Protection of inactivated influenza virus vaccine against lethal influenza virus infection in diabetic mice

Influenza virus infection frequently causes complications and some excess mortality in the patients with diabetes. Vaccination is an effective measure to prevent influenza virus infection. In this paper, antibody response and protection against influenza virus infection induced by vaccination were studied in mouse model of diabetes. Healthy and diabetic BALB/c mice were immunized once or twice with inactivated influenza virus vaccine at various dosages. Four weeks after the first immunization or 1 week after the second immunization, the mice were challenged with influenza virus at a lethal dose. The result showed that the antibody responses in diabetic mice were inhibited. Immunization once with high dose or twice with low dose of vaccine provided full protection against lethal influenza virus challenge in diabetic mice, however, in healthy mice, immunization only once with low dose provided a full protection.     

Cellular immune responses of mice to influenza virus infection

Mice infected with influenza virus develop cytotoxic T lymphocytes (CTL) specific for viral antigens prior to the appearance of virus-specific antibody-forming cells (AFCs). Effector T cells were detected at a time coincident with a precipitous decline in pulmonary virus titer. CTLs of draining lymph nodes and spleen were found to be cross-reactive among H-2 compatible cells infected with influenza type A virus subtypes. AFCs were observed to be primarily hemagglutinin specific. Virus-specific IgA-secreting AFCs were detected in mediastinal lymph nodes of infected mice.     

Protection of neonatal mice against herpes simplex virus infection: probable in vivo antibody-dependent cellular cytotoxicity

Infant mice are extremely susceptible to fatal Herpes simplex virus (HSV) infection. They are unable to produce antibody to HSV, and their leukocytes cannot mediate antibody-dependent cellular cytotoxicity (ADCC) to HSV-infected cells. In order to avoid H-2-dependent effector mechanisms and instead analyze possible in vivo ADCC, a murine model employing adoptive transfer of antibody and human leukocytes was developed. Administration of either human immune globulin or leukocytes i.p. from HSV immune or nonimmune humans could not protect infant C57BL/6 mice from fatal HSV infection. In contrast, a combination of a subneutralizing dilution of globulin and leukocytes from nonimmune or immune human donors, given one day before inoculation, was highly protective against lethal HSV infection. The cells involved included lymphocytes or monocyte-macrophages. At least 5 X 10(6) viable leukocytes (or 1 X 10(6) monocyte-macrophages) and immune serum globulin concentrations as low as 10(-8) were protective. Infected cell monolayer adsorption and DEAE column fractionation demonstrated that the protection by globulin was due to specific antiviral IgG antibody. Protection was n ot seen in animals receiving virus before immune transfer. Protection did not involve synergistic viral neutralization by antibody and cells, as shown by in vitro experiments. Animals receiving globulin and cells, unlike normal infant mice, had circulating antiviral antibody and peritoneal leukocytes able to mediate ADCC to HSV-infected cells. This is the first in vivo evidence for the role of human ADCC. This model also allows for the in vivo evaluation of the ability of cells from immunocompromised humans to curb viral infection.     

Heat-killed Lactobacillus gasseri TMC0356 protects mice against influenza virus infection by stimulating gut and respiratory immune responses

This study investigated whether heat-killed Lactobacillus protects host animal against influenza virus infection and stimulates their immunity. Heat-killed Lactobacillus gasseri TMC0356 was orally administered to BALB/c mice for 19 days; the mice were intranasally infected with Flu A/PR/8/34 (H1N1) on day 14, and clinical symptoms were monitored. After 6 days, the mice were sacrificed, and pulmonary virus titres were determined. Splenic activation of natural killer (NK) cells and the mRNA expression of cytokines and other immune molecules in the lung and Peyer's patch (PP) were analysed. Clinical symptom scores of mice orally fed TMC0356 ameliorated significantly (P < 0.01); their pulmonary virus titres decreased significantly compared with those of control mice (P < 0.05); their mRNA expression of interleukin (IL)-12, IL-15 and IL-21 in PP and the pulmonary mRNA expression of IFN-γ, TNF, IL-12a, IL-12rbl, IL-2rb and perforin 1 increased significantly (P < 0.05). Oral administration of heat-killed lactobacilli may protect against influenza virus infection by stimulating local and systemic immune responses. Cellular components of lactobacilli may be pivotal in protecting against viral infection by enhancing gut and respiratory immune responses.     

