Comparison of vaccination efficacy using live or ultraviolet-inactivated influenza viruses introduced by different routes in a mouse model

Kyeongbin Baek; Sony Maharjan; Madhav Akauliya; Bikash Thapa; Dongbum Kim; Jinsoo Kim; Minyoung Kim; Mijeong Kang; Suyeon Kim; Joon-Yong Bae; Keun-Wook Lee; Man-Seong Park; Younghee Lee; Hyung-Joo Kwon

doi:10.1371/journal.pone.0275722

. 2022 Oct 10;17(10):e0275722. doi: 10.1371/journal.pone.0275722

Comparison of vaccination efficacy using live or ultraviolet-inactivated influenza viruses introduced by different routes in a mouse model

Kyeongbin Baek ^1,^#, Sony Maharjan ^2,^#, Madhav Akauliya ^2,^#, Bikash Thapa ³, Dongbum Kim ², Jinsoo Kim ¹, Minyoung Kim ¹, Mijeong Kang ¹, Suyeon Kim ¹, Joon-Yong Bae ⁴, Keun-Wook Lee ³, Man-Seong Park ⁴, Younghee Lee ⁵, Hyung-Joo Kwon ^1,^2,^*

Editor: Juan Carlos de la Torre⁶

PMCID: PMC9550053 PMID: 36215268

Abstract

Influenza is a major cause of highly contagious respiratory illness resulting in high mortality and morbidity worldwide. Annual vaccination is an effective way to prevent infection and complication from constantly mutating influenza strains. Vaccination utilizes preemptive inoculation with live virus, live attenuated virus, inactivated virus, or virus segments for optimal immune activation. The route of administration also affects the efficacy of the vaccination. Here, we evaluated the effects of inoculation with ultraviolet (UV)-inactivated or live influenza A virus strains and compared their effectiveness and cross protection when intraperitoneal and intramuscular routes of administration were used in mice. Intramuscular or intraperitoneal inoculation with UV-inactivated Influenza A/WSN/1933 provided some protection against intranasal challenge with a lethal dose of live Influenza A/WSN/1933 but only when a high dose of the virus was used in the inoculation. By contrast, inoculation with a low dose of live virus via either route provided complete protection against the same intranasal challenge. Intraperitoneal inoculation with live or UV-inactivated Influenza A/Philippines/2/1982 and intramuscular inoculation with UV-inactivated Influenza A/Philippines/2/1982 failed to produce cross-reactive antibodies against Influenza A/WSN/1933. Intramuscular inoculation with live Influenza A/Philippines/2/1982 induced small amounts of cross-reactive antibodies but could not suppress the cytokine storm produced upon intranasal challenge with Influenza A/WSN/1993. None of the tested inoculation conditions provided observable cross protection against intranasal challenge with a different influenza strain. Taken together, vaccination efficacy was affected by the state and dose of the vaccine virus and the route of administration. These results provide practical data for the development of effective vaccines against influenza virus.

Introduction

Influenza is an acute infection of the respiratory tract mainly caused by the influenza A and B viruses. Since the 1918 influenza outbreak, the emergence of new influenza viruses has caused recurrent pandemic and seasonal epidemic outbreaks resulting in substantial morbidity and mortality worldwide [1, 2]. The fragmented nature of the influenza virus genome enables genetic reassortment and the constant generation of genetically and phenotypically distinct variants. This persistent variation presents a major challenge to the development of influenza vaccines [3]. Over the years, there have been many efforts to develop a universal influenza vaccine, but enduring and broad protective immunity is currently still out of reach. Understanding the mechanisms of cross-reactivity and the immune responses elicited by influenza infection and vaccination is vital to generate more effective vaccines [4].

Antibodies against the head of the influenza hemagglutinin (HA) protein have neutralizing activity against multiple influenza subtypes [5–7]. Neutralizing antibodies induced by infection with the pandemic H1N1 2009 strain that bound to the stem and head regions of HA were largely cross-reactive against other influenza strains [8, 9]. Vaccination with live seasonal FluMist vaccine enhances cross-protective T cell immunity against Influenza H1N1 CA04 by inducing CD4⁺ cells but not CD8⁺ T cells [10]. Notably, a single-dose intranasal administration of γ-A/PR8[H1N1] was reported to induce cross-protective immunity against Influenza H5N1 and other heterotypic infections, which was mediated by memory cytotoxic T cells [11]. Furthermore, it was reported that vaccination with the seasonal Influenza A/H3N2 virus induced protection against Influenza H5N1 and pH1N1 viruses, and this heterosubtypic immunity was also mediated by a strong T cell response [12, 13].

We previously demonstrated that intraperitoneal inoculation of mice with the Influenza H1N1 strain A/WSN/1933 (WSN) induced cross-reactive antibodies that facilitated heterosubtypic immunity to Influenza H3N2 strain A/Hongkong/4801/2014 [14, 15]. Furthermore, we found that intraperitoneal inoculation with live influenza A virus altered immune cell populations at an early stage, resulting in depletion of B cells and macrophages along with immense neutrophil infiltration in the peritoneal cavity and bone marrow [15]. Expansion of the CD8⁺ T cell population in response to intraperitoneal inoculation with live influenza A virus likely played a role in cell-mediated protective immunity.

Globally, various types of influenza vaccines have been used, including vaccines based on live attenuated viruses and whole inactivated viruses [16]. In addition, the route and site of immunization profoundly affect vaccine efficacy [17]. We evaluated the effects of vaccination using UV-inactivated virus or live virus administered at different doses intraperitoneally or intramuscularly. We also examined the cross protection conferred by different modes of vaccination.

Material and methods

Cell line and viruses

Madin-Darby canine kidney (MDCK) cells obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) were grown in minimum essential medium (MEM; Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific), 100 μg/ml streptomycin, and 100 U/ml penicillin in a 5% CO₂ incubator at 37°C. Influenza A virus subtypes A/WSN/1933(H1N1) (WSN) and A/Philippines/2/1982(H3N2) (H3N2 Php) were used in this study.

Virus preparations

Single-passage viruses were used to inoculate the allantoic cavity of 9-day old specific pathogen-free (SPF) embryonated chicken eggs. The inoculated eggs were then incubated in a humidified incubator at 37°C for 48 h. Allantoic fluid was then collected, centrifuged, and stored at -80°C prior to use.

