three doses of inactivated vaccine ¹⁷.However, no reliable data has been documented on the efficacy and rationale of heterologous prime-boost strategies using mRNA and protein platforms.

In this study, we developed an mRNA platform expressing the influenza HA protein and analyzed the immune responses induced by heterologous or homologous immunization. We demonstrated that mRNA-HA priming and protein-HA boosting induced higher levels of IgG2a compared to homologous mRNA-HA vaccination. Our results supported the speculation that the sequence of immunization critically affects vaccine-induced humoral responses. We showed that protein-HA priming and mRNA-HA boosting (P-R) failed to increase IgG2a compared to homologous protein-HA immunization (P-P). In line with this, the HI titers in the P-R groups were lower than those in the R-P groups. Furthermore, vaccine-induced T-cell responses were differentially induced, depending on the immunization sequence. Antigen-specific IFN-γ and TNF-α producing CD4⁺ T cells were increased in the R-P group 7 days after boosting. We also detected a significant increase in antigen-specific IFN-γ and TNF-α producing CD8⁺ T cells in the R-R group. These results suggest that homologous immunization with mRNA vaccines induces stronger CD8⁺ T cell responses than heterologous prime-boosting with protein and mRNA vaccines. However, after the challenge with the PR8 virus, a dramatic increase in IFN-γ and TNF-α producing CD8⁺ T cells was detected in the P-R group, and a lesser extent of increase was detected in the R-P group. Consistent with previous studies, this study suggests that the mRNA vaccine boosting induces a CD8⁺ T cell response ^{18, 19}. However, unlike CD8⁺ T cells, cytokine-producing CD4⁺ T cells dramatically increased in the R-P group. Therefore, the decision on the order of immunization should carefully consider the requirement of CD4⁺ or CD8⁺ T cell responses for protection. In addition, although no difference in clinical symptoms such as body weight, clinical score, and survival rate was observed 1 week after the challenge with the PR8 virus, the viral titers in the R-P group were lower than those in the P-R group. This result suggests that priming with an mRNA vaccine showed a more protective effect against influenza virus infection than priming with a protein vaccine.

Taken together, the present study demonstrated that an mRNA vaccine priming followed by a protein vaccine boosting showed better vaccine efficacy than the reverse immunization order. Studies have shown that mRNA vaccines exhibit more adverse reactions than traditional vaccines. In particular, when inoculation is repeated, mRNA vaccines might cause adverse reactions, such as myocarditis or hypersensitivity reactions, more frequently than traditional protein vaccines ^{20, 21}. However, because of their rapid design and large-scale manufacturing ability, mRNA vaccines could serve as the first line of defense to protect against the emergence of a new pandemic. Nevertheless, expanding the available vaccine options is the need of the hour to prevent the incidence of future pandemics. Non-live vaccines, such as protein subunit vaccines, have a good safety profile compared to other vaccines, even in infants, the elderly, and pregnant women ²². In addition, the low cost ²³ and stability of protein subunit vaccines under normal refrigeration conditions are advantageous ^{24, 25}. Therefore, it is speculated that for continuous vaccination, if required with the emergence of new variants of coronavirus, protein vaccines may impart a safety advantage over mRNA vaccines. Owing to these benefits, several protein subunit type vaccines targeting S or RBD proteins of SARS-CoV-2 are now under clinical trials; some, including Novavax, have obtained FDA approval for emergency use for people above 18 years of age ⁸. Recently, a nanoparticle-based vaccine consisting of a two-protein component with self-assembling and RBD proteins was clinically approved for COVID-19 in Korea ^{26, 27}.

In conclusion, our findings suggest a heterologous vaccination strategy with the first inoculation of an mRNA vaccine to defend against the infectious disease and then performing a secondary or tertiary inoculation with a protein vaccine that requires time to be produced could be the best way to develop a safe and efficient vaccination strategy against the virus.

The protocols of “Hemagglutination inhibition (HI) assay,” “Microneutralization (MN) assay,” “Flow cytometry,” “Transcriptome analysis,” and “Immunohistochemistry” are detailed in the Supplementary material.

Mice

Six-week-old female BALB/c mice were obtained from Daehan Biolink Co. Ltd. (Seoul, South Korea). The mice were acclimatized for 1 week immediately after they were brought to the Catholic University of Korea before starting the experiment. Mice were housed under pathogen-free conditions with a 12/12 h light/dark cycle, a temperature of 23 ± 2°C, and a relative humidity of 50% ± 10%. All animal experimental procedures in this study followed the guidelines of, and were approved by, the Institutional Animal Care and Use Committee of the Catholic University of Korea (CUK-IACUC-2022-020).