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.     

Adjuvant efficacy of mOMV against avian influenza virus infection in mice

Highly pathogenic avian influenza H5N1 viruses are found chiefly in birds and have caused severe disease and death in infected humans. Development of influenza vaccines capable of inducing heterosubtypic immunity against a broad range of influenza viruses is the best option for the preparedness, since vaccination remains the principal method in controlling influenza viral infections. Here, a mOMV-adjuvanted recombinant H5N2 (rH5N2) whole virus antigen vaccine with A/Environment/Korea/W149/06(H5N1)-derived H5 HA and A/Chicken/Korea/ma116/04(H9N2)-derived N2 NA in the backbone of A/Puerto Rico/8/34(H1N1) was prepared and generated by reverse genetics. Groups of mice were vaccinated by a prime-boost regime with the rH5N2 vaccine (1.75 μg of HA with/without 10 μg mOMV or aluminum hydroxide adjuvant for comparison). At two weeks post-immunizations, vaccinated mice were challenged with lethal doses of 10^3.5 EID₅₀/ml of H5N1 or H9N2 avian influenza viruses, and were monitored for 15 days. Both mOMV- and alum-adjuvant vaccine groups had high survival rates after H5N1 infection and low levels of body weight changes compared to control groups. Interestingly, the mOMV-adjuvanted group induced better cross-reactive antibody responses serologically and promoted cross-protectivity against H5N1 and H9N2 virus challenges. Our results suggest that mOMV could be used as a vaccine adjuvant in the development of effective vaccines used to control influenza A virus transmission.     

Protection against influenza infection by cytokine-enhanced aerosol genetic immunization

BACKGROUND: Conventional vaccine development for newly emerging pandemic influenza virus strains would likely take too long to prevent devastating global morbidity and mortality. If DNA vaccines can be distributed and delivered efficiently, genetic immunization could be an attractive solution to this problem, since plasmid DNA is stable, easily engineered to encode new protein antigens, and able to be quickly produced in large quantities. METHODS: We compared two novel genetic immunization methods in a mouse model of influenza to evaluate protective effects: aerosol delivery of polyethylenimine (PEI)-complexed hemagglutinin (HA)-expressing plasmid and intravenous (IV) delivery of the plasmid complexed with macroaggregated albumin/PEI. Serial serum samples were obtained for assay of neutralizing antibodies against HA. Mice were then challenged in the airway with influenza virus, and production of infectious virus in the lungs was titered. RESULTS: Most mice immunized with HA plasmid alone by aerosol and all mice immunized IV developed protective immune responses, whereas none administered control plasmid were protected. Aerosol co-administration of HA plasmid with plasmids encoding the cytokines interleukin 12 (IL12) and granulocyte-macrophage colony stimulating factor (GM-CSF) markedly increased neutralizing antibody responses, so that all aerosol immunized mice were protected from high level virus proliferation. CONCLUSIONS: Cytokine-enhanced aerosol delivery of plasmid vaccines can elicit robust protective immune responses against influenza. Thus, aerosol delivery has the potential to address the need for rapid widespread immunization against new influenza virus strains, and may have applications for other infectious and toxic disease processes.     

Protection of mice against a lethal influenza virus challenge after immunization with yeast-derived secreted influenza virus hemagglutinin.

The A/Victoria/3/75 (H3N2-subtype) hemagglutinin (HA) gene was engineered for expression in Pichia pastoris as a soluble secreted molecule. The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to the Saccharomyces cerevisiae alpha-mating factor secretion signal and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Growth of transformants on methanol-containing medium resulted in the secretion of recombinant non-cleaved soluble hemagglutinin (HA0s). Remarkably, the pH of the induction medium had an important effect on the expression level, the highest level being obtained at pH 8.0. The gel filtration profile and the reactivity against a panel of different HA-conformation specific monoclonal antibodies indicated that HA0s was monomeric. Analysis of the N-linked glycans revealed a typical P. pastoris type of glycosylation, consisting of glycans with 10-12 glycosyl residues. Mice immunized with purified soluble hemagglutinin (HA0s) showed complete protection against a challenge with 10 LD50 of mouse-adapted homologous virus (X47), whereas all control mice succumbed. Heterologous challenge with X31 virus [A/Aichi/2/68 (H3N2-subtype)], resulted in significantly higher survival rates in the immunized group compared with the control group. These results, together with the safety, reliability and economic potential of P. pastoris, as well as the flexibility and fast adaptation of the expression system may allow development of an effective recombinant influenza vaccine.     

