Further Search for Small Molecular Inactivants Capable of Eliciting Respiratory Mucosal Immunogenicity by Modifying Sendai Virus Core RNA

Five groups of 32 chemicals were examined regarding their immunological functions as modifier inactivants to make inactivated Sendai nasal vaccine using a contact exposure experiment, direct immunofluorescent method, and serum HI titer. (1) Five of the nine reactive groups of reactive dyes (2-chloropyridine, 2, 4, 6-trichloropyrimidine, vinylsulfonic acid, epichlorohydrin and beta-chloroethylamine) induced complete or almost complete defense in the entire respiratory tract, and the four other vaccines brought about slight infection in the respiratory tracts. There was no marked rise in serum HI titers post-exposure, despite uneven development. (2) Of the four sizable substituted AS naphthol vaccines, naphthol AS-IRG and AS-G vaccines elicited nearly complete defense, but the two other vaccines, inactivated with more elongated molecules, invited rare and successive infections. The three immune groups produced invariably high serum HI titers. (3) Of the six naphthalene derivative vaccines, two (3-hydroxy-2-naphthoic acid methylester and 2-naphthol-6-sulfonic acid) induced complete or almost complete protection. But two vaccines brought about less protection, and the remaining two vaccines caused heavy infections. (4) Of the six benzene derivative vaccines, both m-nitrobenzenesulfonic acid and isatoic anhydride induced complete protection. Three vaccines permitted slight infections but 2, 4, 6-trinitrobenzenesulfonic acid vaccine caused severe infection. (5) Of the seven food dye vaccines, only orange I induced complete or nearly complete defense, while the other dye vaccines were inferior. In effect, twelve inactivated Sendai nasal vaccines modified the ribose and/or phosphate groups of the virus core RNA through five groups of small-sized molecules with specially fixed side chains, and elicited complete or almost complete respiratory mucosal defense. The viral stabilization requiring the least alteration of the configuration will be involved in the chemical modification.     

Inducement of Respiratory Mucosal Immunogenicity for Nasal Vaccine Due to Amine- and Amide-Inactivation of Parainfluenza Virus Type 1, Sendai

The protective effects in mice by nasal vaccination of amine- and amide-inactivated Sendai viruses were investigated by a contact exposure experiment, immunofluorescent examination of the entire respiratory tract, and checking the serum HI antibody development. Of 10 monoamines, ethanolamine and 2-methoxyethylamine vaccines induced complete protection, and methylamine, ethylamine, n-propylamine, n-butylamine, 2-ethoxyethylamine, diethylamine and triethylamine vaccines brought about almost complete protection or lower respiratory infection. The methoxyamine-treated mouse conferred the least protection. Of 5 diamines, 1, 3-diaminopropane vaccine inhibited completely the infection, but hydrazine, ethylenediamine, putrescine, and cadaverine vaccines produced regional infection. Two polyamines, spermine and spermidine, did not inactivate the virus. Of 4 amides, only semicarbazide vaccine conferred complete mucosal defense, while acetamide, propionamide, and isonicotinic acid hydrazide vaccines lead to regional infection. Serum HI titers developed by vaccination were low on the whole, following their slight rise, fall or maintenance postexposure. In effect, the 4 vaccines inactivated by a best-suited interstrand crosslink between phosphate groups in helix of viral RNA brought about the strongest protection, and showed the necessity of a definite length of molecules for inactivants.     

Comparative evaluation on mouse nasal immunogenicity of arylmethane-, xanthene-, quinone-imine-, and acridine-dye-inactivated Sendai virus vaccines

Twenty-seven kinds of organic dye-inactivated Sendai virus vaccines were prepared by treatment in dark at 23 C for 2 months or more, and selected with the high HA titers as a guide. Their nasal immunogenicities were examined in mice by contact infection and immunofluorescent method, and the relative merits of the dye-inactivants were determined. The strongest protection was elicited with acriflavine-, auramine O-, eosin Y-, neutral red-, night blue-, patent blue V-, thymol blue-, uranin-, and xylene cyanol FF-treated vaccines. Middling protective efficacy was induced by use of erio green B-, malachite green-, methyl green-, proflavine-, pyronin B-, and thionin-inactivated vaccines. Dye-inactivated vaccines that resulted in the weakest protection were Bindschedler's green-, bromothymol blue-, erythrosin B-, ethyl violet-, gallein-, light green SF yellowish-, methyl violet-, new methylene blue N-, phenol red-, rhodamine 6G-, spirit blue- and victoria blue B-treated ones. Serum HI titers developed by nasal vaccination were variable, and rose still more in most vaccinated groups postexposure. Elicitation of the most effective nasal immunogenicity in dye-inactivated vaccines appeared to depend on selective modification of capsid protein or ribose in viral core with dyes possessing definite functions, despite the different molecular structures.     