MDCK cell monolayers were washed with PBS, infected with influenza A virus at an MOI of 0.01 in MEM containing 1 μg/ml L-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich, Saint Louis, MO, USA), and incubated at 37°C for 1 h. The inoculum was then removed, and the cells were grown in MEM containing 0.3% BSA for 72 h. Supernatants were then collected and centrifuged at 2,000 rpm for 10 min at 4°C to remove the cell debris. The presence of amplified influenza A viruses in the collected supernatant was verified by plaque assay. All work related to virus propagation and cell culture in this study was performed in Biosafety level 2 conditions.

Plaque assay

Plaque assays were performed using MDCK cells as described previously [18]. Briefly, 7×10⁵ cells/well were seeded on six-well plates and incubated at 37°C for 18 h. The confluent MDCK cell monolayer was then washed with PBS, inoculated with tenfold serial dilutions of virus stocks and lung homogenates, and incubated for 1 h at 37°C with periodic shaking at 15 min intervals. Unabsorbed virus was then removed, and the cells were overlaid with 2 ml DMEM/F12 medium (Thermo Fisher Scientific) containing 4% BSA, 10 mM HEPES, 2 mM glutamine, 50 mg/ml DEAE dextran, 2.5% sodium bicarbonate, 1 μg/ml TPCK-treated trypsin, 100 μg/ml streptomycin, 100 U/ml penicillin, and 0.6% immunodiffusion-grade agar. After incubation for 72 h at 37°C in a 5% CO₂ incubator, the cells were stained with 0.1% crystal violet, plaques were counted, and the virus titers were calculated.

Ultraviolet inactivation of influenza A virus

Inactivation of influenza virus was carried out as previously described [19]. Briefly, viruses were irradiated with 254 nm UV light at distance of 5 cm from a UV light source for 15 min. Inactivation of the viruses was confirmed by plaque assays showing no plaque-forming units after the UV exposure.

Ethics statement

All animal studies were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Veterinary Research & Quarantine Service of Korea. The Institutional Animal Care and Use Committee (IACUC) of Hallym University (Permit Number: Hallym2021-70) approved the animal experiments in this study. Research staff involved in animal care or handling took an education course for the users of experimental animal facility in Laboratory Animal Resources Center of Hallym University. They also took an education course for Biosafety Level 3 at Korea Human Resource Development Institute for Health & Welfare (KOHI). Exposure to 1–2% isoflurane (Pharmaceutical, Seoul, Korea) was used to anesthetize mice for virus infection. To collect lungs and blood by cardiac puncture, mice were anesthetized by intraperitoneal injection of 0.2 ml 2,2,2-tribromoethanol in tert-amyl alcohol (Avertin; Sigma-Aldrich). Health and behavior of experimental animals were monitored daily, and the bedding was changed once a week, which ensures a regulatory compliance for the welfare of laboratory animals. Humane endpoints were planned to euthanize the mice by CO₂ inhalation in accordance to the approved IACUC protocol when the mice lose 30% of adult body weight or exhibit evidence of debilitation, pain or distress such as a hunched posture, rough haircoat, reduced food consumption, emaciation, inactivity, difficulty ambulating, respiratory problems. Among 510 mice used, 120 mice were found dead before they reached the endpoint criteria. After experiments were terminated, remnant mice (n = 390) were euthanized by CO₂ inhalation, and all efforts were made to minimize animal pain and suffering.

Mice immunization and infection

Four-week-old female BALB/c (H-2^b) mice were obtained from Nara Biotech, Inc. (Seoul, Korea) and maintained in environmentally controlled SPF rooms with a 12-h light/dark cycle at 20–25°C with 40–45% humidity and ad libitum access to food and water. All animal experiments involving virus infection were performed under animal biosafety level 2 conditions in the Research Institute of Medical-Bio Convergence of Hallym University in accordance with the recommendation of the Institutional Biosafety Committee of Hallym University. During the experimental period, mice were maintained in an individually ventilated cage (IVC) under a 12-h light/dark cycle at 20–25°C with 40–45% humidity and ad libitum access to food and water. Mice were inoculated intraperitoneally or intramuscularly with live or UV-inactivated WSN or H3N2 Php at a dose of 5×10⁶ pfu or 5 × 10⁷ pfu per mouse. The live influenza virus used here is not a live attenuated vaccine. At 14 days post inoculation (dpi), the mice (n = 10/group) were challenged intranasally with 10 LD₅₀ of live WSN virus as described previously [19]. The mice were monitored daily for clinical signs and body weight for up to 10 days.

Sample collection

To examine virus-specific antibody production and cytokine production, blood samples (n = 5/group) were collected via retro-orbital bleeding 14 days after intraperitoneal or intramuscular inoculation and via cardiac puncture 5 days after intranasal challenge. Serum samples were prepared and stored at -80°C. The mice were sacrificed 5 days after intranasal challenge, and the lungs were removed for analysis. Lungs (n = 5/group) were weighed and homogenized using Tissue Lyser II (Qiagen, Hilden, Germany), and virus titers were determined by plaque assay. For histopathologic examination, lungs (n = 5/group) were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 μm thickness. The specimens were then stained with Gill’s Hematoxylin V (Muto Pure Chemicals, Tokyo, Japan) and Eosin Y solution (Sigma-Aldrich).

Measurement of cytokines in mouse lungs and sera

Cytokines in sera and lung homogenates were measured using a Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 Cytokine Kit (BD Biosciences, San Jose, CA, USA. Catalog No: 560485). The CBA experiments were performed in accordance with the manufacturer’s instructions as described previously [14]. The CBA kit contained a mixture of seven different capture beads with distinct fluorescent intensities coated with antibodies specific for IL-2, IL-4, IL-6, IL-10, IL-17A, TNF, and IFN-γ. The lung homogenates and sera were analyzed using FACSCalibur (BD Bioscience), and the levels of cytokines were quantified using the LEGENDplex^™ software, version 7.0 (BioLegend, San Diego, CA, USA).

