Search

Adult females of reproductive age develop greater antibody responses to inactivated influenza vaccines (IIV) than males. How sex, age, and sex steroid concentrations impact B cells and durability of IIV-induced immunity and protection over 4 months post-vaccination (mpv) was analyzed. Vaccinated adult females had greater germinal center B cell and plasmablast frequencies in lymphoid tissues, higher neutralizing antibody responses 1-4 mpv, and better protection against live H1N1 challenge than adult males. Aged mice, regardless of sex, had reduced B cell frequencies, less durable antibody responses, and inferior protection after challenge than adult mice, which correlated with diminished estradiol among aged females. To confirm that greater IIV-induced immunity was caused by sex hormones, four core genotype (FCG) mice were used, in which the testes-determining gene, Sry , was deleted from chromosome Y (ChrY) and transferred to Chr3 to separate gonadal sex (i.e., ovaries or testes) from sex chromosome complement (i.e., XX or XY complement). Vaccinated, gonadal female FCG mice (XXF and XYF) had greater numbers of B cells, higher antiviral antibody titers, and reduced pulmonary virus titers following live H1N1 challenge than gonadal FCG males (XYM and XXM). To establish that lower estradiol concentrations cause diminished immunity, adult and aged females received either a placebo or estradiol replacement therapy prior to IIV. Estradiol replacement significantly increased IIV-induced antibody responses and reduced morbidity after the H1N1 challenge among aged females. These data highlight that estradiol is a targetable mechanism mediating greater humoral immunity following vaccination among adult females.IMPORTANCEFemales of reproductive ages develop greater antibody responses to influenza vaccines than males. We hypothesized that female-biased immunity and protection against influenza were mediated by estradiol signaling in B cells. Using diverse mouse models ranging from advanced-age mice to transgenic mice that separate sex steroids from sex chromosome complement, those mice with greater concentrations of estradiol consistently had greater numbers of antibody-producing B cells in lymphoid tissue, higher antiviral antibody titers, and greater protection against live influenza virus challenge. Treatment of aged female mice with estradiol enhanced vaccine-induced immunity and protection against disease, suggesting that estradiol signaling in B cells is critical for improved vaccine outcomes in females., Competing Interests: The authors declare no conflict of interest.

Inactivated influenza vaccines induce greater antibody responses in females than males among both humans and mice. To test the breadth of protection, we used recombinant mouse-adapted A/California/2009 (maA/Cal/09) H1N1 viruses containing mutations at one (1M), two (2M), or three (3M) antigenic sites, in addition to a virus containing the 1M mutation and a substitution of the Ca2 antigenic site (Sub) with one derived from an H5 hemagglutinin (HA) to challenge mice of both sexes. Following maA/Cal/09 vaccination, females produced greater virus-specific, class-switched total IgG and IgG2c antibodies against the vaccine and all mutant viruses, and antibodies from females recognized a greater number of unique, linear HA epitopes than did antibodies from males. While females had greater neutralizing antibody titers against the vaccine virus, both sexes showed a lower neutralization capacity against mutant viruses. After virus challenge, vaccinated females had lower pulmonary virus titers and reduced morbidity than males for the 1M and 2M viruses, but not the Sub virus. Females generated greater numbers of germinal center (GC) B cells containing superior somatic hypermutation (SHM) frequencies than vaccinated males. Deletion of activation-induced cytidine deaminase ( Aicda ) eliminated female-biased immunity and protection against the 2M virus. Harnessing methods to improve GC B cell responses and frequencies of SHM, especially in males, should be considered in the development of universal influenza vaccines. IMPORTANCE Adult females develop greater antibody responses to influenza vaccines than males. We hypothesized that female-biased immunity and protection would be dependent on the extent of virus diversity as well as molecular mechanisms in B cells which constrain the breadth of epitope recognition. We developed a panel of mouse-adapted (ma) A/Cal/09 viruses that had mutations in the immunodominant hemagglutinin. Following vaccination against maA/Cal/09, females were better able to neutralize maA/Cal/09 than males, but neutralization of mutant maA/Cal/09 viruses was equally poor in both sexes, despite vaccinated females being better protected against these viruses. Vaccinated females benefited from the greater production of class-switched, somatically hypermutated antibodies generated in germinal center B cells, which increased recognition of more diverse maA/Cal/09 hemagglutinin antigen epitopes. Female-biased protection against influenza infection and disease after vaccination is driven by differential mechanisms in males versus females and should be considered in the design of novel vaccine platforms.