Design and synthesis of mRNA-HA

The DNA template for the mRNA vaccine was a DNA fragment encoding the HA protein of the influenza A virus (A/Puerto Rico/8/1934). DNA templates of the mRNA vaccine were cloned into a plasmid vector with backbone sequence elements (T7 promoter, 5′ and 3′ UTR, 100 nucleotide poly(A) tail) interrupted by a linker (A50LA50, 20 nucleotides) to improve RNA stability and translational efficiency. The DNA was purified, spectrophotometrically quantified, and in vitro-transcribed with an EZ™ T7 High Yield In Vitro Transcription kit (Enzynomics, Daejeon, South Korea) and a Cap 1 capping analog (SMARTCAP®, ST PHARM, Seoul, South Korea) and with N1-methylpseudouridine-5′-triphosphate (m1ΨTP; TriLink, CA, USA) to replace uridine-5′-triphosphate (UTP).

After transcription, RNA was purified by lithium chloride precipitation. dsRNA was eliminated by cellulose-based purification ²⁸. RNA integrity was assessed using gel electrophoresis, and the concentration, pH, and endotoxin levels of the solution were determined.

mRNA transfection and western blot

Vero cells were seeded at a density of 1 × 10⁶ cells/well in 6 well plates and incubated overnight. Afterward, 10 µg mRNA was transfected into each well using Lipofectamine2000™ (Invitrogen, MA, USA) according to the manufacturer's instructions. HA protein was detected using western blotting with influenza A H1N1 hemagglutinin antibody (Cat:11684-R107; SinoBiological, Inc., Beijing, China). Primary antibodies were diluted as 1:1,000 in phosphate-buffered solution containing 0.1% Tween20.

LNP formulation of the mRNA-HA

LNPs were prepared as reported protocol ²⁹. Briefly, all lipid components were dissolved in ethanol at a molar ratio of 25:25:10:38.5:1.5 (SM-102; 6,6’-trehalose dioleate; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamin (DOPE); butyl lithocholate; and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000)) and mRNAs were dissolved at a charge ratio of N/P = 3 in 50 mM sodium citrate buffer (pH 4) solution ³⁰. LNPs were formulated using NanoAssemblr® Ignite™ (Precision Nanosystems, BC, Canada) by mixing the aqueous and organic solutions at a ratio of 3:1 and a total flow rate of 10 mL/min. The solution of LNPs was concentrated by ultrafiltration using Amicon Ultra centrifugal Filter (UFC9030, Merck Millipore, MA, USA) following the manufacturer’s instructions.

Characterization of LNP

The size and zeta potential of the LNPs were determined using ZetaSizer Ultra (Malvern Panalytical, Malvern, UK). All the size and zeta potential data were obtained in triplicates (Supplementary Table 1). The mRNA encapsulation efficiency was analyzed using the Quant-iT RiboGreen RNA kit (Thermo Fisher Scientific, MA, USA). Briefly, mRNA-LNPs were lysed with 0.5% Triton-X or left untreated, followed by treatment with RiboGreen reagent following the manufacturer’s instructions. The quantity of mRNA in the samples was measured using a microplate reader (Spark®, TECAN, Mannedorf, Switzerland). The calculated encapsulation efficiency of mRNA was approximately 87%.

Immunization

BALB/c mice were immunized intramuscularly in the upper thigh twice (prime and boost) at an interval of 2 weeks, with 1 µg HA protein or 5 µg mRNA-HA. HA protein (Cat:11684-V08H; SinoBiological, Inc.) was formulated with AddaVax™ (1:1 ratio, v/v, InvivoGen, CA, USA), and mRNA-HA was formulated with LNP. The negative control group was injected with saline solution. For the T-cell analysis experiment, mice were immunized at 2-week intervals and sacrificed 1 week after boosting.

Elisa

Antigen-specific antibody levels were measured using ELISA. Briefly, 50 ng HA protein was coated onto 96-well transparent plates. To identify the endpoint, sera were diluted 1:50 to 1:819,200; the secondary antibody was diluted 1:5,000; total IgG was measured. The dilution factor was set to 1:10,000, and antigen-specific IgG1 and IgG2a levels were measured; IgA levels were measured in the sera at a 1:50 dilution.

ELISpot assay

ELISpot was conducted using an ELISpot Plus mouse IFN-γ (ALP) kit (3321-4APW; Mabtech, OH, USA). Approximately 2.5 × 10⁵ splenocytes were seeded in 48-well cell culture plates and stimulated with 2 µg/well HA-specific T cell epitope peptide mixture (IYSTVASSL, LYEKVKSQL, DYEELREQL, SFERFEIFPKE, HNTNGVTAACSH, KLKNSYVNKKGK, NAYVSVVTSNYNRRF, and CPKYVRSAKLRM) for 24 h at 37°C cell incubator. After 24 h, each step was performed following the manufacturer’s instructions.

Viruses

The A/H1N1 virus, A/Puerto Rico/8/1934, used for viral infection challenges, was generously provided by Dr. Seong BL of Yonsei University, Seoul, Korea.

Virus challenge

The heterologously immunized mice were challenged intranasally with 1 × 10³ plaque-forming units (PFU) of mouse-adapted A/Puerto Rico/8/1934 H1N1 virus in saline (50 µL) using a pipette. After the challenge, the body weight, survival, and clinical illness of mice were assessed. Clinical illness was scored using the following scale:0 = no visible signs of disease; − 1 = slight ruffling of fur; − 2 = ruffled fur, reduced mobility; − 3 = ruffled fur, reduced mobility, and rapid breathing; and − 4 = ruffled fur, minimal mobility, huddled appearance, and rapid and/or labored breathing. Animals were sacrificed when their body weight decreased by more than 25% of their original body weight.