Protection against Mesocestoides corti infection in mice treated with zymosan or Salmonella enteritidis 11RX

Toye P. G. and Jenkin C. R. 1982. Protection against Mesocestoides corti infection in mice treated with zymosan or Salmonella enteritidis 11RX. International Journal for Parasitology12: 399–402. Zymosan and Salmonella enteritidis 11RX were found to partially protect mice against infection with the cestode Mesocestoides corti. Thus, mice previously infected with S. enteritidis 11RX contained fewer parasites in the peritoneal cavity compared to normal mice. Mice pretreated with zymosan contained fewer parasites in the peritoneal cavity and in the liver compared to normal mice and this protection was enhanced by the passive transfer of serum from mice chronically infected with M. corti. Examination of mice in the initial stages of infection revealed that the administration of zymosan led to an alteration in parasite location from the peritoneal cavity to the liver.     

Activity of recombinant human alpha interferon against influenza virus infection in mice

The in vivo antiviral activity of recombinant human leukocyte hybrid interferon, HuIFN-alpha AD, was examined. Results showed that this material in highly purified form did not protect mice against a lethal dose of influenza virus, although administration of natural MuIFN-alpha/beta to mice infected with a lethal dose of influenza virus had a marked protective effect. The effect of alveolar macrophages treated with IFN on influenza virus replication was examined in vitro. The antiviral activity of alveolar macrophages treated with HuIFN-alpha AD was lower than that of MuIFN-alpha/beta. It is concluded that HuIFN-alpha AD is effective in direct inhibition of influenza virus, but not in indirect inhibition mediated by alveolar macrophages or in protection of mice from influenza virus infection.     

Protection of rats against pseudorabies virus infection by gamma-interferon

Administration of recombinant rat gamma-interferon to rats conferred complete protection against an otherwise lethal intraperitoneal pseudorabies virus (PRV) infection. The primary target cell of the virus has been identified as the serosal cell of the peritoneum. Histologic examination showed that after infection of the underlying adventitia, the virus replicates in the myenteric and submucosal plexuses of the gastrointestinal tract; this is followed by centripetal spread to the autonomous and central nervous system. In recombinant rat gamma-interferon-treated rats, viral antigen was absent in the primary target cells and was not detected in any other organ. In interferon-treated cultures of peritoneal fibroblasts, which represent another primary target cell population in vivo, complete inhibition of PRV replication was observed. The peritoneal macrophage is not susceptible to PRV, as was shown by coculture and immunocytochemical studies. Peritoneal cells from gamma-interferon-treated rats showed enhanced major histocompatibility class II antigen expression and extrinsic antiviral activity in PRV-susceptible rat embryo fibroblasts. The results presented in this study indicate that protection by the lymphokine is likely to be based on direct inhibition of viral replication in serosal cells.     

Neuropathogenesis of influenza virus infection in mice

The neurovirulent WSN strain of influenza A virus, introduced into the olfactory bulb of C57BL/6 mice, selectively attacks several brain nuclei which are highly implicated in the pathogenesis of neuropsychiatric disturbances. The virus-infected neurons are eradicated through apoptotic neurodegeneration. On the other hand, activated microglia serve the neuroprotection against virus infection.     