ELISA

Ninety-six-well immunoplates (Nunc^™, Roskilde, Denmark) were coated with live WSN or H3N2 Php in carbonate buffer (pH 9.6) and incubated overnight at 4°C. After blocking with 1% BSA, threefold dilutions of sera in PBST were added to the plates, which were then incubated for 2 h at room temperature. The plates were then washed three times with PBST, and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (1:500 dilution; Catalogue No: 5300–05, Southern Biotechnology Associates, Inc., Birmingham, AL, USA) was added. After 1 h incubation at room temperature, the plates were washed three times with PBST and developed colorimetrically using TMB (3,3’,5,5’-tetramethylbenzidine) substrate solution (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA). The absorbance at 450 nm was then measured using a SpectraMax 250 microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Hemagglutination inhibition (HI) assay

Ninety-six-well V-bottom plates (Costar, Corning, NY, USA) were used for the HI assay as described previously [15]. Receptor-destroying enzyme-treated serum samples were serially diluted two-fold with PBS and then incubated with an equal volume of 4 hemagglutination units (4HA) of WSN or H3N2 Php for 30 min. After incubation, an equal volume of 0.5% chicken red blood cells (Innovative Research, Novi, MI, USA) were added to the wells and incubated for 30 min at room temperature, and HI titers were measured.

Statistical analysis

Results are shown as the mean ± standard deviation. Differences between two samples were evaluated using Student’s t-test with P < 0.05 as the threshold for statistical significance.

Results

Intraperitoneal immunization with a high dose of UV-inactivated Influenza A/WSN/1933 induced a prophylactic effect against Influenza A/WSN/1933 challenge

To determine whether immunization with UV-inactivated WSN (UV-WSN) has a protective effect against subsequent challenge with live WSN, mice were intraperitoneally inoculated with a low (5 × 10⁶ pfu) or high (5 × 10⁷ pfu) dose of UV-WSN without adjuvants and challenged intranasally 14 days later with live WSN. The mice were then monitored for a further 10 days. The low-dose inoculation did not confer any protection in terms of reduced weight loss or increased survival after the intranasal challenge with the live virus, whereas the high-dose inoculation improved survival and reduced weight loss after the intranasal challenge (Fig 1A and 1B). We checked the production of WSN-specific antibody (IgG) in the sera of the mice by ELISA and found that the high-dose intraperitoneal inoculation produced a higher level of WSN-specific IgG than the low-dose intraperitoneal inoculation (Fig 1C). These results showed that the high-dose inoculation with UV-WSN protected mice against a subsequent lethal dose of live WSN.

Fig 1 — BALB/c mice were inoculated intraperitoneally with 5 × 10⁶ pfu (low dose, LD) or 5 × 10⁷ pfu (high dose, HD) of UV-inactivated Influenza A/WSN/1933 (UV-WSN). At day 14 post inoculation, the mice were challenged intranasally with 10 LD₅₀ of live Influenza A/WSN/1933 (WSN). (A, B) Survival (A) and body weight (B) were evaluated over a 10-day period after the challenge (n = 10/group). (C) Blood samples were collected at day 14 after initial intraperitoneal inoculation or at day 5 after intranasal challenge, and amounts of WSN-specific IgG in sera were determined by ELISA (n = 5/group). ***p < 0.001.

Intraperitoneal inoculation with live Influenza A/WSN/1933, but not live Influenza A/Philippines/2/1982, provided protection against Influenza A/WSN/1933 challenge

Cross protection against different strains of influenza is desirable for effective vaccines. To study the cross-protective effect of inoculation with different influenza strains, we injected mice intraperitoneally with PBS or a low dose (5 × 10⁶ pfu/mouse) of either live WSN or live H3N2 Php and challenged the mice 14 days later with intranasal administration of a lethal dose (10 LD₅₀) of live WSN. In contrast to low-dose inoculation with UV-WSN, low-dose inoculation with live WSN virus provided protection against subsequent intranasal challenge with a lethal dose of the same virus, as shown by a 100% survival rate and no observable weight loss (Fig 2A and 2B), no obvious lung pathology (Fig 2C and 2E), and evidence of viral clearance in plaque assays of lung homogenates (Fig 2D). In contrast, inoculation with live H3N2 Php did not confer any protection against weight loss or mortality after intranasal challenge with WSN (Fig 2A and 2B). Morbidity was further confirmed in the mice inoculated with H3N2 Php by an abnormal husky red color, increased viral load, severe alveolar damage, and invasion by inflammatory cells within lung tissues (Fig 2C–2E). The serum levels of WSN-specific IgG 5 days after the intranasal challenge were higher in the mice that were pre-inoculated with WSN than in control mice that were injected with PBS prior to the intranasal challenge (Fig 2F, left panel). By contrast, no WSN-specific IgG was detected in the mice that were pre-inoculated with H3N2 Php (Fig 2F, left panel), although these mice did produce high levels of H3N2 Php-specific IgG (Fig 2F, right panel). When the mice pre-inoculated with H3N2 Php was intranasally challenged with WSN, WSN-specific IgG was produced with a level similar to that of the WSN-inoculated mice. Importantly, WSN challenge alone did not induce detectable production of WSN-specific IgG in the PBS control mice. Therefore, H3N2 Php inoculation is presumed to activate the immune system even though it can’t induce WSN-specific antibody production. Production of H3N2 Php-specific IgG was further increased by intranasal challenge with WSN suggesting that WSN can activate production of H3N2 Php-specific IgG possibly via cellular immunity. Importantly, the serum of WSN-inoculated mice inhibited hemagglutination mediated by WSN, but did not show any cross-reactivity to inhibit hemagglutination by H3N2 Php (Fig 2G). Taken together, these results indicate that intraperitoneal inoculation with live H3N2 Php did not induce any detectable WSN-binding antibody or protect against subsequent intranasal exposure to WSN.

Fig 2 — BALB/c mice were inoculated intraperitoneally with 5 × 10⁶ pfu of live Influenza A/WSN/1933 (WSN) or live Influenza A/Philippines/2/1982 (H3N2 Php). At day 14 post inoculation, the mice were challenged intranasally with 10 LD₅₀ of WSN. (A, B) Survival (A) and body weight (B) were evaluated over a 10-day period after the intranasal challenge (n = 10/group). (C) Lungs were observed macroscopically 5 days after the intranasal challenge n = 5/group. (D) Viral titers in lung homogenates were determined by plaque assay 5 days after the intranasal challenge. (E) H&E staining of the paraffin-embedded lung sections collected 5 days after the intranasal challenge. Scale bars: 25 μm. (F) The level of WSN-specific total IgG (left) and H3N2 Php-specific total IgG (right) in sera were determined by ELISA (n = 5/group). (G) Hemagglutination inhibition (HI) assay. HI titers of each serum sample were determined with 4 hemagglutination units (4HA) of WSN (left) or H3N2 Php (right). *p<0.05, **p<0.01, ***p < 0.001.