Real-time polymerase chain reaction (PCR) for virus titration

Total RNA from the lungs and bronchoalveolar lavage fluid (BALF) was extracted using TRIzol® reagent (Favorgen, Ping-Tung, Taiwan). The PCR reaction mix (25 µL) comprised 12.5 µL 2X SuperScript III Platinum Master Mix (Invitrogen), 2 µL of the mixture comprising forward primer (10 µM), reverse primer (10 µM), and dual-labeled probe (5 pmol), 0.5 µL SuperScript III Taq polymerase (Invitrogen), and 10 µL template RNA, standard, or negative control. Real-time PCR was performed on a Bio-Rad thermocycler CFX96 (Bio-Rad Laboratories Inc., CA, USA). The PCR conditions were as follows: 30 min at 50°C and 5 min at 95°C, followed by 45 cycles of 20 s at 95°C, and 1 min at 55°C. For virus detection, we used two pairs of influenza virus-specific primers (forward 5′-GACCRATCCTGTCACCTCTGAC-3′, reverse 5′-AGGGACTTYTGGACAAAKCGTCTA-3′) and TaqMan probes (5′-FAM-TGCAGTCCTCGCTCACTGGGCACG-BHQ1) designed based on the conserved matrix gene region of influenza A virus.

BALF collection

Mouse lungs were lavaged using a 22-gauge catheter and 1 mL saline by flushing the airway compartment three times. The BALF was centrifuged at 20,000 × g for 10 min at 4°C.

Histopathological analysis

Sectioned lungs and spleens from experimental mice were submerged in 10% neutral buffered formalin, dehydrated, paraffin-embedded, and sliced into 4 µm thick sections for histopathological examination. Histological images were obtained and evaluated using the Aperio ImageScope version 12.4 (Leica Biosystems Pathology Imaging, Buffalo Grove, IL, USA). The severity of histological changes was determined using a 5-point scoring system ³¹ as follows: 0, no abnormality detected (NAD); 1 = minimal; 2 = mild; 3 = moderate; 4 = moderately severe; and 5, severe. The distribution was recorded as focal, multifocal, and diffused. Recruitment of inflammatory cells and morphological alterations in the lungs and spleen were assessed after hematoxylin and eosin (H&E) staining under a light microscope.

Statistical analysis

One-way ANOVA with the Bonferroni post hoc test was performed for multiple-group comparisons, and Mann–Whitney U test was used to compare the two groups. Differences were considered statistically significant at *p < 0.05, **p < 0.01, ***p < 0.005. Data are expressed as the mean ± standard deviation (SD). All statistical analyses were performed using GraphPad Prism 9 (GraphPad Software Inc., CA, USA).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

DATA AVAILABILITY

The data that support the findings of this study are available from the corresponding author upon reasonable requests.

ACKNOWLEDGMENTS

Jae-Hwan Nam was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. NRF-2021M3E5E3080558) and the Ministry of Food and Drug Safety (No. 22213MFDS421). Eun-Kyoung Bang was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (NRF-2021M3E5E3080563), National Research Council of Science and Technology (CAP20012-100), and KIST Institutional Program (2E21512, 2E31502).

AUTHOR CONTRIBUTIONS

J.-H. N. conceived and supervised the study and designed the experiments. H.-J. P., Y.-J. B., S. P. K., W. K., S.-I. P., G. R., J.-Y. K., E.-K. B., S. Y., H.-W. K., S.-H.B., Y. K., D. K., and G. K. contributed to the data acquisition. S.-H. H., H.-J. P., E.-K. B., and J.-H. N. contributed to manuscript writing.

COMPETING INTERESTS

The authors declare no competing interest.

Data availability statement

Data availability statements should provide a statement about the availability of the minimal dataset that would be necessary to interpret, replicate and build upon the methods or findings reported in the article. Data availability statements should be provided as a separate section after the Methods section before the References, under the heading "Data Availability". For further guidance, please refer to the data availability and data citations policy information and Frequently Asked Questions (FAQs).

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Analysis of vaccine-induced immune responses according to the immunization sequences of mRNA and protein vaccines

Status:

Version 1

Abstract

Figures

Introduction

Results

mRNA-HA priming and protein-HA boosting elicited balanced IgG1/IgG2a and high hemagglutination inhibition (HI) titers

mRNA-HA priming and protein-HA boosting induced strong T-cell responses

Heterologous prime-boost regime enabled the timely activation of distinct immune response

Discussion

Materials And Methods

Mice

Design and synthesis of mRNA-HA

mRNA transfection and western blot

LNP formulation of the mRNA-HA

Characterization of LNP

Immunization

Elisa

ELISpot assay

Viruses

Virus challenge

Real-time polymerase chain reaction (PCR) for virus titration

BALF collection

Histopathological analysis

Statistical analysis

Reporting summary

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1