Protective effects of nasal immunization in mice with various kinds of inactivated Sendai virus vaccines

The immunoprophylactic effects of nasal vaccination with 13 different kinds of inactivated Sendai virus vaccines were compared by contact exposure to infector mice. Efficacies of the vaccines were evaluated on the basis of the presence of virus-infected cells by immunofluorescent examination of the entire respiratory tract, including the nasal mucosa. A single or double inoculations of B-propiolactone (0.5%)-vaccine promoted the infection in the respiratory tract, particularly in the nasal mucosa, whereas three inoculations of B-propiolactone (0.2%)-vaccine provided considerable protection throughout the respiratory tract with only slight development of serum HI titer. Formalin (0.1%)-vaccine and UV irradiated-vaccine strongly protected the nasal mucosa from infection, but did not sufficiently safeguard the lower respiratory tract even with three vaccinations despite adequate development of serum antibody. Nearly complete protection of the entire respiratory tract was induced with six to eight inoculations of a vaccine treated excessively with both UV rays and 1% formalin, without significant development of serum antibody. Out of eight thermal vaccines, five (inactivated at 23 C, 30 C, 37 C and 7 C, and 30 C and 7 C) provided strong protection against infection when inoculated three times. The others inactivated at higher temperatures (37 C, 50 C, or 60 C) were not so protective. High serum HI titers developed, on the whole, with the drop in the temperature required for inactivating the virus. In eight immune mouse groups in which infection was strongly suppressed in the entire respiratory tract, most of the mice harbored less than 50 viral antigen-positive cells in their nasal mucosa in the postexposure period. The number of the cells was assumed to be a useful criterion for evaluation of vaccine efficacy.     

Efficacy of DNA and fowlpox virus priming/boosting vaccines for simian/human immunodeficiency virus

Further advances are required in understanding protection from AIDS by T-cell immunity. We analyzed a set of multigenic simian/human immunodeficiency virus (SHIV) DNA and fowlpox virus priming and boosting vaccines for immunogenicity and protective efficacy in outbred pigtail macaques. The number of vaccinations required, the effect of DNA vaccination alone, and the effect of cytokine (gamma interferon) coexpression by the fowlpox virus boost was also studied. A coordinated induction of high levels of broadly reactive CD4 and CD8 T-cell immune responses was induced by sequential DNA and fowlpox virus vaccination. The immunogenicity of regimens utilizing fowlpox virus coexpressing gamma interferon, a single DNA priming vaccination, or DNA vaccines alone was inferior. Significant control of a virulent SHIV challenge was observed despite a loss of SHIV-specific proliferating T cells. The outcome of challenge with virulent SHIV(mn229) correlated with vaccine immunogenicity except that DNA vaccination alone primed for protection almost as effectively as the DNA/fowlpox virus regimen despite negligible immunogenicity by standard assays. These studies suggest that priming of immunity with DNA and fowlpox virus vaccines could delay AIDS in humans.     

Selection of the most efficacious of twenty-two inactivated Sendai virus nasal vaccines by determination of the protection index in mice.

BACKGROUND AND PURPOSE: Sendai virus nasal vaccines inactivated with various chemicals induce complete protection against contact-challenge exposure with the Nagoya strain. The study reported here was to reevaluate the efficacy of the inactivants by determining the protective index (PI) in mice, using the more virulent MN strain. METHODS: Mice were given each of 22 inactivated vaccines intranasally three times. After challenge exposure with 10(-2) to 10(6) MID50 of virus, infection of cells of the respiratory tract was determined by immunofluorescence. RESULTS: Twelve vaccines induced PI > or = 2.0 in the nasal mucosa and were classified as group 1. The first half of the preceding vaccines that induced PI > or = 3.2 in the larynx were classified subgroup a, and the rest were classified subgroup b. Of the other 10 vaccines, 6 that induced PI < or = 2.0 in the larynx and 4 that induced intermediate PI in the nasal mucosa and larynx were ranked as groups 3 and 2, respectively; PI of the trachea decreased by numeric order of groups. Serum hemagglutination inhibition titer induced by intranasal vaccination was low in general. CONCLUSION: On the basis of PI values, 6 of the 22 nasal vaccines provided the strongest defense in the respiratory tract.     