Intramuscular inoculation with UV-inactivated Influenza A/WSN/1933 conferred a small prophylactic effect against Influenza A/WSN/1933 challenge

The most common immunization route in humans is intramuscular injection, which is generally convenient and effective [20]. We therefore investigated the protective effect of intramuscular injections of low (5 × 10⁶ pfu/mouse) and high (5 × 10⁷ pfu/mouse) doses of UV-WSN or UV-inactivated H3N2 Php (UV-H3N2 Php) against intranasal challenge with a lethal dose of live WSN (10 LD₅₀). Intramuscular inoculation with the low dose of either UV-inactivated strain did not confer protective or cross-protective effects against the subsequent intranasal challenge, as all the mice succumbed to infection, exhibited weight loss, and died within 7 days post challenge (Fig 3A and 3B). Macroscopic features, histological features, and viral load within the lungs were similar between the mice with low-dose intramuscular inoculations and control mice that were not inoculated prior to intranasal challenge (Fig 3C–3E). In contrast, the high-dose intramuscular inoculation with UV-WSN elicited a moderate prophylactic effect, as shown by less weight loss and 40% survival after the intranasal challenge (Fig 4A and 4B). Additionally, the lungs of the mice inoculated with the high dose of UV-WSN appeared normal on macroscopic examination, with histological evidence of moderate pathology in lung sections and a slight reduction in viral load within the lungs (Fig 4C–4E). By contrast, high-dose immunization with UV-H3N2 Php did not show any protective effects in comparison with control mice that were not inoculated prior to intranasal challenge (Fig 4B–4E). Notably, the high-dose intramuscular inoculation with UV-WSN did not produce IgG specific for WSN or H3N2 Php in the mice (Fig 4F).

Fig 3 — BALB/c mice were inoculated intramuscularly with 5 × 10⁶ pfu of UV-inactivated Influenza A/WSN/1933 (UV-WSN) or UV-inactivated Influenza A/Philippines/2/1982 (UV-H3N2 Php). At day 14 after inoculation, the mice were challenged intranasally with 10 LD₅₀ of live Influenza A/WSN/1933 (WSN). (A, B) Survival (A) and body weight (B) were evaluated over a 10-day period after the intranasal challenge (n = 10/group). (C–E) Lung tissues and blood were collected 5 days after the intranasal challenge (n = 5/group). (C) Macroscopic features of the lung were examined. (D) Viral titers in lung homogenates were determined by plaque assay. (E) Paraffin-embedded sections of lung tissue were stained with H&E. Scale bars: 25 μm.

Fig 4 — BALB/c mice were intramuscularly inoculated with 5 × 10⁷ pfu of UV-inactivated Influenza A/WSN/1933 (UV-WSN) or UV-inactivated Influenza A/Philippines/2/1982 (UV-H3N2 Php). After 14 days, the mice were challenged intranasally with 10 LD₅₀ of live Influenza A/WSN/1933 (WSN). (A, B) Survival (A) and body weight (B) were measured for 10 days after the intranasal challenge (n = 10/group). (C–F) Lungs and sera were collected 5 days after the intranasal challenge (n = 5/group). (C) Lungs were evaluated macroscopically. (D) The viral load in the lungs was measured by plaque assay. (E) Paraffin-embedded lung sections were stained with H&E. Scale bars: 25 μm. (F) The amounts of WSN-specific (left) and H3N2 Php-specific (right) total IgG in the sera were determined by ELISA (n = 5/group). ND, not detected. **p < 0.01.

Intramuscular inoculation with live Influenza A/WSN/1933, but not live Influenza A/Philippines/2/1982, protected against subsequent intranasal challenge with Influenza A/WSN/1933

Because intramuscular inoculation with UV-inactivated virus was only moderately effective to prevent morbidity and mortality after subsequent intranasal exposure to live virus, we tested the protective effects of intramuscular inoculation with low doses of live virus. Mice were intramuscularly inoculated with 5 × 10⁶ pfu live WSN or live H3N2 Php and then challenged intranasally with a lethal dose (10 LD₅₀) of live WSN. Intramuscular inoculation with live WSN conferred complete protection against the subsequent intranasal challenge, as the inoculated mice displayed no weight loss and 100% survival after the challenge (Fig 5A and 5B) and had macroscopic features, viral load, and tissue histology of the lungs that were similar to those in uninfected mice (Fig 5C–5E). In contrast to inoculation with UV-inactivated virus, the inoculation with live WSN induced a high level of WSN-specific serum IgG production, which explains the complete protection against the intranasal challenge (Fig 5F, left panel). In contrast, intramuscular inoculation with live H3N2 Php did not improve weight loss, mortality, gross lung inflammation, lung histology, or viral load after the intranasal challenge in comparison with control mice that were inoculated with PBS prior to the intranasal challenge (Fig 5A–5E).

Fig 5 — BALB/c mice were intramuscularly inoculated with 5 × 10⁶ pfu of live Influenza A/WSN/1933 (WSN) or live Influenza A/Philippines/2/1982 (H3N2 Php). After 14 days, the mice were challenged intranasally with 10 LD₅₀ of live WSN. (A, B) Survival (A) and body weight (B) were measured for 10 days after the intranasal challenge (n = 10/group). (C–F) Lungs and blood were collected 5 days after the intranasal challenge (n = 5/group). (C) The lungs were examined macroscopically. (D) Viral titers in the lungs were measured by plaque assay. (E) H&E staining was performed on formalin-fixed, paraffin-embedded tissue sections of the lungs. Scale bars: 25 μm. (F) The amounts of WSN-specific (left) and H3N2 Php-specific (right) total IgG in sera were determined by ELISA (n = 5/group). (G) Hemagglutination inhibition (HI) assay. HI titers of each serum sample were determined with 4 hemagglutination units (4HA) of WSN (left) or H3N2 Php (right). *p<0.05, ***p<0.001.