Protection by polyI:polyC against infection with herpes simplex virus in mice pretreated with Corynebacterium parvum

The effect of Corynebacterium parvum on the protection by polyinosinic-polycytidylic acid (polyI:polyC) against lethal infection with Herpes simplex virus type 1 (HSV) was studied in mice. Pretreatment with C. parvum resulted in prolonged survival times in all experiments. One third of the mice survived an infection with 100 LD50, whereas all mice died when treated with polyI:polyC alone. Increased protection was observed up to 6 weeks after pretreatment and only seen when both C. parvum and polyI:polyC were given at the same site of injection (intraperitoneally). Protection against HSV correlated with increased interferon (IFN) activities induced by polyI:polyC in the peritoneal cavity of C. parvum-pretreated mice. In these mice, natural killer cell activity of peritoneal exudate cells (PEC) was also augmented in response to polyI:polyC. Protection was markedly decreased by intraperitoneal injection of silica or of an antiserum against murine IFN. It appears that increased local levels of IFN presumably produced by macrophages in response to polyI:polyC in C. parvum-pretreated mice play the major role in the antiviral defence in our model and that activation of NK cells may be a secondary effect of IFN.     

Protection of mice against lethal infection with highly pathogenic H7N7 influenza A virus by using a recombinant low-pathogenicity vaccine strain

In 2003, an outbreak of highly pathogenic avian influenza occurred in The Netherlands. The avian H7N7 virus causing the outbreak was also detected in 88 humans suffering from conjunctivitis or mild respiratory symptoms and one person who died of pneumonia and acute respiratory distress syndrome. Here we describe a mouse model for lethal infection with A/Netherlands/219/03 isolated from the fatal case. Because of the zoonotic and pathogenic potential of the H7N7 virus, a candidate vaccine carrying the avian hemagglutinin and neuraminidase proteins produced in the context of the high-throughput vaccine strain A/PR/8/34 was generated by reverse genetics and tested in the mouse model. The hemagglutinin gene of the recombinant vaccine strain was derived from a low-pathogenicity virus obtained prior to the outbreak from a wild mallard. The efficacy of a classical nonadjuvanted subunit vaccine and an immune stimulatory complex-adjuvanted vaccine was compared. Mice receiving the nonadjuvanted vaccine revealed low antibody titers, lack of clinical protection, high virus titers in the lungs, and presence of virus in the spleen, liver, kidneys, and brain. In contrast, mice receiving two doses of the immune stimulatory complex-adjuvanted vaccine revealed high antibody titers, clinical protection, approximately 1,000-fold reduction of virus titers in the lungs, and rare detection of the virus in other organs. This is the first report of an H7 vaccine candidate tested in a mammalian model. The data presented suggest that vaccine candidates based on low-pathogenicity avian influenza A viruses, which can be prepared ahead of pandemic threats, can be efficacious if an effective adjuvant is used.     

Protection against divergent influenza H1N1 virus by a centralized influenza hemagglutinin

Influenza poses a persistent worldwide threat to the human population. As evidenced by the 2009 H1N1 pandemic, current vaccine technologies are unable to respond rapidly to this constantly diverging pathogen. We tested the utility of adenovirus (Ad) vaccines expressing centralized consensus influenza antigens. Ad vaccines were produced within 2 months and protected against influenza in mice within 3 days of vaccination. Ad vaccines were able to protect at doses as low as 10(7) virus particles/kg indicating that approximately 1,000 human doses could be rapidly generated from standard Ad preparations. To generate broadly cross-reactive immune responses, centralized consensus antigens were constructed against H1 influenza and against H1 through H5 influenza. Twenty full-length H1 HA sequences representing the main branches of the H1 HA phylogenetic tree were used to create a synthetic centralized gene, HA1-con. HA1-con minimizes the degree of sequence dissimilarity between the vaccine and existing circulating viruses. The centralized H1 gene, HA1-con, induced stronger immune responses and better protection against mismatched virus challenges as compared to two wildtype H1 genes. HA1-con protected against three genetically diverse lethal influenza challenges. When mice were challenged with 1934 influenza A/PR/8/34, HA1-con protected 100% of mice while vaccine generated from 2009 A/TX/05/09 only protected 40%. Vaccination with 1934 A/PR/8/34 and 2009 A/TX/05/09 protected 60% and 20% against 1947 influenza A/FM/1/47, respectively, whereas 80% of mice vaccinated with HA1-con were protected. Notably, 80% of mice challenged with 2009 swine flu isolate A/California/4/09 were protected by HA1-con vaccination. These data show that HA1-con in Ad has potential as a rapid and universal vaccine for H1N1 influenza viruses.     