To determine if biological sex and age intersect to affect universal influenza vaccine-induced immunity, adult and aged male and female C57BL/6 mice were sequentially immunized with a chimeric-hemagglutinin (cHA) stalk-based H1 vaccine. Adult mice developed greater quantity and quality of H1-stalk antibodies, that were more cross-reactive with other group 1, but not group 2, influenza viruses, than aged mice. The vaccine did not induce neutralizing or hemagglutination inhibition antibodies, but rather antibody-dependent cellular cytotoxicity, which was greater in adult than aged mice. Vaccinated adult mice were better protected than aged mice after challenge with 2009 H1N1 virus, experiencing less morbidity and having lower pulmonary virus titers. The age-associated decline in immunity and protection was consistently greater among females than males, with the reduction in immunity and protection for aged as compared with adult females often being the sole comparison driving the overall age-associated significant differences. The age-associated reduction in stalk-based immunity in females was not, however, associated with changes in estradiol. To determine if the better antibodies in adults could be utilized to protect aged mice, serum was passively transferred from vaccinated adult mice into naïve sex-matched aged mice. Even with transferred serum from young adult mice, aged females still suffered greater morbidity than aged males. These data suggest there are sex-dependent effects of aging on cHA-based universal influenza virus vaccine-induced immunity that cannot be reversed through transfer of serum from young animals. The lack of consideration of sex-specific effects of aging on immunity could hinder efforts toward universal vaccines., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Mount Sinai has filed IP on universal influenza virus vaccine candidates and FK is listed as inventor.]., (Copyright © 2020. Published by Elsevier Ltd.)

Healthcare institutions with mandatory influenza vaccination policies have over 90% vaccination rates among healthcare workers (HCWs) resulting in a population that has received the influenza vaccine in many, consecutive years. This study explored the impact of sex and other host factors in pre- and post-vaccination neutralizing antibody (nAb) titers and seroconversion against the H1N1 and H3N2 influenza A viruses (IAVs) among HCWs enrolled into a cross-sectional serosurvey during the annual Johns Hopkins Hospital employee vaccination campaign in the 2017-18 and 2018-19 seasons. The study enrolled 111 participants (male = 38, female = 73) in 2017-18 and 163 (male = 44, female = 119) in 2018-19. Serum samples were collected immediately prior to vaccination and approximately 28 days later and nAb titers to vaccine strains determined. An intersectional approach was used to disaggregate the combined effects of sex with age and body mass index (BMI) in the nAb response. Differences between the pre- or post-vaccination geometric mean nAb titers between male and female HCWs were not observed. Male HCWs were 2.86 times more likely to seroconvert compared to female HCWs in 2017-2018, but the same trend was not observed in the following year. When data were disaggregated by age and sex, older female HCWs had higher H1N1 pre- and post-vaccination nAb titers compared to male HCWs in the same age group for both vaccination campaign seasons. In both years, the decline in H3N2 pre-vaccination titers with increasing BMI was greater in female than male HCW. The sex-specific effects of age and BMI on nAb responses to seasonal influenza vaccines require greater consideration., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Swine influenza is a highly contagious respiratory disease of pigs caused by influenza A viruses (IAV-S). IAV-S causes significant economic losses to the swine industry and poses challenges to public health given its zoonotic potential. Thus effective IAV-S vaccines are needed and highly desirable and would benefit both animal and human health. Here, we developed two recombinant orf viruses, expressing the hemagglutinin (HA) gene (OV-HA) or the HA and the nucleoprotein (NP) genes of IAV-S (OV-HA-NP). The immunogenicity and protective efficacy of these two recombinant viruses were evaluated in pigs. Both OV-HA and OV-HA-NP recombinants elicited robust virus neutralizing antibody response in pigs, with higher levels of neutralizing antibodies (NA) being detected in OV-HA-NP-immunized animals pre-challenge infection. Although both recombinant viruses elicited IAV-S-specific T-cell responses, the frequency of IAV-S-specific proliferating CD8+ T cells upon re-stimulation was higher in OV-HA-NP-immunized animals than in the OV-HA group. Importantly, IgG1/IgG2 isotype ELISAs revealed that immunization with OV-HA induced Th2-biased immune responses, whereas immunization with OV-HA-NP virus resulted in a Th1-biased immune response. While pigs immunized with either OV-HA or OV-HA-NP were protected when compared to non-immunized controls, immunization with OV-HA-NP resulted in incremental protection against challenge infection as evidenced by a reduced secondary antibody response (NA and HI antibodies) following IAV-S challenge and reduced virus shedding in nasal secretions (lower viral RNA loads and frequency of animals shedding viral RNA and infectious virus), when compared to animals in the OV-HA group. Interestingly, broader cross neutralization activity was also observed in serum of OV-HA-NP-immunized animals against a panel of contemporary IAV-S isolates representing the major genetic clades circulating in swine. This study demonstrates the potential of ORFV-based vector for control of swine influenza virus in swine., Competing Interests: DD has patent on orf virus vector (patent no. 11013798). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Joshi, Knudsen, Piñeyro, Dhakal, Renukaradhya and Diel.)