Intramuscular inoculation with live H3N2 Php induced production of WSN-specific IgG in the serum; however, the amount was smaller than that produced by intramuscular inoculation with live WSN (Fig 5F, left panel). Production of the WSN-specific antibody was further increased in the H3N2 Php-inoculated mice by the intranasal challenge with live WSN; however, the highest measured antibody level was comparable to that in control mice that were inoculated intramuscularly with live WSN without further challenge, which explains the absence of cross protection from the intramuscular inoculation. As expected, intramuscular inoculation with live H3N2 Php also induced production of H3N2 Php-specific IgG; however, the intranasal challenge with live WSN did not significantly enhance the production of that antibody. Conversely, intramuscular inoculation with live WSN induced production of H3N2 Php-specific IgG (Fig 5F, right panel), and the production of that antibody was enhanced by the intranasal challenge with live WSN. The serum of WSN-inoculated mice inhibited hemagglutination mediated by WSN but didn’t have any inhibitory effect against hemagglutination induced by H3N2 Php (Fig 5G) as shown for intraperitoneal inoculated mice in Fig 2G. Taken together, these results suggest that low-dose intramuscular inoculation with live virus confers complete protection against subsequent infection with the same virus but provides no cross protection against other viral subtypes.

Cytokine profiles in the sera of mice inoculated intramuscularly with live virus

During influenza infection, the level of cytokine storm determines the severity of the disease [21]. Therefore, we examined the cytokine levels in sera and lung tissues at various time points of immunization and infection. Cytokine levels in the sera and lungs of mice inoculated intramuscularly with live WSN or live H3N2 Php were similar to those in control mice injected with PBS (Fig 6A–6E); only IFN-γ was slightly increased at day 5 in the sera of the mice intramuscularly inoculated with live H3N2 Php (Fig 6B). These results indicate that intramuscular inoculation with live virus did not significantly affect systemic inflammation, which suggests that it would be safe for healthy individuals.

Fig 6 — BALB/c mice (n = 5) were intramuscularly injected with PBS, 5 × 10⁶ pfu of live Influenza A/WSN/1933 (WSN) (A, D) or live Influenza A/Philippines/2/1982 (H3N2 Php) (B, E). Blood (A, B) and lungs (D, E) were collected after mice were sacrificed at the indicated time points after inoculation. Sera and lung homogenates were prepared, and levels of cytokines were quantified using a cytokine bead array. (C, F) BALB/c mice (n = 5) were intramuscularly inoculated with PBS, live WSN or live H3N2 Php. After 14 days, the mice were challenged intranasally with PBS or 10 LD₅₀ of WSN. Sera (C) and lung homogenates (F) were prepared at day 5 after the challenge, and levels of cytokines were measured by cytokine bead array. ***p < 0.001.

Mice that were inoculated intramuscularly with live WSN and then challenged intranasally 14 days later with the same virus had the same cytokine levels as uninfected control mice (Fig 6C), whereas mice that were pre-inoculated with live H3N2 Php prior to intranasal challenge with live WSN had increased IFN-γ and IL-6 levels in the sera and increased IL-6 levels in the lungs compared with uninfected control mice (Fig 6C and 6F). Mice that were inoculated with PBS prior to the intranasal challenge had INF-γ and IL-6 levels in the lungs that were similar to those in the uninfected control mice (Fig 6F). These results suggest that the reduced morbidity and increased survival observed in mice that were intramuscularly inoculated with live WSN can be attributed to enhanced IgG production and suppression of inflammatory cytokines. Conversely, the absence of cross protection against H3N2 Php in WSN-inoculated mice might be related to the absence of functionally cross-reactive antibodies and failure to suppress inflammatory cytokine levels. Additionally, the cytokine levels in sera were not different than those in uninfected mice when the mice were intranasally challenged with live WSN 14 days after intraperitoneal inoculation with live H3N2 Php (S1 Fig). This suggests that the effect of the vaccine on cytokine regulation depends on vaccination route.

Discussion

Reemerging influenza virus subtypes cause high morbidity and mortality and represent a perpetual global threat [22]. Vaccination is one of the most cost-effective public health interventions against seasonal and pandemic influenza outbreaks. Currently, the standard vaccines protect against influenza by eliciting a neutralizing-antibody response to the viral HA and neuraminidase proteins, but they are unable to protect against new subtypes [23, 24]. One of the limitations to efficient vaccination arises from the fact that influenza viruses continuously undergo changes via antigenic drift and shift. Moreover, the traditional method of manufacturing influenza vaccines using eggs is time consuming and expensive [25]. A vaccine that induces cross protection against unpredictable virus strains and can be manufactured promptly and economically is urgently required. Therefore, we examined factors that might be expected to impact on the effectiveness of vaccination. We first examined the abilities of inoculation with live and UV-inactivated influenza viruses to protect against subsequent lethal influenza virus challenge in a mouse model. We then determined cross-protective efficacy using inoculation and challenge with two different influenza strains. In addition, we compared the breadth of protection provided by different routes of immunization.

Previously, we reported that intraperitoneal inoculation of mice with WSN induced cross protection against a lethal intraperitoneal dose of Influenza A/Hongkong/4801/2014, which was likely due to an increase in the CD8⁺ T cell population and cell-mediated protective immunity in response to the intraperitoneal inoculation [15]. In addition, we found that intraperitoneal inoculation with WSN induced immunity against intranasal exposure to Influenza A/Hongkong/4801/2014 [14].

Live-attenuated vaccines have been associated with highly successful global vaccination campaigns [26]. Both live-attenuated vaccines and inactivated-virus vaccines have been shown to be efficient and safe [27–32]; however, better protection is occasionally observed with live-attenuated vaccines [33–36]. Immunization with del-NS1 live-attenuated vaccine in mice provided protection against pandemic H1N1, H5N1, and H7N9 influenza viruses [37]. Studies in mice and ferrets using Influenza A/Ann Arbor/6/60 cold-adapted (ca) donor strain (H2N2) and Influenza A/Ann Arbor/6/60 ca-based 2009 pandemic H1N1 vaccine demonstrated the protective efficacy of live reassortant virus vaccines against H1N1 and H5N1 viruses [38, 39].