Pathogenicity and vaccine efficacy of different clades of Asian H5N1 avian influenza A viruses in domestic ducks

Waterfowl represent the natural reservoir of all subtypes of influenza A viruses, including H5N1. Ducks are especially considered major contributors to the spread of H5N1 influenza A viruses because they exhibit diversity in morbidity and mortality. Therefore, as a preventive strategy against endemic as well as pandemic influenza, it is important to reduce the spread of H5N1 influenza A viruses in duck populations. Here, we describe the pathogenicity of dominant clades (clades 1 and 2) of H5N1 influenza A viruses circulating in birds in Asia. Four representatives of dominant clades of the viruses cause symptomatic infection but lead to different profiles of lethality in domestic ducks. We also demonstrate the efficacy, cross-protectiveness, and immunogenicity of three different inactivated oil emulsion whole-virus H5 influenza vaccines (derived by implementing reverse genetics) to the viruses in domestic ducks. A single dose of the vaccines containing 1 μg of hemagglutinin protein provides complete protection against a lethal A/Duck/Laos/25/06 (H5N1) influenza virus challenge, with no evidence of morbidity, mortality, or shedding of the challenge virus. Moreover, two of the three vaccines achieved complete cross-clade or cross-subclade protection against the heterologous avian influenza virus challenge. Interestingly, the vaccines induce low or undetectable titers of hemagglutination inhibition (HI), cross-HI, and/or virus neutralization antibodies. The mechanism of complete protection in the absence of detectable antibody responses remains an open question.     

Anthrax vaccine design: strategies to achieve comprehensive protection against spore,bacillus, and toxin

The successful use of Bacillus anthracis as a lethal biological weapon has prompted renewed research interest in the development of more effective vaccines against anthrax. The disease consists of three critical components: spore, bacillus, and toxin, elimination of any of which confers at least partial protection against anthrax. Current remedies rely on postexposure antibiotics to eliminate bacilli and pre- and postexposure vaccination to target primarily toxins. Vaccines effective against toxin have been licensed for human use, but need improvement. Vaccines against bacilli have recently been developed by us and others. Whether effective vaccines will be developed against spores is still an open question. An ideal vaccine would confer simultaneous protection against spores, bacilli, and toxins. One step towards this goal is our dually active vaccine, designed to destroy both bacilli and toxin. Existing and potential strategies towards potent and effective anthrax vaccines are discussed in this review.     

Complete Protection of Mice against Lethal Murine Cytomegalovirus Challenge by Immunization with DNA Vaccines Encoding Envelope Glycoprotein Complex III Antigens gH,gL and gO

Human cytomegalovirus infects the majority of humanity which may lead to severe morbidity and mortality in newborns and immunocompromised adults. Humoral and cellular immunity are critical for controlling CMV infection. HCMV envelope glycoprotein complexes (gC I, II, III) represent major antigenic targets of antiviral immune responses. The gCIII complex is comprised of three glycoproteins, gH, gL, and gO. In the present study, DNA vaccines expressing the murine cytomegalovirus homologs of the gH, gL, and gO proteins were evaluated for protection against lethal MCMV infection in the mouse model. The results demonstrated that gH, gL, or gO single gene immunization could not yet offer good protection, whereas co-vaccination strategy apparently showed effects superior to separate immunization. Twice immunization with gH/gL/gO pDNAs could provide mice complete protection against lethal salivary gland-derived MCMV (SG-MCMV) challenge, while thrice immunization with pgH/pgL, pgH/pgO or pgL/pgO could not provide full protection. Co-vaccination with gH, gL and gO pDNAs elicited robust neutralizing antibody and cellular immune responses. Moreover, full protection was also achieved by simply passive immunization with anti-gH/gL/gO sera. These data demonstrated that gCIII complex antigens had fine immunogenicity and might be a promising candidate for the development of HCMV vaccines.     

Induction of interferon in man by vaccines.

