Efficacy of Influenza Haemagglutinin and Nucleoprotein as Protective Antigens against Influenza Virus Infection in Mice

Influenza nucleoprotein (NP)-specific cytotoxic T lymphocytes (CTL) stimulated by immunization of mice with VV-PR8-NP6, a recombinant vaccinia virus expressing A/PR/8/34 NP, did not protect mice against challenge with A/PR/8/34 4 days later. Neither were secondary NP-specific CTL stimulated by reimmunization able to protect mice. These results contrast with the ability of transferred, in vitro-cultured and stimulated, NP-specific CTL to protect recipient mice from challenge with A/PR/8/34. Immunization of mice with a recombinant vaccinia virus expressing A/PR/8/34 HA protected mice challenged 4 days later, either via the small amount of antibody already present, or via HA-specific CTL that would have to be more efficient than NP-specific CTL in either trafficking to the infected lung or in effector function.     

基于M2e的广谱流感疫苗及其免疫保护机制研究进展

疫苗接种是预防流感最有效和经济的手段.当前使用的流感疫苗主要是作用于特异性的流感病毒表面糖蛋白血凝素,只能对和疫苗株匹配的流行株提供保护,因此每年需要对疫苗株进行更新.研发能够预防不同亚型流感病毒的广谱疫苗成为流感疫苗研究的热点.M2e是甲型流感病毒保守片段,可作为广谱流感疫苗的候选抗原,是目前广谱流感疫苗研究的热点....     

Recombinant Vaccine Vector-Induced Protection of Athymic, Nude Mice from Influenza A Virus Infection. Analysis of Protective Mechanisms

Athymic, nude mice, which normally succumb to virus infection, can resolve infection with recombinant vaccinia virus (rVV) engineered to express IL-2. We have demonstrated that interferon-gamma (IFN-gamma) produced by natural killer (NK) cells and other immunocytes in response to the virus-encoded interleukin-2 (IL-2) is crucial to recovery. Here, we extend this work to show that nude mice, when primed intravenously with rVV co-expressing both IL-2 and an influenza virus haemagglutinin (HA) gene, are also protected following challenge with a lethal dose of homologous influenza virus. A substantial increase in the number of influenza virus-reactive antibody-secreting cells producing antibody of the IgM isotype, but not of the IgG or IgA isotypes, was found in spleens and lungs of the protected mice. Treatment with monoclonal antibodies to IFN-gamma or to the NK marker, as GM1, at challenge and thereafter, led to their death however, though the specific IgM antibody response was unaffected. These data suggest that both specific antibody and non-specific antiviral reactivity are important elements of the protective response and show that this immunization strategy may be used to protect severely immunocompromised individuals.     

DNA and Modified Vaccinia Virus Ankara Vaccines Encoding Multiple Cytotoxic and Helper T-Lymphocyte Epitopes of Human Immunodeficiency Virus Type 1 (HIV-1) Are Safe but Weakly Immunogenic in HIV-1-Uninfected,Vaccinia Virus-Naive Adults

We evaluated a DNA plasmid-vectored vaccine and a recombinant modified vaccinia virus Ankara vaccine (MVA-mBN32), each encoding cytotoxic and helper T-lymphocyte epitopes of human immunodeficiency virus type 1 (HIV-1) in a randomized, double-blinded, placebo-controlled trial in 36 HIV-1-uninfected adults using a heterologous prime-boost schedule. HIV-1-specific cellular immune responses, measured as interleukin-2 and/or gamma interferon production, were induced in 1 (4%) of 28 subjects after the first MVA-mBN32 immunization and in 3 (12%) of 25 subjects after the second MVA-mBN32 immunization. Among these responders, polyfunctional T-cell responses, including the production of tumor necrosis factor alpha and perforin, were detected. Vaccinia virus-specific antibodies were induced to the MVA vector in 27 (93%) of 29 and 26 (93%) of 28 subjects after the first and second immunizations with MVA-mBN32. These peptide-based vaccines were safe but were ineffective at inducing HIV-1-specific immune responses and induced much weaker responses than MVA vaccines expressing the entire open reading frames of HIV-1 proteins.     