Previous studies in mice showed that intraperitoneal inoculation with live influenza virus confers protection against subsequent intranasal infection [40, 41]. We inoculated mice intraperitoneally with low and high doses of UV-WSN and then challenged them with a lethal intranasal dose of live WSN. The results showed that the high-dose inoculation with UV-inactivated virus provided substantial protection (~40%) against the intranasal exposure, whereas the low-dose inoculation did not confer any protection. Furthermore, we inoculated mice intraperitoneally with a low dose of live WSN or live H3N2 Php and then challenged them intranasally with a lethal dose of live WSN. The inoculation with live WSN conferred significantly more protection than the inoculation with UV-WSN, which is in line with previous findings that live virus is more effective than UV-inactivated virus [15]. In contrast, the intraperitoneal inoculation with live H3N2 Php did not provide any cross protection against intranasal challenge with live WSN.

The vaccine composition and route of administration are important parameters that affect the quality of vaccine response. The vast majority of licensed vaccines are administered via the intramuscular route [42], because conventional vaccination with aluminum-salt adjuvant led to severe adverse reactions when subcutaneous injection was used [20, 43]. Intramuscular injection of influenza vaccines was found to be more immunogenic than subcutaneous injection in elderly adults [44]. We found that intramuscular immunization with a high dose of UV-inactivated virus was only moderately effective to prevent morbidity and mortality due to subsequent intranasal infection. Similar to intraperitoneal inoculation, low-dose intramuscular inoculation did not confer any cross protection against intranasal infection with a different influenza strain, although it provided complete protection against intranasal infection with the same strain. Considering our previous results that intraperitoneal inoculation with WSN provided protection against subsequent challenge with Influenza A/Hongkong/4801/2014 [14, 15], cross-reactivity among different virus strains probably depends on the degree of homology of the viral gene sequences. In this study, we mainly revealed antibody production and compared outcome of the vaccinated mice after intranasal challenge. Considering that cellular immunity plays an important role in regulating the humoral responses and cellular responses toward the conserved genes of viruses may be critical for effective cross protection against heterologous viruses [4, 24], further investigation on cellular immunity including T cell responses is required.

The cytokine storm produced in response to influenza infection is known to determine the severity of disease [21]. Although higher cytokine levels during primary immunization were previously shown to enhance protection against a secondary challenge with influenza virus in mice [45], failure to lower the cytokine levels over the course of infection can result in severe damage to organs and eventually death [21]. We found that mice inoculated with H3N2 Php had elevated levels of IFN-γ and IL-6 after subsequent challenge with WSN, even at 5 days after the challenge. Hence, an inability to suppress the cytokine storm might have contributed to the lack of cross protection afforded by the initial inoculation. Further studies using various doses and different subtypes of live virus will provide more data on potential cross protection.

In summary, our study provides experimental evidence of the prophylactic effect of intraperitoneal and intramuscular immunizations with UV-inactivated or live influenza virus against subsequent intranasal exposure to live influenza virus. Overall, inoculation with live virus was more protective than inoculation with UV-inactivated virus, and the intramuscular and intraperitoneal routes of administration provided similar levels of protection when live virus was used.

Supporting information

S1 Fig. Cytokine levels in the sera after intraperitoneal inoculation with live virus and/or intranasal virus challenge.

BALB/c mice (n = 5) were intraperitoneally inoculated with PBS, live WSN or live H3N2 Php. After 14 days, the mice were challenged intranasally with PBS or 10 LD₅₀ of WSN. Sera were prepared at day 5 after the challenge, and levels of cytokines were measured by cytokine bead array.

(TIF)

Click here for additional data file.^{(80.5KB, tif)}

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

This research was supported by grants from the National Research Foundation (NRF-2020R1A2B5B02001806) funded by the Ministry of Science and ICT in South Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0275722.r001

Decision Letter 0

Juan Carlos de la Torre

8 Aug 2022

PONE-D-22-17990Comparison of vaccination efficacy using live or UV-inactivated influenza viruses introduced by different routes in a mouse modelPLOS ONE

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Reviewer #1: Kyeongbin Baek et al. investigated the protective effects of live and UV-inactivated

influenza viruses via intraperitoneal and intramuscular administration. Cross-

protection against different subtypes of influenza virus was also investigated. The

compositions and administration routes of vaccination are important issue to

consider in order to enhance the efficacy of vaccines. In the case of the UV-

inactivated A/WSN/1993 (H1N1) virus, only a high dose of intraperitoneal

administration induced some degree of protection against intranasal challenge with

live A/WSN/1993 virus. However, low dose of live A/WSN/1993 virus via both routes

of administration showed complete protective effects. Intraperitoneal or

intramuscular administration with live- or UV-inactivated A/Philippines/2/1982 (H3N2)

virus produced no cross-protective antibodies, and resulted in no cross-protection

against intranasal challenge with A/WSN/1993. This information can be used as a

practical data in order to develop an effective influenza vaccine for cross-protection.

The experiments are well conducted with appropriate data analysis. Therefore, I

recommend the manuscript to be accepted for publication, while it is necessary to

finish the revision as follows:

1. The authors used ELISA to measure the amount of influenza A virus-specific

IgG. It will be necessary to describe whether the WSN or H3N2 Php virus

coated on 96-well plates is a live virus or a UV-inactivated virus.

2. In Figure 5, cross-reactive IgG was produced by intramuscular administration

of live viruses. However, cross-protection against different subtypes of

influenza virus was not observed. Could you add some more discussion with

the concept of threshold?

3. On line 316-318, there is a sentence ‘‘Additionally, the cytokine levels in sera

and lungs were not different than those in uninfected mice when the mice

were intranasally challenged with live WSN 14 days after intraperitoneal

inoculation with live H3N2 Php (data not shown).’’ Please show the results in

the supplementary data.

Reviewer #2: Here, Baek et al. reported a study on evaluating the impact of vaccination administration routes on the vaccine efficacy using both inactivated and live influenza viruses. They compared the Intramuscular and intraperitoneal inoculation. They assessed antibody responses (ELISA) and protective immune responses (viral lethal challenge study). Moreover, they tested the breath of immune responses induced by these routes of vaccination. They found that there was a protective immunity induced by either route of vaccination but only when a high does was used. Finally, they tested the breath of immune responses triggered by their immunization.