Current influenza vaccines, live attenuated or inactivated, do not protect against antigenically novel influenza A viruses (IAVs) of pandemic potential, which has driven interest in the development of universal influenza vaccines. Universal influenza vaccine candidates targeting highly conserved antigens of IAV nucleoprotein (NP) are promising as vaccines that induce T cell immunity, but concerns have been raised about the safety of inducing robust CD8 T cell responses in the lungs. Using a mouse model, we systematically evaluated effects of recombinant adenovirus vectors (rAd) expressing IAV NP (A/NP-rAd) or influenza B virus (IBV) NP (B/NP-rAd) on pulmonary inflammation and function after vaccination and following live IAV challenge. After A/NP-rAd or B/NP-rAd vaccination, female mice exhibited robust systemic and pulmonary vaccine-specific B cell and T cell responses and experienced no morbidity (e.g., body mass loss). Both in vivo pulmonary function testing and lung histopathology scoring revealed minimal adverse effects of intranasal rAd vaccination compared with unvaccinated mice. After IAV challenge, A/NP-rAd-vaccinated mice experienced significantly less morbidity, had lower pulmonary virus titers, and developed less pulmonary inflammation than unvaccinated or B/NP-rAd-vaccinated mice. Based on analysis of pulmonary physiology using detailed testing not previously applied to the question of T cell damage, mice protected by vaccination also had better lung function than controls. Results provide evidence that, in this model, adenoviral universal influenza vaccine does not damage pulmonary tissue. In addition, adaptive immunity, in particular, T cell immunity in the lungs, does not cause damage when restimulated but instead mitigates pulmonary damage following IAV infection. IMPORTANCE Respiratory viruses can emerge and spread rapidly before vaccines are available. It would be a tremendous advance to use vaccines that protect against whole categories of viruses, such as universal influenza vaccines, without the need to predict which virus will emerge. The nucleoprotein (NP) of influenza virus provides a target conserved among strains and is a dominant T cell target. In animals, vaccination to NP generates powerful T cell immunity and long-lasting protection against diverse influenza strains. Concerns have been raised, but not evaluated experimentally, that potent local T cell responses might damage the lungs. We analyzed lung function in detail in the setting of such a vaccination. Despite CD8 T cell responses in the lungs, lungs were not damaged and functioned normally after vaccination alone and were protected upon subsequent infection. This precedent provides important support for vaccines based on T cell-mediated protection, currently being considered for both influenza and SARS-CoV-2 vaccines., (Copyright © 2021 American Society for Microbiology.)

Parenteral administration of killed/inactivated swine influenza A virus (SwIAV) vaccine in weaned piglets provides variable levels of immunity due to the presence of preexisting virus specific maternal derived antibodies (MDA). To overcome the effect of MDA on SwIAV vaccine in piglets, we developed an intranasal deliverable killed SwIAV antigen (KAg) encapsulated chitosan nanoparticles called chitosan-based NPs encapsulating KAg (CS NPs-KAg) vaccine. Further, to target the candidate vaccine to dendritic cells and macrophages which express mannose receptor, we conjugated mannose to chitosan (mCS) and formulated KAg encapsulated mCS nanoparticles called mannosylated chitosan-based NPs encapsulating KAg (mCS NPs-KAg) vaccine. In MDA-positive piglets, prime-boost intranasal inoculation of mCS NPs-KAg vaccine elicited enhanced homologous (H1N2-OH10), heterologous (H1N1-OH7), and heterosubtypic (H3N2-OH4) influenza virus-specific secretory IgA (sIgA) antibody response in nasal passage compared to CS NPs-KAg vaccinates. In vaccinated upon challenged with a heterologous SwIAV H1N1, both mCS NPs-KAg and CS NPs-KAg vaccinates augmented H1N2-OH10, H1N1-OH7, and H3N2-OH4 virus-specific sIgA antibody responses in nasal swab, lung lysate, and bronchoalveolar lavage (BAL) fluid; and IgG antibody levels in lung lysate and BAL fluid samples. Whereas, the multivalent commercial inactivated SwIAV vaccine delivered intramuscularly increased serum IgG antibody response. In mCS NPs-KAg and CS NPs-KAg vaccinates increased H1N2-OH10 but not H1N1-OH7 and H3N2-OH4-specific serum hemagglutination inhibition titers were observed. Additionally, mCS NPs-KAg vaccine increased specific recall lymphocyte proliferation and cytokines IL-4, IL-10, and IFNγ gene expression compared to CS NPs-KAg and commercial SwIAV vaccinates in tracheobronchial lymph nodes. Consistent with the immune response both mCS NPs-KAg and CS NPs-KAg vaccinates cleared the challenge H1N1-OH7 virus load in upper and lower respiratory tract more efficiently when compared to commercial vaccine. The virus clearance was associated with reduced gross lung lesions. Overall, mCS NP-KAg vaccine intranasal immunization in MDA-positive pigs induced a robust cross-reactive immunity and offered protection against influenza virus., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Renu, Feliciano-Ruiz, Patil, Schrock, Han, Ramesh, Dhakal, Hanson, Krakowka and Renukaradhya.)