Structural Changes in the Brain of Mice Infected with Influenza A/H5N1 Virus

Structural changes in the brain of outbred mice were studied after infection with infuenza A/H5N1 strain isolated in the Novosibirsk region. High mortality was observed after intranasal infection. Examination of brain specimens revealed vasculopathies with thrombosis of the microcirculatory vessels, pericellular and perivascular edema with multifocal ischemic necrosis, hyperplasia of glial cells, caspase-dependent apoptosis of neurons caused by the cytopathic effect of the virus, and hypercytokinemia.     

Live Victoria/75-ts-1[E] Influenza A Virus Vaccines in Adult Volunteers: Role of Hemagglutinin Immunity in Protection Against Illness and Infection Caused by Influenza A Virus

To explore the relationship between neuraminidase immunity and the degree of attenuatíon of live influenza A virus vaccines, a comparative evaluation of three Victoria/75-ts-1[E] (Vic/75-ts-1[E]) recombinant viruses in serum hemagglutination-inhibiting-negative (titer, 相似文献   

Influence of Individual Mutations in Genes Coding Internal Proteins of the Influenza A Virus on Formation of Humoral and Cellular Immune Response in Mice

This study addresses a current problem of vaccinal prevention—the development of approaches to increase the immunogenicity of influenza vaccines—and it is directed at studying the effect of mutations responsible for the degree of attenuation of influenza viruses on the formation of an immune response. We conducted the analysis of the humoral and cellular immune response in mice with the introduction of strains of the influenza A virus containing single and combined point mutations alike typical for the attenuation donor A/Leningrad/134/17/57 (H2N2), which is used at present for the preparation of domestic live influenza vaccine. In the study, 13 mutant strains were compared to the attenuation donor (containing all these mutations) and its “wild” predecessor (without mutations). It has been shown that the presence in internal genes of the “wild” virus of single mutations that are typical for the attenuation donor, as well as their combinations, can affect not only the quantitative indices of the humoral immune response, but also the rate of accumulation of virus-specific serum and secretory antibodies. A group of viruses with mutations in the M1 gene was isolated based on the ability to stimulate T cell response. A promising approach to development of ways to increase the immunogenic properties of live influenza vaccines will be a further search for a balanced combination of mutations in M1 and NS2 genes that enhance the stimulation of most of the immuneresponse factors studied, with mutations in genes of polymerase complex that impart attenuating properties to strains.     

Comparison of the Long-Term Immunogenicity of Two Pandemic Influenza A/H1N1 2009 Vaccines,the MF59-Adjuvanted and Unadjuvanted Vaccines,in Adults

Since the first reports of the A/H1N1 virus in April 2009, the pandemic influenza virus spread globally and circulated for a long time. The primary method for the control of influenza is vaccination, but levels of influenza vaccine-induced antibody are known to decline rapidly during a 6-month period. In adults aged 18 to 64 years, we compared the long-term immunogenicity of two of the influenza A/H1N1 2009 monovalent vaccines, 3.75-μg MF59-adjuvanted vaccine and 15-μg unadjuvanted vaccine. The serum hemagglutinin inhibition (HI) titers were determined prevaccination and at 1, 6, and 10 months after vaccination. One hundred six (88.3%) of the 120 subjects were monitored for the entire 10-month period after receiving the influenza A/H1N1 2009 monovalent vaccine. There were 60 patients who received the unadjuvanted vaccine and 46 patients who received the MF59-adjuvanted vaccine. The seroprotection rates, seroconversion rates, and the geometric mean titer (GMT) folds fulfilled the criteria of the European Medicines Agency (EMA) for influenza A/California/7/2009 (H1N1) at 1 month after vaccination irrespective of the vaccine composition. Although the GMTs at 1 month postvaccination were somewhat higher in the unadjuvanted vaccine recipients than in the MF59-adjuvanted vaccine recipients, the difference was not significant (P = 0.29). The seroprotection rates at 6 and 10 months postvaccination were preserved above 70% but only in the MF59-adjuvanted vaccine recipients. In conclusion, low-dose MF59-adjuvanted influenza vaccine, even with 3.75 μg hemagglutinin antigen, might induce excellent long-term immunity that is comparable to the conventional dose of unadjuvanted vaccine among healthy adults aged 18 to 64 years.     