Overall, this manuscript targeted on an important topic, the impact of vaccination route on efficacy. The entire story will benefit from a more stringent experimental design, including critical information and more precise description.

Major points:

1. Given that they used IM and IP as their vaccination route, they induced humoral responses through systematic immune responses. Thus, cellular immunity should play an important role in regulating the humoral responses. To strengthen their conclusion, it is important to provide the audience some experimental information about cellular immune responses. Particularly, they tested the immune responses towards a heterologous virus. This is likely because of cellular responses towards the internal genes, which are more conserved between the viruses they tested. Therefore, it is important to present data about T cells responses after their vaccination.

2. In the manuscript, they evaluated the antibody by ELISA. This only gave the information about the level of specific antibodies, but not potency for those specific antibodies. HI or microneutralization assay should be adequate to address this concern.

3. The manuscript will benefit from a more precise description. For example, the live influenza virus used here is not a live attenuated vaccine. Another example would be the description of dosage used for challenge. Instead of using specific PFU, a much more meaningful way would use an equivalent MLD50 value. In such a way, the readers will be better informed about their challenge stringency.

Minor points:

1. Line 28, it is a typo for “Influenza A/WSN/1993”

2. There is a lack of consistency in experimental design. For example, in Figure 3, in this set of in vivo studies, they only observed animals 7 days post challenge. In their other in vivo studies, they observed animal 10 days post challenge.

3. The amount of protein (ug) would be a more proper unit for describing the dosage used for IM inoculation.

4. There is no indication to authenticate their inactivation.

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2022 Oct 10;17(10):e0275722. doi: 10.1371/journal.pone.0275722.r002

Author response to Decision Letter 0

19 Sep 2022

We adjusted the format of our manuscript according to PLoS One 's style requirements. We indicated that “the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript”. We also clarified the issue of data availability as follows: All relevant data are within the manuscript and its Supporting information file. We don’t have any other repository information for our data. We are sorry to make you confused. We inserted S1 Fig for the data not shown in the original manuscript.

Point-to-point responses

Reviewer #1: Kyeongbin Baek et al. investigated the protective effects of live and UV-inactivated

influenza viruses via intraperitoneal and intramuscular administration. Cross-protection against different subtypes of influenza virus was also investigated. The compositions and administration routes of vaccination are important issue to consider in order to enhance the efficacy of vaccines. In the case of the UV-inactivated A/WSN/1993 (H1N1) virus, only a high dose of intraperitoneal administration induced some degree of protection against intranasal challenge with live A/WSN/1993 virus. However, low dose of live A/WSN/1993 virus via both routes of administration showed complete protective effects. Intraperitoneal or intramuscular administration with live- or UV-inactivated A/Philippines/2/1982 (H3N2) virus produced no cross-protective antibodies, and resulted in no cross-protection against intranasal challenge with A/WSN/1993. This information can be used as a practical data in order to develop an effective influenza vaccine for cross-protection. The experiments are well conducted with appropriate data analysis. Therefore, I recommend the manuscript to be accepted for publication, while it is necessary to finish the revision as follows:

1. The authors used ELISA to measure the amount of influenza A virus-specific IgG. It will be necessary to describe whether the WSN or H3N2 Php virus coated on 96-well plates is a live virus or a UV-inactivated virus.

Response : We used live viruses for ELISA to measure the amount of influenza A virus-specific IgG. Therefore, we described it in Line 174.

2. In Figure 5, cross-reactive IgG was produced by intramuscular administration of live viruses. However, cross-protection against different subtypes of influenza virus was not observed. Could you add some more discussion with the concept of threshold?

Response : As you pointed, the concept of threshold may explain our finding considering that the amounts of cross-reactive antibodies are small. In this revision, we additionally performed HI assays and found that the cross-reactive antibodies can’t neutralize another virus (Fig 2G and Fig 5G). Therefore, we became to know that the absence of cross-protection may be related with the functional properties of the specific antibodies induced by different subtypes rather than the quantity of the antibodies. Thanks.

3. On line 316-318, there is a sentence ‘‘Additionally, the cytokine levels in sera and lungs were not different than those in uninfected mice when the mice were intranasally challenged with live WSN 14 days after intraperitoneal inoculation with live H3N2 Php (data not shown).’’ Please show the results in the supplementary data.

Response : According to your comments, the data were presented in Supporting information as S1 Fig.

Reviewer #2: Here, Baek et al. reported a study on evaluating the impact of vaccination administration routes on the vaccine efficacy using both inactivated and live influenza viruses. They compared the Intramuscular and intraperitoneal inoculation. They assessed antibody responses (ELISA) and protective immune responses (viral lethal challenge study). Moreover, they tested the breath of immune responses induced by these routes of vaccination. They found that there was a protective immunity induced by either route of vaccination but only when a high dose was used. Finally, they tested the breath of immune responses triggered by their immunization.

Major points:

Responses:

As you commented, cellular responses are definitely important for vaccination effect. When we intranasally challenged with WSN after IP or IM inoculation with WSN or H3N2Php, the levels of virus-specific antibodies and their HI titers were significantly increased. Therefore, cellular immunity should be involved. However, to investigate the change of cellular immunity including T cell responses at this moment is difficult for us. Previously, we reported change of cell population including the increase of CD8+ cells as well as CD4+ T cells in response to IP injection with WSN (Frontiers in Immunology, 2019;10:1160. doi: 10.3389/fimmu.2019.01160). We wrote this finding in the introduction as follows in Line 67-74.

“We previously demonstrated that intraperitoneal inoculation of mice with the Influenza H1N1 strain A/WSN/1933 (WSN) induced cross-reactive antibodies that facilitated heterosubtypic immunity to Influenza H3N2 strain A/Hongkong/4801/2014 [14,15]. Furthermore, we found that intraperitoneal inoculation with live influenza A virus altered immune cell populations at an early stage, resulting in depletion of B cells and macrophages along with immense neutrophil infiltration in the peritoneal cavity and bone marrow [15]. Expansion of the CD8+ T cell population in response to intraperitoneal inoculation with live influenza A virus likely played a role in cell-mediated protective immunity.”