We designed the killed swine influenza A virus (SwIAV) H1N2 antigen (KAg) with polyriboinosinic:polyribocytidylic acid [(Poly(I:C)] adsorbed corn-derived Nano-11 particle based nanovaccine called Nano-11-KAg+Poly(I:C), and evaluated its immune correlates in maternally derived antibody (MDA)-positive pigs against a heterologous H1N1 SwIAV infection. Immunologically, in tracheobronchial lymph nodes (TBLN) detected enhanced H1N2-specific cytotoxic T-lymphocytes (CTLs) in Nano-11-KAg+Poly(I:C) vaccinates, and in commercial vaccinates detected CTLs with mainly IL-17A + and early effector phenotypes specific to both H1N2 and H1N1 SwAIV. In commercial vaccinates, activated H1N2- and H1N1-specific IFNγ + &TNFα + , IL-17A + and central memory T-helper/Memory cells, and in Nano-11-KAg+Poly(I:C) vaccinates H1N2-specific central memory, IFNγ + and IFNγ + &TNFα + , and H1N1-specific IL-17A + T-helper/Memory cells were observed. Systemically, Nano-11-KAg+Poly(I:C) vaccine augmented H1N2-specific IFNγ + CTLs and H1N1-specific IFNγ + T-helper/Memory cells, and commercial vaccine boosted H1N2- specific early effector CTLs and H1N1-specific IFNγ + &TNFα + CTLs, as well as H1N2- and H1N1-specific T-helper/Memory cells with central memory, IFNγ + &TNFα + , and IL-17A + phenotypes. Remarkably, commercial vaccine induced an increase in H1N1-specific T-helper cells in TBLN and naive T-helper cells in both TBLN and peripheral blood mononuclear cells (PBMCs), while H1N1- and H1N2-specific only T-helper cells were augmented in Nano-11-KAg+Poly(I:C) vaccinates in both TBLN and PBMCs. Furthermore, the Nano-11-KAg+Poly(I:C) vaccine stimulated robust cross-reactive IgG and secretory IgA (SIgA) responses in lungs, while the commercial vaccine elicited high levels of serum and lung IgG and serum hemagglutination inhibition (HI) titers. In conclusion, despite vast genetic difference (77% in HA gene identity) between the vaccine H1N2 and H1N1 challenge viruses in Nano-11-KAg+Poly(I:C) vaccinates, compared to over 95% identity between H1N1 of commercial vaccine and challenge viruses, the virus load and macroscopic lesions in the lungs of both types of vaccinates were comparable, but the Nano-11-KAg+Poly(I:C) vaccine cleared the virus from the nasal passage better. These data suggested the important role played by Nano-11 and Poly(I:C) in the induction of polyfunctional, cross-protective cell-mediated immunity against SwIAV in MDA-positive pigs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Patil, Renu, Feliciano-Ruiz, Han, Ramesh, Schrock, Dhakal, HogenEsch and Renukaradhya.)