Comparative Antigenicity and Immunogenicity of A/USSR/77 Influenza Vaccines in Normal and Primed Mice

In response to the threat of wide-spread epidemics of influenza in the United States due to the A/USSR/77 strain of virus, vaccines containing A/USSR/77 virus were prepared by four manufacturers. The vaccines were standardized by immunological measurements of viral hemagglutinin and were tested for their ability to induce serum hemagglutination-inhibiting antibodies in mice and to protect the animals against challenge infection with A/USSR/77 virus. Whole-virus and subunit virus vaccines were found to be equally efficacious, in contrast to our previous findings with vaccines prepared from other influenza virus strains. The effect of priming animals by infection with representative viruses of earlier eras on their response to A/USSR/77 vaccines was also studied. Enhanced responses were noted to both subunit and whole-virus A/USSR/77 vaccines in animals primed with viruses prevalent before 1957; higher antibody titers were induced with the subunit vaccine. A high degree of heterologous protection to A/USSR/77 challenge infection was found in mice primed with virus strains having hemagglutinin antigens unlike those of A/USSR/77 virus. Comparison of the responses of mice with those of humans inoculated with the same vaccines showed a similarity in many instances, with higher responses in those individuals primed with H0N1 and H1N1 viruses than in younger vaccinees.     

Cytotoxic T Cells in the Lungs of Mice Infected with an Influenza A Virus

Cytotoxic T cells are present in the lungs and the bronchoalveolar washings of mice infected intravenously (i.v.) or intranasally (i.n.) with live influenza A/WSN virus. After i.v. injection, cytotoxic T cell activity in both spleens and lungs reaches a peak at 6 days when the level of infectious virus recovered from the lungs falls sharply and the mice do not die. If a lethal dose of virus is given intranasally, very high levels of virus appear rapidly in the lungs, and the development of lung consolidation follows slightly behind the appearance of cytotoxic T cells there. When a non-lethal dose of virus is given intranasally, lower levels of virus are found in the lung and the appearance of cytotoxic T cells is delayed. These results suggest that the cytotoxic T cells play a protective role if the level of virus in the lungs does not reach very high levels. After injection of antithymocyte serum, the subsequent level of cytotoxic T cell activity in the lungs was greatly reduced, suggesting that the T cells recovered in lungs had at an earlier stage been circulating cells. However, splenectomized mice develop high levels of cytotoxic T cell activity, after intranasal infection of mice, indicating that the spleen did not contribute substantially to the T cells recovered in the lungs.     

Cells Mediating Delayed-Type Hypersensitivity in the Lungs of Mice Infected with an Influenza A Virus

Effector cells that demonstrate delayed-type hypersensitivity (DTH) on transfer with antigen to naive mice can he recovered from the lungs of mice inoculated intranasally 6 days earlier with a lethal dose (usually 5 × 10⁴EID₅₀) of influenza A virus. The activity recovered was proportional to the dose of virus instilled intranasally and the extent of lung consolidation observed. Active cells could also be recovered from the draining lymph nodes and from the peripheral blood. The effector cells were identified as T lymphocytes of Ly I phenotype and required I-region sharing between donor and recipient for activity to be elicited. They were cross-reactive within the A group of influenza viruses. Two experiments are reported in which immune cell preparations that expressed DTH activity but had very little cytotoxic T cell activity were transferred to mice inoculated 1 or 2 days earlier with a lethal dose of virus. The mice were not protected from death, and in both experiments, the recipient mice died more rapidly than the controls. These results contrast with earlier results in which cell preparations with high cytotoxic T-cell activity were shown to protect recipient infected mice from death.     