The purpose of this study was to extend the spectrum of vaccines with interferon-inducing potential in man. The vaccines selected for study were the commercially available attenuated poliomyelitis vaccine type 2 (Sabin strain) and the new live attenuated influenza A/England/42/72 (H3N2) vaccine ("Alice" strain). Five subjects, two of whom had low or undetectable polio type 2 neutralizing antibody levels were given the type 2 vaccine (10-4.7 TCID50) in the standard manner orally. Even though the two individuals with low titers experienced a fourfold or greater antibody rise and one of them shed the virus in his stool, neither they nor the remaining three volunteers developed detectable levels of interferon in their sera obtained at very closely spaced intervals from day 0 to day 25 following immunization. Fifteen subjects were given approximately 10-7.5 TCID50 of influenza A/England/42/72 (H3N2) by nasal drops. Specimens consisting of sera and nasal washings were obtained at closely timed intervals for 23 days, starting with day 3 following immunization. Interferon could be detected in three of nine (33.3%) subjects who had fourfold or greater HI antibody rises. No interferon was detected in nasal washings, however. It is concluded that poliomyelitis is not a good interferon inducers in man. Live attenuated influenza vaccine does induce an interferon response in subjects with low initial serum antibody titers. This response is at best modest. The latter finding also suggests that the attenuation of the Alice strain of influenza A vaccine is not dependent on its interferon inducing potential.     

Immunogenicity and Protective Efficacy in Mice of Influenza B Virus Vaccines Grown in Mammalian Cells or Embryonated Chicken Eggs

The immunogenicity and protective efficacy of formalin-inactivated influenza B/Memphis/1/93 virus vaccines propagated exclusively in Vero cells, MDCK cells, or embryonated chicken eggs (hereafter referred to as eggs) were investigated. Mammalian cell-grown viruses differ from the egg-grown variant at amino acid position 198 (Pro/Thr) in the hemagglutinin gene. The level of neuraminidase activity was highest in egg-grown virus, while MDCK and Vero cell-derived viruses possessed 70 and 90% less activity, respectively. After boosting, each of the vaccines induced high levels of hemagglutinin-inhibiting, neuraminidase-inhibiting, and neutralizing antibodies that provided complete protection from MDCK-grown virus challenge. Mammalian cell-derived virus vaccines induced serum antibodies that were more cross-reactive, while those induced by egg-grown virus vaccines were more specific to the homologous antigen. Enzyme-linked immunospot analysis indicated that cell-grown virus vaccines induced high frequencies of immunoglobulin G (IgG)-producing cells directed against both cell- and egg-grown virus antigens, whereas egg-grown virus vaccine induced higher frequencies of IgG- and IgM-producing cells reacting with homologous antigen and low levels of IgG-producing cells reactive with cell-grown viruses. These studies indicate that influenza B virus variants selected in different host systems can elicit different immune responses, but these alterations had no detectable influence on the protective efficacy of the vaccines with the immunization protocol used in this study.     

Evaluation of the cell culture based and the mouse brain derived inactivated vaccines against Crimean-Congo hemorrhagic fever virus in transiently immune-suppressed (IS) mouse model

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus in the Nairoviridae family within the Bunyavirales order of viruses. Crimean-Congo hemorrhagic fever (CCHF) is the most widespread among tick-borne human viral diseases. It is endemic in many areas of Africa, Asia, the Middle East, in the Balkans, Russia and countries of the former Soviet Union. The confirmed CCHF cases were seen in Spain in 2016 to signify expansion of the virus into new geographical areas. CCHFV causes a viral human disease characterized by sudden onset of fever, headache, abdominal pain, nausea, hypotension, hemorrhage, and hepatic dysfunction with fatality rates up to 30%. Currently, there are no spesific treatments or licensed vaccines available for CCHFV. The absence of a susceptible animal model for CCHFV infection was severely hindered work on the development of vaccines. However, several animal models of CCHFV infection have been recently developed and used to assess vaccine efficacy. In this study, we have used the transiently immune-suppressed (IS) mouse model that MAb-5A3 was used to block IFN-I signaling in immune intact, wild-type mice at the time of CCHFV infection to evaluate the immune response and efficacy of the cell culture based and the mouse brain derived inactivated vaccines against CCHFV. Both vaccine preparations have provided complete protection but the cell culture based vaccine more effectively induced to CCFHV spesific antibodies and T cell responses. This is the first comparison of the cell culture based and the mouse brain derived vaccines for assessing the protective efficacy and the immunogenicity in the IS mouse CCHFV model.     