To improve the innate and adaptive immune responses elicited by a killed/inactivated swine influenza virus antigen (KAg)-loaded chitosan nanoparticles (CS NPs-KAg), we used the adjuvant, poly(I:C). The formulated CS NPs-KAg and CS NPs-poly(I:C) had a net surface charge of +30.7 mV and +25.1 mV, respectively. The CS NPs-KAg was coadministered with CS NPs-poly(I:C) (chitosan nanovaccine) as intranasal mist. Vaccinations enhanced homologous (H1N2-OH10) and heterologous (H1N1-OH7) hemagglutination inhibition (HI) titers in both vaccinated and virus-challenged animals compared to the control soluble poly(I:C) vaccinated pigs. In addition, the chitosan nanovaccine induced the proliferation of antigen-specific IFNγ secreting T-helper/memory and γδ T cells compared to control poly(I:C) group; and an increased Th1 (IFNγ, IL-6 and IL-2) and Th2 (IL-10 and IL-13) cytokines mRNA expression in the tracheobronchial lymph nodes compared to lymphoid tissues obtained from pigs given commercial influenza vaccine. The virus load in nasal passages and microscopic lung lesions were partially reduced by both chitosan nanovaccine and commercial vaccine. The HA gene homology between the vaccine and challenge viruses indicated that the chitosan nanovaccine induced a cross-protective immune response. In conclusion, coadministration of CS NPs-poly(I:C) with CS NPs-KAg augmented the cross-reactive specific HI titers and the cell-mediated immune responses in pigs., Competing Interests: Declaration of Competing Interest The authors have no financial conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Influenza is a global public health problem. Current seasonal influenza vaccines have highly variable efficacy, and thus attempts to develop broadly protective universal influenza vaccines with durable protection are under way. While much attention is given to the virus-related factors contributing to inconsistent vaccine responses, host-associated factors are often neglected. Growing evidences suggest that host factors including age, biological sex, pregnancy, and immune history play important roles as modifiers of influenza virus vaccine efficacy. We hypothesize that host genetics, the hormonal milieu, and gut microbiota contribute to host-related differences in influenza virus vaccine efficacy. This review highlights the current insights and future perspectives into host-specific factors that impact influenza vaccine-induced immunity and protection. Consideration of the host factors that affect influenza vaccine-induced immunity might improve influenza vaccines by providing empirical evidence for optimizing or even personalizing vaccine type, dose, and use of adjuvants for current seasonal and future universal influenza vaccines., (Copyright © 2019 American Society for Microbiology.)

Development of a broadly reactive influenza vaccine that can provide protection against emerging type A influenza viruses is a big challenge. We previously demonstrated that a vaccine displaying the extracellular domain of the matrix protein 2 (M2e) on the surface loops of norovirus P-particle (M2eP) can partially protect chickens against several subtypes of avian influenza viruses. In the current study, a chimeric vaccine containing a conserved peptide from the subunit 2 of hemagglutinin (HA) glycoprotein (HA2) and Arabidopsis thaliana cyanase protein (AtCYN) (HA2-AtCYN vaccine) was evaluated in 2-weeks-old chickens. Depending on the route of administration, the HA2-AtCYN vaccine was shown to induce various levels of HA2-specific IgA in tears as well as serum IgG, which were associated with partial protection of chickens against tracheal shedding of a low pathogenicity H5N2 challenge virus. Furthermore, intranasal administration with a combination of HA2-AtCYN and M2eP vaccines resulted in enhanced protection compared to each vaccine alone. Simultaneous intranasal administration of the vaccines did not interfere with secretory IgA induction by each vaccine. Additionally, significantly higher M2eP-specific proliferative responses were observed in peripheral blood mononuclear cells of all M2eP-vaccinated groups when compared with the mock-vaccinated group. Although tripling the number of M2e copies did not enhance the protective efficacy of the chimeric vaccine, it significantly reduced immunodominance of P-particle epitopes without affecting the robustness of M2e-specific immune responses. Taken together, our data suggests that mucosal immunization of chickens with combinations of mechanistically different cross-subtype-conserved vaccines has the potential to enhance the protective efficacy against influenza virus challenge., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Influenza (flu) is a constant threat to humans and animals, and vaccination is one of the most effective ways to mitigate the disease. Due to incomplete protection induced by current flu vaccines, development of novel flu vaccine candidates is warranted to achieve greater efficacy against constantly evolving flu viruses., Methods: In the present study, we used liposome nanoparticle (<200 nm diameter)-based subunit flu vaccine containing ten encapsulated highly conserved B and T cell epitope peptides to induce protective immune response against a zoonotic swine influenza A virus (SwIAV) H1N1 challenge infection in a pig model. Furthermore, we used monosodium urate (MSU) crystals as an adjuvant and co-administered the vaccine formulation as an intranasal mist to flu-free nursery pigs, twice at 3-week intervals., Results: Liposome peptides flu vaccine delivered with MSU adjuvant improved the hemagglutination inhibition antibody titer and mucosal IgA response against the SwIAV challenge and also against two other highly genetically variant IAVs. Liposomal vaccines also enhanced the frequency of peptides and virus-specific T-helper/memory cells and IFN-γ response. The improved specific cellular and mucosal humoral immune responses in adjuvanted liposomal peptides flu vaccine partially protected pigs from flu-induced fever and pneumonic lesions, and reduced the nasal virus shedding and viral load in the lungs., Conclusion: Overall, our study shows great promise for using liposome and MSU adjuvant- based subunit flu vaccine through the intranasal route, and provides scope for future, pre-clinical investigations in a pig model for developing potent human intranasal subunit flu vaccines., Competing Interests: Disclosure The authors report a pending patent for a liposomal subunit flu vaccine formulation: US patent application no 15/723,793, 2017. The authors report no other conflicts of interest in this work.

