{'fraction_non_alphanumeric': 0.03464545699649501, 'fraction_numerical': 0.01712051765974656, 'mean_word_length': 5.255480607082631, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 1, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Nasal Immunization with a Vaccinia Virus-based Pentavalent H5N1 Avian Influenza Vaccine Induces Cellular Immunity in the Respiratory Tract of Mice

A previous study showed that vaccinia virus-vectored pentavalent H5N1 influenza vaccine adjuvanted with IL-15 injected via the parenteral route induced broadly effective protective immunity. However, influenza virus is known to infect the mucosal surfaces of the respiratory tract, and nasal immunization induces effective protective immunity against respiratory tract infection. Therefore, we explored the vaccine potential of nasal immunization with the vaccinia virus-vectored multivalent pandemic influenza vaccine candidate. BALB/c mice were nasally immunized with the Wyeth strain of vaccinia virusbased pentavalent H5N1 vaccine adjuvanted with IL-15 or Wyeth virus. Four weeks after the first immunization, the mice received a booster, and antigen(Ag)-specific antibody (Ab)and cellular immune responses induced in the vaccinated mice were analyzed 3 weeks later. Nasal immunization of the pentavalent Wyeth-based H5N1 vaccine induced broadly reactive Ag-specific systemic Ab responses compared with Wyeth-vaccinated mice. The secretory IgA Ab response was induced in the lower respiratory tract, although the degree of induction was marginal. The number of IFN-producing cells, and the frequencies of IFN+ CD4+, TNF+ CD4+ and TNF+ CD8+ T cells were significantly increased in the lungs of mice immunized with Wyeth-based H5N1 vaccine. The results of this study suggested that although the immunogenicity of Wyeth-based pentavalent H5N1 vaccine expressing IL-15 was insufficient as a mucosal vaccine, cellular immunity was preferentially induced in the respiratory mucosal compartment, which is the site of influenza virus infection, when the vaccine was delivered via the nasal mucosa.


Introduction
Influenza is a contagious acute respiratory disease caused by influenza virus infection. Influenza epidemics include seasonal influenza and pandemics (1). The appearance of novel and highly pathogenic influenza virus strains may lead to respiratory disorders with significant morbidity and high mortality, and may cause pandemics. H10N7. Although the pathogenicity of these subtypes is generally low, the H5N1 and H7N9 subtypes are highly pathogenic; the development of effective vaccines against such highly pathogenic influenza strains is urgently required in preparation for possible future outbreaks (3,4).
Correspondence to : Tomomi Hashizume-Takizawa E-mail : hashizume.tomomi@nihon-u.ac.jp An inactivated, detergent-extracted H5N1 vaccine demonstrated potency, and has been accepted as a pandemic vaccine (5). However, the immunogenicity of this vaccine may be poor and high levels of antigens(Ags)and adjuvant are required for vaccination. Therefore, it is necessary to develop a vaccine with a more efficient delivery system, and a proper adjuvant and delivery route. The efficacy of various H5N1 vaccines has been studied, and live attenuated H5N1 vaccine and live vector-delivered H5N1 vaccines were shown to be effective compared with inactivated subunit or split vaccines (6,7 (8). This pandemic influenza vaccine candidate was shown to induce effective Ag-specific antibody (Ab) responses in serum against diverse subtypes of influenza viruses, and also produced cellular immune responses and protected against H5N1 infection when injected subcutaneously (8).
Vaccination via the mucosal route induces both systemic and local mucosal immune responses, resulting in two layers of protection against infectious diseases (9). As influenza is a respiratory disease and influenza virus infects humans primarily through the mucosal route (1), induction of influenza-specific immune responses in the bronchial mucosa is a logical means of preventing influenza virus infection. Nasal immunization is known to induce mucosal immunity in the bronchial compartment (10,11).
The present study was performed to assess the Agspecific immune responses induced by nasal immunization with Wyeth, expressing five H5N1-derived Ags along with IL-15 (Wyeth/IL-15/5Flu), and to evaluate the vaccine potential of this influenza vaccine candidate.

Mice
Female BALB/c mice were obtained from Japan SLC, Inc. Lakes, NJ, USA)and the lungs were washed with 0.6 mL of PBS to obtain bronchoalveolar lavage samples, as described elsewhere (12).
Further, Wyeth/IL-15/5Flu-immunized mice showed elevated anti-H5 IgG Ab responses (Fig. 1B). In contrast, H5specific secretory (S) IgA Abs were produced at low but detectable levels in the lung (Fig. 1C). However, SIgA anti- Splenic and lung MNCs were isolated from vaccinated mice, and restimulated with H1N1 virus in vitro followed by FACS analysis to determine intracellular cytokine production. Our results showed that the frequency of TNF-+ CD4 + cells was significantly increased in the spleen of Wyeth/IL-15/5Flu vaccinated mice (Fig. 1A). Furthermore, the frequencies of TNF-+ CD4 + , IFN-+ CD4 + , and IFN-+ CD8 + T cells were significantly increased in the lungs of vaccinated mice (Fig. 1B). These results indicated that the levels of cellular immunity-related cytokines were increased in the lungs compared with the spleen of mice vaccinated with Wyeth/Il-15/5Flu via the nasal mucosal route.

Discussion
In this study, we evaluated the efficacy of vaccinia virus- Vaccination via the parenteral route induced protective immunity only in the systemic compartment (14). However, influenza virus infection occurs mainly via the mucosal surface of the respiratory tract(1)and nasal delivery of Ags is the best way to induce protective immunity in the respiratory tract (10,11). Therefore, we explored the potential of this Wyeth-based vaccine as a mucosal vaccine.
The results of the present study indicated that intranasal delivery of the Wyeth-based H5N1 vaccine candidate induced broadly reactive humoral systemic IgG Ab re-sponses( Fig. 1A and B). However, the induction levels of these IgG Ab responses were lower than those in mice immunogenicity (11). Therefore, we employed the cytokine IL-15, which contributes to enhancement of CD8 + T cellmediated cellular immunity (13), as an immune stimulator.
Furthermore, the introduction of IL-15 genes into the vector virus enhanced Ag-specific Ab production against coexpressed vaccine Ags (8,19). A previous study showed that IL-15 plays an important role in intestinal IgA induction by B1 cells (20). However, the present study indicated that IL-15 did not enhance mucosal IgA responses following nasal immunization with IL-15-expressing Wyeth-based influenza vaccine (Fig. 1C). Our results showed that nasal Wyeth-vectored pentavalent influenza vaccine induced cellular immunity, including IFN-production, via both CD4 + and CD8 + T cells, and TNF-production by CD4 + T cells in the lung. In contrast, cellular immunity-related cytokine production in the spleen was not enhanced by nasal immunization, with the exception of TNF-production by CD4 + T cells. (Figs. 2 and 3). These results indicated that cellular immunity was preferentially induced in the respiratory tract when vaccine was delivered via the intranasal route. In this regard, the main sites where protective immunity was induced differed depending on the route of Ag delivery, and intranasal immunization induced superior immune responses in the respiratory tract compared with oral immunization (10,23