Carbohydrate vaccines that induce antibodies against cancer. 2. Previous experience and future plans

In conclusion  The primary function of antibodies is the elimination of circulating viral or bacterial pathogens from the blood-stream, lymphatics and interstitial spaces, and so, once induced, antibodies should be ideally suited for eliminating tumor cells and micrometastases from these spaces as well. Natural or tumor-induced and vaccine-induced antibodies against human cancer-associated antigens have been correlated with an improved clinical outcome. In the mouse, passive administration of monoclonal antibodies against cell-surface antigens 1–4 days after tumor challenge, and active induction of antibodies with vaccines, has resulted in prolonged survival or complete protection from tumor growth. This is a setting similar to the adjuvant setting in humans. Carbohydrates are the most abundant antigens at the cell surface of cancer cells, where they play important roles in cell-cell interactions, proliferation and the metastatic process. They have been shown to be excellent targets for immune attack by antibodies against human cancers, especially in the adjuvant setting. Vaccines containing these carbohydrate antigens covalently attached to immunogenic carrier proteins, such as KLH, plus potent immunological adjuvants, such as QS-21, effectively induce antibodies against these antigens in patients, which can result in complement-mediated lysis of antigen-positive tumor cells. Phase III trials with KLH conjugate vaccines have been initiated in the adjuvant setting against two carbohydrate antigens, the ganglioside GM2 and the blood-group-related antigen sTn. As the immunogenicity of additional vaccines is confirmed in small pilot trials, trials with polyvalent vaccines against two to five different antigens tailored for particular cancer types are planned.     

Thermosensitive and mucoadhesive delivery systems of mucosal vaccines

Mucosal vaccination is emerging as a potential administration route for eliciting antigen-specific mucosal and systemic immunogenicity. Most mucosal vaccines have been administered in a phosphate-buffered saline vehicle that may limit the exposure of antigens to the mucosal surfaces and result in poor immunogenicity. To improve the potency of the mucosal vaccines, we have developed mucosal vaccine delivery systems that might prevent leakage and increase retention of vaccines on mucosal surfaces. Thermosensitive polymers have been used to reduce the leakage problems of nasal or vaginal vaccines, while mucoadhesive polymers have been employed to increase the mucosal contact of the vaccines. Here, we describe the formulation and delivery methods of mucosal vaccines using thermosensitive and mucoadhesive polymers.     

Immunogenic and protective properties of the first kazakhstan vaccine against pandemic influenza A (H1N1) pdm09 in Ferrets

This paper presents the results of a pre-clinical study of the immunogenicity and efficacy of an egg-derived, inactivated, whole-virion adjuvanted vaccine (Refluvac®) on ferret models. For this purpose, groups of eight ferrets (6 to 7 months old) were injected with 0.5 mL of vaccine specimens containing 3.75, 7.5 or 15.0 μg of virus hemagglutinin. Administration was intramuscular and given either as a single dose or as two doses 14 days apart. All vaccine specimens manifested immunogenicity in ferrets for single (HI titer, from 51 ± 7 to 160 ± 23) and double (HI titer, from 697 ± 120 to 829 ± 117) administrations. To assess the protective effects of the vaccine, ferrets from the vaccinated and control groups were infected intranasally with pandemic virus A/California/7/09 (H1N1) pdm09 at a dose of 10⁶ EID₅₀/0.5 mL. Fourteen days post-infection, the ferrets inoculated with single or double vaccines containing 3.75, 7.5 or 15.0 μg of hemagglutinin per dose showed no signs of influenza infection, weight loss, or body temperature rise, and no premature deaths occurred. The number of vaccinated ferrets shedding the virus via the upper airway, as well as the amount of virus shed after infection, was significantly reduced in comparison with animals from the control group. Based on our results, we suggest that a single vaccination at a dose of 3.75 or 7.5 μg hemagglutinin be used for Phase I clinical trials.     

Pre- and postexposure protection by passive immunoglobulin but no enhancement of infection with a flavivirus in a mouse model.

Antibody-dependent enhancement of flavivirus infection, which except for dengue virus is without clear proof in vivo, is still under debate. Recently, postexposure immunoglobulin prophylaxis against tick-borne encephalitis virus, a flavivirus, was claimed to possibly have worsened the outcome of infection due to antibody-dependent enhancement. In the present study, antibody-dependent enhancement and pre- or postexposure protection by passive administration of tick-borne encephalitis virus immunoglobulin were evaluated in a mouse model. Preexposure treatment with homologous murine or heterologous human immunoglobulin provided complete protection against lethal challenge with tick-borne encephalitis virus. For postexposure treatment with antibody, the degree of protection correlated with the amount of immunoglobulin administered and was inversely related to the time interval between infection and treatment. Indications of enhancement of infection would have been increased lethality or reduced mean survival time, but neither was observed under the conditions used in our experiments despite the broad range of immunoglobulin and virus challenge doses applied. In contrast to these in vivo results, antibody-dependent enhancement of tick-borne encephalitis virus infection of murine peritoneal macrophages was readily demonstrable in vitro. Thus, antibody-dependent enhancement of viral infection in vitro does not necessarily predict enhancement in vivo.     