Intranasal vaccination of pigs with poly lactic-co-glycolic acid and polyanhydride nanoparticles delivered inactivated influenza virus provides cross-reactive T-cell response, but not antibody response, resulting in incomplete protection and no reduction in nasal virus shedding. Expression of BAFF and Th2 transcription factor GATA-3 were downregulated in lungs of pigs vaccinated with influenza nanovaccine, but in mice it upregulated the expression of BAFF and cytokine TGFβ in cervical lymph nodes. However, the intranasal iNKT cell adjuvant, α-Galctosylceramide upregulates the expression of BAFF in pig lungs. In conclusion, expression of BAFF is differentially regulated by intranasal nanovaccine and α-Galctosylceramide in pig respiratory tract., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Annually, swine influenza A virus (SwIAV) causes severe economic loss to swine industry. Currently used inactivated SwIAV vaccines administered by intramuscular injection provide homologous protection, but limited heterologous protection against constantly evolving field viruses, attributable to the induction of inadequate levels of mucosal IgA and cellular immune responses in the respiratory tract. A novel vaccine delivery platform using mucoadhesive chitosan nanoparticles (CNPs) administered through intranasal (IN) route has the potential to elicit strong mucosal and systemic immune responses in pigs. In this study, we evaluated the immune responses and cross-protective efficacy of IN chitosan encapsulated inactivated SwIAV vaccine in pigs. Killed SwIAV H1N2 (δ-lineage) antigens (KAg) were encapsulated in chitosan polymer-based nanoparticles (CNPs-KAg). The candidate vaccine was administered twice IN as mist to nursery pigs. Vaccinates and controls were then challenged with a zoonotic and virulent heterologous SwIAV H1N1 (γ-lineage). Pigs vaccinated with CNPs-KAg exhibited an enhanced IgG serum antibody and mucosal secretory IgA antibody responses in nasal swabs, bronchoalveolar lavage (BAL) fluids, and lung lysates that were reactive against homologous (H1N2), heterologous (H1N1), and heterosubtypic (H3N2) influenza A virus strains. Prior to challenge, an increased frequency of cytotoxic T lymphocytes, antigen-specific lymphocyte proliferation, and recall IFN-γ secretion by restimulated peripheral blood mononuclear cells in CNPs-KAg compared to control KAg vaccinates were observed. In CNPs-KAg vaccinated pigs challenged with heterologous virus reduced severity of macroscopic and microscopic influenza-associated pulmonary lesions were observed. Importantly, the infectious SwIAV titers in nasal swabs [days post-challenge (DPC) 4] and BAL fluid (DPC 6) were significantly ( p  < 0.05) reduced in CNPs-KAg vaccinates but not in KAg vaccinates when compared to the unvaccinated challenge controls. As well, an increased frequency of T helper memory cells and increased levels of recall IFNγ secretion by tracheobronchial lymph nodes cells were observed. In summary, chitosan SwIAV nanovaccine delivered by IN route elicited strong cross-reactive mucosal IgA and cellular immune responses in the respiratory tract that resulted in a reduced nasal viral shedding and lung virus titers in pigs. Thus, chitosan-based influenza nanovaccine may be an ideal candidate vaccine for use in pigs, and pig is a useful animal model for preclinical testing of particulate IN human influenza vaccines.