Protection of Suckling Mice from Experimental Cholera by Maternal Immunization: Comparison of the Efficacy of Whole-Cell, Ribosomal-Derived, and Enterotoxin Immunogens

The susceptibility of suckling mice to oral infection with several different Vibrio cholerae was determined. Mice up to 10 days of age were uniformly susceptible to oral infection with 10(8) colony-forming units of virulent organisms. Age-dependent resistance occurred thereafter to a maximum at about 15 days of age. The efficacy of selected vaccines was compared by oral challenge of 8-day-old, passively immunized CFW mice. An Ogawa-derived ribosomal antigen was found to be superior to a commercial whole-cell vaccine or to purified cholera enterotoxin. The ribosomal antigen was 50- to 100-fold more protective than the other vaccines on a weight basis against otherwise lethal challenge with Ogawa, Inaba, or El Tor Ogawa serotypes.     

A Point Mutation in the Sendai Virus Accessory C Proteins Attenuates Virulence for Mice,but Not Virus Growth in Cell Culture

A mutant Sendai virus (SeV^MVC), which grows much better than its progenitor virus (SeV^M) in cell culture, but, in strong contrast to SeV^M, is totally avirulent for mice, has been described. SeV^MVCcontains two amino acid substitutions relative to SeV^M, namely, F170S in the C protein and E2050A in the L protein. We have examined which substitutions were responsible for the above phenotypes by exchanging the C gene of our reference strain Z with those of SeV^H(another reference strain), SeV^M, and SeV^MVC, in turn. We have found that the F170S mutation in the C^MVCprotein is responsible both for enhanced replication in cell culture and for avirulence in mice. Avirulence appeared to be due to restricted viral replication primarily after day 1, implicating some aspect of innate immunity in this process. The SeV C proteins thus appear to be required for multiple cycles of replication in mice.     

Immunogenicity and Protective Efficacy of Prime-Boost Regimens with Recombinant ΔureC hly+ Mycobacterium bovis BCG and Modified Vaccinia Virus Ankara Expressing M. tuberculosis Antigen 85A against Murine Tuberculosis

In the light of the recent emergence of multidrug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis, the epidemic of tuberculosis (TB) in populations coinfected with human immunodeficiency virus, and the failure of Mycobacterium bovis bacillus Calmette-Guerin (BCG) to protect against disease, new vaccines against TB are urgently needed. Two promising new vaccine candidates are the recombinant ΔureC hly⁺ BCG (recBCG), which has been developed to replace the current BCG vaccine strain, and modified vaccinia virus Ankara (MVA) expressing M. tuberculosis antigen 85A (MVA85A), which is a leading candidate vaccine designed to boost the protective efficacy of BCG. In the present study, we examined the effect of MVA85A boosting on the protection afforded at 12 weeks postchallenge by BCG and recBCG by using bacterial CFU as an efficacy readout. recBCG-immunized mice were significantly better protected against aerosol challenge with M. tuberculosis than mice immunized with the parental strain of BCG. Intradermal boosting with MVA85A did not reduce the bacterial burden any further. In order to identify a marker for the development of a protective immune response against M. tuberculosis challenge, we analyzed splenocytes after priming or prime-boosting by using intracytoplasmic cytokine staining and assays for cytokine secretion. Boosting with MVA85A, but not priming with BCG or recBCG, greatly increased the antigen 85A-specific T-cell response, suggesting that the mechanism of protection may differ from that against BCG or recBCG. We show that the numbers of systemic multifunctional cytokine-producing cells did not correlate with protection against aerosol challenge in BALB/c mice. This emphasizes the need for new biomarkers for the evaluation of TB vaccine efficacy.     

Intranasal Immunization of Mice with Inactivated Virus and Mast Cell Activator C48/80 Elicits Protective Immunity against Influenza H1 but not H5

Vaccination represents the most economic and effective strategy of preventing influenza pandemics. We previously demonstrated that intranasal immunization of mice with recombinant hemagglutinin and the mast cell activator C48/80 elicited protective immunity against challenge with the 2009 pandemic H1N1 influenza in mice, demonstrating that the novel C48/80 mucosal adjuvant was safe and effective. The present study demonstrated that intranasal immunization with inactivated H1N1 virus and C48/80 elicited protective immunity against lethal challenge with homologous virus, however, when the immunogen was replaced with inactivated H5N1 virus protection was lost. These observations suggested that the adjuvant effects conferred by C48/80 were virus subtype specific and that its use as a broad-spectrum adjuvant for use in immunizations against all influenza viruses needs to be further analyzed.