季节性流感疫苗对小鼠感染 H7N9 禽流感病毒的保护效力简

目的 评价季节性流感裂解疫苗对流感病毒H7N9的免疫保护效力.方法 用我国2012～2013年度季节性流感裂解疫苗,以腹腔注射方式免疫BALB/c小鼠,并设PBS免疫模型组,末次免疫14 d后以5 LD50 A/Anhui/1（H7N9）进行攻试验.感染后观察记录小鼠临床表现,体重变化,并分别于第2天和第4天每组处死3只小鼠,取肺组织和鼻甲骨测病毒滴度和载量.结果 感染后疫苗与模型组小鼠体重下降明显,疫苗组存活率为10%,模型组全部死亡.感染后第4天疫苗组鼻甲骨滴度显著低于模型组.血凝抑制试验及中和实验表明免疫小鼠血清无中和H7N9病毒抗体.结论 季节性流感疫苗在小鼠中对于H7N9流感病毒感染无明显保护作用.     

Protection against lethal lymphocytic choriomeningitis virus (LCMV) infection by immunization of mice with an influenza virus containing an LCMV epitope recognized by cytotoxic T lymphocytes.

The reverse genetics system has made it possible to modify the influenza virus genome. By this method, we were able to assess influenza virus as a vaccine vector for protecting BALB/c mice against otherwise lethal lymphocytic choriomeningitis virus (LCMV) infection. A single dose of influenza virus [A/WSN/33 (H1N1)] bearing a cytotoxic T-lymphocyte-specific epitope of the LCMV nucleoprotein (residues 116 to 127) in the neuraminidase stalk protected mice against LCMV challenge for at least 4 months. The immunity was mediated by cytotoxic T lymphocytes and was haplotype specific, indicating that the observed protective response was solely a consequence of prior priming with the H-2d LCMV nucleoprotein epitope expressed in the recombinant influenza virus. We also found that as many as 58 amino acids could be inserted into the neuraminidase stalk without loss of viral function. These findings demonstrate the potential of influenza virus as a vaccine vector, with the neuraminidase stalk as a repository for foreign epitopes.     

Protective cellular immunity: cytotoxic T-lymphocyte responses against dominant and recessive epitopes of influenza virus nucleoprotein induced by DNA immunization.

DNA immunization offers a novel means to induce cellular immunity in a population with a heterogeneous genetic background. An immunorecessive cytotoxic T-lymphocyte (CTL) epitope in influenza virus nucleoprotein (NP), residues 218 to 226, was identified when mice were immunized with a plasmid DNA encoding a full-length mutant NP in which the anchor residues for the immunodominant NP147-155 epitope were altered. Mice immunized with wild-type or mutant NP DNA were protected from lethal cross-strain virus challenge, and the protection could be adoptively transferred by immune splenocytes, indicating the role of cell-mediated immunity in the protection. DNA immunization is capable of eliciting protective cellular immunity against both immunodominant and immunorecessive CTL epitopes in the hierarchy seen with virus infection.     

Oral administration of lactobacilli from human intestinal tract protects mice against influenza virus infection

Aims: Our study was conducted to evaluate the potent protective effects of oral administration of probiotic Lactobacillus strains against influenza virus (Flu) infection in a mouse model. Method and Results: Lyophilized Lactobacillus rhamnosus GG (LGG) and Lactobacillus gasseri TMC0356 (TMC0356) were orally administered to BALB/c mice for 19 days. The test mice were intranasally infected with Flu A/PR/8/34 (H1N1) on day 14, and any changes in clinical symptoms were monitored. After 6 days of infection, the mice were killed and pulmonary virus titres were determined. The clinical symptom scores of mice administered oral LGG and TMC0356 were significantly ameliorated, compared to those of the control mice (P < 0·01). The pulmonary virus titres of the mice fed LGG and TMC0356 were also significantly decreased compared to those of control mice (P < 0·05). Conclusions: These results indicate that oral administration of lactobacilli, such as LGG and TMC0356, might protect a host animal against Flu infection. Significance and Impact of the Study: These results demonstrate that oral administration of selected lactobacilli might protect host animals from Flu infection by interactions with gut immunity.