We have recently demonstrated the effectiveness of an influenza A virus (IAV) subunit vaccine based on biodegradable polyanhydride nanoparticles delivery in mice. In the present study, we evaluated the efficacy of ∼200nm polyanhydride nanoparticles encapsulating inactivated swine influenza A virus (SwIAV) as a vaccine to induce protective immunity against a heterologous IAV challenge in pigs. Nursery pigs were vaccinated intranasally twice with inactivated SwIAV H1N2 (KAg) or polyanhydride nanoparticle-encapsulated KAg (KAg nanovaccine), and efficacy was evaluated against a heterologous zoonotic virulent SwIAV H1N1 challenge. Pigs were monitored for fever daily. Local and systemic antibody responses, antigen-specific proliferation of peripheral blood mononuclear cells, gross and microscopic lung lesions, and virus load in the respiratory tract were compared among the groups of animals. Our pre-challenge results indicated that KAg nanovaccine induced virus-specific lymphocyte proliferation and increased the frequency of CD4 + CD8αα + T helper and CD8 + cytotoxic T cells in peripheral blood mononuclear cells. KAg nanovaccine-immunized pigs were protected from fever following SwIAV challenge. In addition, pigs immunized with the KAg nanovaccine presented with lower viral antigens in lung sections and had 6 to 8-fold reduction in nasal shedding of SwIAV four days post-challenge compared to control animals. Immunologically, increased IFN-γ secreting T lymphocyte populations against both the vaccine and challenge viruses were detected in KAg nanovaccine-immunized pigs compared to the animals immunized with KAg alone. However, in the KAg nanovaccine-immunized pigs, hemagglutination inhibition, IgG and IgA antibody responses, and virus neutralization titers were comparable to that in the animals immunized with KAg alone. Overall, our data indicated that intranasal delivery of polyanhydride-based SwIAV nanovaccine augmented antigen-specific cellular immune response in pigs, with promise to induce cross-protective immunity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Swine influenza virus (SwIV) is one of the important zoonotic pathogens. Current flu vaccines have failed to provide cross-protection against evolving viruses in the field. Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable FDA approved polymer and widely used in drug and vaccine delivery. In this study, inactivated SwIV H1N2 antigens (KAg) encapsulated in PLGA nanoparticles (PLGA-KAg) were prepared, which were spherical in shape with 200 to 300nm diameter, and induced maturation of antigen presenting cells in vitro. Pigs vaccinated twice with PLGA-KAg via intranasal route showed increased antigen specific lymphocyte proliferation and enhanced the frequency of T-helper/memory and cytotoxic T cells (CTLs) in peripheral blood mononuclear cells (PBMCs). In PLGA-KAg vaccinated and heterologous SwIV H1N1 challenged pigs, clinical flu symptoms were absent, while the control pigs had fever for four days. Grossly and microscopically, reduced lung pathology and viral antigenic mass in the lung sections with clearance of infectious challenge virus in most of the PLGA-KAg vaccinated pig lung airways were observed. Immunologically, PLGA-KAg vaccine irrespective of not significantly boosting the mucosal antibody response, it augmented the frequency of IFN-γ secreting total T cells, T-helper and CTLs against both H1N2 and H1N1 SwIV. In summary, inactivated influenza virus delivered through PLGA-NPs reduced the clinical disease and induced cross-protective cell-mediated immune response in a pig model. Our data confirmed the utility of a pig model for intranasal particulate flu vaccine delivery platform to control flu in humans., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The current inactivated influenza vaccines provide satisfactory protection against homologous viruses but limited cross-protection against antigenically divergent strains. Consequently, there is a need to develop more broadly protective vaccines. The highly conserved extracellular domain of the matrix protein 2 (M2e) has shown promising results as one of the components of a universal influenza vaccine in different animal models. As an approach to overcome the limited, strain specific, protective efficacy of inactivated influenza vaccine (IIV), a combination of recombinant M2e expressed on the surface of norovirus P particle (M2eP) and IIV was tested in chickens. Co-immunization of birds with both vaccines did not affect the production of M2e-specific IgG antibodies compared to the group vaccinated with M2eP alone. However, the co-immunized birds developed significantly higher pre-challenge hemagglutination inhibition antibody titers against the homologous IIV antigen and heterologous challenge virus. These combined vaccine groups also had cross reactive antibody responses against different viruses (H5, H6, and H7 subtypes) compared to the IIV alone vaccinated group. Upon intranasal challenge with homologous and heterologous viruses, the combined vaccine groups showed greater reduction in viral shedding in tracheal swabs compared to those groups receiving IIV alone. Moreover, M2eP antisera from vaccinated birds were able to bind to the native M2 expressed on the surface of whole virus particles and infected cells, and inhibit virus replication in vitro. Our results support the potential benefit of supplementing IIV with M2eP, to expand the vaccine cross protective efficacy., Competing Interests: The authors have declared that no competing interests exist.