In addition, we added discussion as follows in Line 377-381. Thanks for your thoughtful comments.

“In this study, we mainly revealed antibody production and compared outcome of the vaccinated mice after intranasal challenge. Considering that cellular immunity plays an important role in regulating the humoral responses and cellular responses toward the conserved genes of viruses may be critical for effective cross protection against heterologous viruses [4,24], further investigation on cellular immunity including T cell responses is required.”

Response : According to your comment, we performed HI assays and presented the data in Fig 2G and Fig 5G. We found that antibodies induced by one virus effectively inhibit hemagglutination mediated by the same virus but had no cross-protective activity to neutralize the other virus. As a result, we became to know that the absence of cross-protection may be related with the functional properties of the specific antibodies induced by different subtypes rather than the quantity of the antibodies. Thanks for your suggestion.

Response : We inserted the information regarding “live virus” in the materials and methods section, “Mice immunization and infection” as you pointed (Line 148-149). I adopted your expression as it is. Thanks.

“The live influenza virus used here is not a live attenuated vaccine.”

We used 10 LD50 WSN virus for intranasal challenge as a lethal dosage. As your comment, we described the amounts of virus as the 10 LD50 WSN virus in Line 150-151. Thanks.

Minor points:

1. Line 28, it is a typo for “Influenza A/WSN/1993”

Response : We corrected as you pointed. Thanks.

Response : Actually, we observed the mice for 10 days in Figure 3 and 5. However, all the challenged mice died 7 days after challenge, therefore we made the graph up to 7 days. Considering your comment and to show the data more clearly, we changed the graph and showed the results up to 10 days. Thanks.

3. The amount of protein (ug) would be a more proper unit for describing the dosage used for IM inoculation.

Response : We used live or UV-inactivated virus solution for IM and IP inoculation. We used cell culture supernatants including viruses rather than purified virus, therefore we believe that pfu is more rational than the amount of protein (ug) for our experiments. Thanks for your consideration.

4. There is no indication to authenticate their inactivation.

Response : We have indicated the authentication of the inactivation in the section “Ultraviolet inactivation of influenza A virus” of the material and methods as follows (line 114-117). “Inactivation of the viruses was confirmed by plaque assays showing no plaque-forming units after the UV exposure.” We marked the part for your convenience.

Attachment

Submitted filename: Response to Reviewers.pdf

Click here for additional data file.^{(123.9KB, pdf)}

PLoS One. doi: 10.1371/journal.pone.0275722.r003

Decision Letter 1

Juan Carlos de la Torre

23 Sep 2022

Comparison of vaccination efficacy using live or ultraviolet-inactivated influenza viruses introduced by different routes in a mouse model

PONE-D-22-17990R1

Dear Dr. Kwon,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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Kind regards,

Juan Carlos de la Torre, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

In this revised version of their paper, the authors have adequately addressed most of the comments and concerns raised by the reviewers.

An issue that was not addressed relates to the T cell responses. However, the authors have provided a reasonable argument about the reasons why these data have not been incorporated into the revised version of their paper.

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0275722.r004

Acceptance letter

Juan Carlos de la Torre

30 Sep 2022

PONE-D-22-17990R1

Comparison of vaccination efficacy using live or ultraviolet-inactivated influenza viruses introduced by different routes in a mouse model

Dear Dr. Kwon:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Juan Carlos de la Torre

Academic Editor

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Cytokine levels in the sera after intraperitoneal inoculation with live virus and/or intranasal virus challenge.

(TIF)

Click here for additional data file.^{(80.5KB, tif)}

Attachment

Submitted filename: Response to Reviewers.pdf

Click here for additional data file.^{(123.9KB, pdf)}

Data Availability Statement

All relevant data are within the paper and its Supporting information files.

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PERMALINK

Comparison of vaccination efficacy using live or ultraviolet-inactivated influenza viruses introduced by different routes in a mouse model

Kyeongbin Baek

Sony Maharjan

Madhav Akauliya

Bikash Thapa

Dongbum Kim

Jinsoo Kim

Minyoung Kim

Mijeong Kang

Suyeon Kim

Joon-Yong Bae

Keun-Wook Lee

Man-Seong Park

Younghee Lee

Hyung-Joo Kwon

Roles

Abstract

Introduction

Material and methods

Cell line and viruses

Virus preparations

Plaque assay

Ultraviolet inactivation of influenza A virus

Ethics statement

Mice immunization and infection

Sample collection

Measurement of cytokines in mouse lungs and sera

ELISA

Hemagglutination inhibition (HI) assay

Statistical analysis

Results

Intraperitoneal immunization with a high dose of UV-inactivated Influenza A/WSN/1933 induced a prophylactic effect against Influenza A/WSN/1933 challenge

Fig 1. Intraperitoneal inoculation with a high dose of UV-inactivated Influenza A/WSN/1933 provided partial protection against lethal challenge with Influenza A/WSN/1933.

Intraperitoneal inoculation with live Influenza A/WSN/1933, but not live Influenza A/Philippines/2/1982, provided protection against Influenza A/WSN/1933 challenge

Fig 2. Intraperitoneal inoculation with a low dose of live Influenza A/WSN/1933 provides complete protection against intranasal challenge with Influenza A/WSN/1933.

Intramuscular inoculation with UV-inactivated Influenza A/WSN/1933 conferred a small prophylactic effect against Influenza A/WSN/1933 challenge

Fig 3. Intramuscular immunization with a low dose of UV-inactivated virus had no protective effect against intranasal challenge with live virus.

Fig 4. Intramuscular inoculation with a high dose of UV-inactivated virus provided partial protection against challenge with live virus.

Intramuscular inoculation with live Influenza A/WSN/1933, but not live Influenza A/Philippines/2/1982, protected against subsequent intranasal challenge with Influenza A/WSN/1933

Fig 5. Intramuscular inoculation with live virus protected against intranasal challenge with the same virus.

Cytokine profiles in the sera of mice inoculated intramuscularly with live virus

Fig 6. Cytokine levels in the sera and lung homogenates after intramuscular inoculation with live virus and/or intranasal virus challenge.

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Juan Carlos de la Torre

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Juan Carlos de la Torre

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Juan Carlos de la Torre

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