国产腮腺炎和进口麻风腮疫苗免后腮腺炎HI抗体比较

报道了用国产流行腮腺炎减毒活疫苗和美国产麻－风－腮减毒活疫苗接种８～９岁小学生后,采血进行ＨＩ抗体测定比较,其抗体阳转率均在８０％左右,ＧＭＴ为７．３５～１０．０３,说明两种疫苗均有较好的免疫应答     

Comparison of hemagglutination inhibition and hemagglutinin pseudovirus neutralization titres in relation to protection against influenza in a mouse model

The hemagglutination inhibition (HI) test has long been used as a standard measure of antibody response for inactivated influenza vaccines. However, the HI test has limitations, such as insensitivity when using some H3N2 virus strains and failure to detect neutralizing antibodies that target regions distant from the receptor binding site. We therefore examined a hemagglutinin pseudovirus neutralization (PVN) test as a possible supplement or alternative to the HI test. We evaluated the association of HI or PVN titres with protection against influenza infection in mice based on morbidity (where the illness was defined as 25% body weight loss). We assessed this relationship using dose–response models incorporating HI or PVN titres as a variable. The morbidity was correlated with the pre-exposure titres, and such a correlation was well described by a modified dose–response model. The mathematical modelling suggests that PVN titres consistently show a stronger association with in vivo protection as compared to HI titres in mice. Given our findings, the PVN test warrants further investigation as a tool for evaluating antibody responses to influenza vaccines containing hemagglutinin. The resulting models may also be useful for analyzing human clinical data to identify potentially protective antibody titres against influenza illness.     

Vaccination to Conserved Influenza Antigens in Mice Using a Novel Simian Adenovirus Vector,PanAd3, Derived from the Bonobo Pan paniscus

Among approximately 1000 adenoviruses from chimpanzees and bonobos studied recently, the Pan Adenovirus type 3 (PanAd3, isolated from a bonobo, Pan paniscus) has one of the best profiles for a vaccine vector, combining potent transgene immunogenicity with minimal pre-existing immunity in the human population. In this study, we inserted into a replication defective PanAd3 a transgene expressing a fusion protein of conserved influenza antigens nucleoprotein (NP) and matrix 1 (M1). We then studied antibody and T cell responses as well as protection from challenge infection in a mouse model. A single intranasal administration of PanAd3-NPM1 vaccine induced strong antibody and T cell responses, and protected against high dose lethal influenza virus challenge. Thus PanAd3 is a promising candidate vector for vaccines, including universal influenza vaccines.     

Protection against influenza virus infection in polymeric Ig receptor knockout mice immunized intranasally with adjuvant-combined vaccines

The role of secretory IgA in conferring cross-protective immunity was examined in polymeric (p)IgR knockout (KO) mice immunized intranasally with different inactivated vaccines prepared from A/PR/8/34 (H1N1), A/Yamagata/120/86 (H1N1), A/Beijing/262/95 (H1N1), and B/Ibaraki/2/85 viruses and infected with the A/PR/8/34 virus in the upper respiratory tract (RT)-restricting volume. In wild-type mice, immunization with A/PR/8/34 or its variant (A/Yamagata/120/86 and A/Beijing/262/95) vaccines conferred complete protection or partial cross-protection against infection, while the B-type virus vaccine failed to provide protection. The protection or cross-protection was accompanied by an increase in the nasal A/PR/8/34 hemagglutinin-reactive IgA concentration, which was estimated to be >30 times the serum IgA concentration and much higher than the nasal IgG concentration. In contrast, the blockade of transepithelial transport of dimeric IgA in pIgR-KO mice reduced the degree of protection or cross-protection, in parallel with the marked increase in serum IgA concentration and the decrease in nasal IgA concentration (about 20 and 0.3 times those in wild-type mice, respectively). The degree of the reduction of protection or cross-protection was moderately reversed by the low but non-negligible level of nasal IgA, transudates from the accumulated serum IgA. These results, together with the absence of the IgA-dependent cross-protection in the lower RT and the unaltered level of nasal or serum IgG in wild-type and pIgR-KO mice, confirm that the actively secreted IgA plays an important role in cross-protection against variant virus infection in the upper RT, which cannot be substituted by serum IgG.