Pigs are believed to be one of the important sources of emerging human and swine influenza viruses (SwIV). Influenza virus conserved peptides have the potential to elicit cross-protective immune response, but without the help of potent adjuvant and delivery system they are poorly immunogenic. Biodegradable polylactic-co-glycolic acid (PLGA) nanoparticle (PLGA-NP) based vaccine delivery system enhances cross-presentation of antigens by the professional antigen presenting cells. In this study, Norovirus P particle containing SwIV M2e (extracellular domain of the matrix protein 2) chimera and highly conserved two each of H1N1 peptides of pandemic 2009 and classical human influenza viruses were entrapped in PLGA-NPs. Influenza antibody-free pigs were vaccinated with PLGA-NPs peptides cocktail vaccine twice with or without an adjuvant, Mycobacterium vaccae whole cell lysate, intranasally as mist. Vaccinated pigs were challenged with a virulent heterologous zoonotic SwIV H1N1, and one week later euthanized and the lung samples were analyzed for the specific immune response and viral load. Clinically, pigs vaccinated with PLGA-NP peptides vaccine had no fever and flu symptoms, and the replicating challenged SwIV was undetectable in the bronchoalveolar lavage fluid. Immunologically, PLGA-NP peptides vaccination (without adjuvant) significantly increased the frequency of antigen-specific IFNγ secreting CD4 and CD8 T cells response in the lung lymphocytes, despite not boosting the antibody response both at pre- and post-challenge. In summary, our data indicated that nanoparticle-mediated delivery of conserved H1N1 influenza peptides induced the virus specific T cell response in the lungs and reduced the challenged heterologous virus load in the airways of pigs.

Pigs are considered as the source of some of the emerging human flu viruses. Inactivated swine influenza virus (SwIV) vaccine has been in use in the US swine herds, but it failed to control the flu outbreaks. The main reason has been attributed to lack of induction of strong local mucosal immunity in the respiratory tract. Invariant natural killer T (iNKT) cell is a unique T cell subset, and activation of iNKT cell using its ligand α-Galactosylceramide (α-GalCer) has been shown to potentiate the cross-protective immunity to inactivated influenza virus vaccine candidates in mice. Recently, we discovered iNKT cell in pig and demonstrated its activation using α-GalCer. In this study, we evaluated the efficacy of an inactivated H1N1 SwIV coadministered with α-GalCer intranasally against a homologous viral challenge. Our results demonstrated the potent adjuvant effects of α-GalCer in potentiating both innate and adaptive immune responses to SwIV Ags in the lungs of pigs, which resulted in reduction in the lung viral load by 3 logs compared to without adjuvant. Immunologically, in the lungs of pigs vaccinated with α-GalCer an increased virus specific IgA response, IFN-α secretion and NK cell-cytotoxicity was observed. In addition, iNKT cell-stimulation enhanced the secretion of Th1 cytokines (IFN-γ and IL-12) and reduced the production of immunosuppressive cytokines (IL-10 and TGF-β) in the lungs of pigs⋅ In conclusion, we demonstrated for the first time iNKT cell adjuvant effects in pigs to SwIV Ags through augmenting the innate and adaptive immune responses in the respiratory tract., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Intranasal vaccination elicits secretory IgA (SIgA) antibodies in the airways, which is required for cross-protection against influenza. To enhance the breadth of immunity induced by a killed swine influenza virus antigen (KAg) or conserved T cell and B cell peptides, we adsorbed the antigens together with the TLR3 agonist poly(I:C) electrostatically onto cationic alpha-D-glucan nanoparticles (Nano-11) resulting in Nano-11-KAg-poly(I:C) and Nano-11-peptides-poly(I:C) vaccines. In vitro, increased TNF-α and IL-1ß cytokine mRNA expression was observed in Nano-11-KAg-poly(I:C)-treated porcine monocyte-derived dendritic cells. Nano-11-KAg-poly(I:C), but not Nano-11-peptides-poly(I:C), delivered intranasally in pigs induced high levels of cross-reactive virus-specific SIgA antibodies secretion in the nasal passage and lungs compared to a multivalent commercial influenza virus vaccine administered intramuscularly. The commercial and Nano-11-KAg-poly(I:C) vaccinations increased the frequency of IFNγ secreting T cells. The poly(I:C) adjuvanted Nano-11-based vaccines increased various cytokine mRNA expressions in lymph nodes compared to the commercial vaccine. In addition, Nano-11-KAg-poly(I:C) vaccine elicited high levels of virus neutralizing antibodies in bronchoalveolar lavage fluid. Microscopic lung lesions and challenge virus load were partially reduced in poly(I:C) adjuvanted Nano-11 and commercial influenza vaccinates. In conclusion, compared to our earlier study with Nano-11-KAg vaccine, addition of poly(I:C) to the formulation improved cross-protective antibody and cytokine response. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]