Protection against respiratory syncytial virus by inactivated influenza virus carrying a fusion protein neutralizing epitope in a chimeric hemagglutinin.

A desirable vaccine against respiratory syncytial virus (RSV) should induce neutralizing antibodies without eliciting abnormal T cell responses to avoid vaccine-enhanced pathology. In an approach to deliver RSV neutralizing epitopes without RSV-specific T cell antigens, we genetically engineered chimeric influenza virus expressing RSV F262-276 neutralizing epitopes in the globular head domain as a chimeric hemagglutinin (HA) protein. Immunization of mice with formalin-inactivated recombinant chimeric influenza/RSV F262-276 was able to induce RSV protective neutralizing antibodies and lower lung viral loads after challenge. Formalin-inactivated RSV immune mice showed high levels of pulmonary inflammatory cytokines, macrophages, IL-4-producing T cells, and extensive histopathology. However, RSV-specific T cell responses and enhancement of pulmonary histopathology were not observed after RSV infection of inactivated chimeric influenza/RSV F262-276. This study provides evidence that an inactivated vaccine platform of chimeric influenza/RSV virus can be developed into a safe RSV vaccine candidate without priming RSV-specific T cells and immunopathology. FROM THE CLINICAL EDITOR: Respiratory syncytial virus (RSV) is a major cause of respiratory tract illness and morbidity in children. Hence, there is a need to develop an effective vaccine against this virus. In this article, the authors engineered chimeric influenza virus to express RSV neutralizing epitopes. The positive findings in in-vivo experiments provide a beginning for future clinical trials and perhaps eventual product realization.

Superior molecularly altered influenza virus hemagglutinin peptide 308-317 inhibits collagen-induced arthritis by inducing CD4+ Treg cell expansion.

OBJECTIVE: To investigate the inhibitory effect and possible mechanism of a novel influenza virus hemagglutinin 308-317 peptide (altered HA308-317 peptide) in collagen-induced arthritis (CIA). METHODS: CIA was induced in DBA/1 mice by immunization with type II collagen (CII). Altered HA308-317 peptide, wild HA308-317 peptide, wild CII263-272 peptide, and irrelevant peptide were administered intranasally beginning at arthritis onset. Clinical and histologic scores were assessed, and cytokine levels were determined in the serum or in supernatants from splenocytes. Characteristics of T cell subsets in response to different peptides were analyzed both in vivo and in vitro. RESULTS: Intranasal administration of wild CII263-272 peptide, wild HA308-317 peptide, or altered HA308-317 peptide could significantly ameliorate CIA, but altered HA308-317 peptide showed greater therapeutic effects than wild CII263-272 peptide and wild HA308-317 peptide. The effect of altered HA308-317 peptide was associated with a substantial decrease in production of interleukin-17 (IL-17) and interferon-γ (IFNγ) and with a marked increase in production of IL-10 and transforming growth factor ß, both in serum and in supernatants from splenocytes treated with altered HA308-317 peptide. Both the number and function of CD4+ Treg cells were significantly up-regulated by altered HA308-317 peptide, with a decreased induction of Th1 cells (CD4+IFNγ+) and Th17 cells (CD4+IL-17+). Adoptive transfer of CD4+CD25+ T cells from altered HA308-317 peptide-treated mice resulted in greater suppressive capacity in ameliorating CIA severity than did adoptive transfer of CD4+CD25+ T cells from wild HA308-317 peptide-treated, wild CII263-272 peptide-treated, or irrelevant peptide-treated mice. CONCLUSION: Intranasal administration of altered HA308-317 peptide potently suppressed the severity of CIA by increasing the number and function of CD4+ Treg cells, suggesting that altered HA308-317 peptide might be a promising candidate for treatment of rheumatoid arthritis.

A lipopeptide based on the M2 and HA proteins of influenza A viruses induces protective antibody.

A conserved 15 amino-acid residue sequence of the ectodomain of the M2 protein of influenza A virus (M2e) induces a strong antibody (Ab) response when incorporated into a synthetic lipopeptide vaccine candidate containing a T-helper epitope from influenza A hemagglutinin and the dendritic cell-targeting lipid moiety S-[2,3-bis(palmitoyloxy)propyl]cysteine (Pam2Cys). Abs elicited by the truncated M2e sequence were specific for the M2 protein of influenza A virus and were also capable of binding to cells that were infected with influenza A viruses of different subtypes. The Ab titres against the lipopeptide were similar in magnitude to those elicited by the full-length (23 residue) M2e peptide when administered in Freund's adjuvant. Abs to the truncated M2e sequence were also able to significantly reduce the viral load in airways of BALB/c mice after challenge with live influenza virus.

Use of viral fusogenic membrane glycoproteins as novel therapeutic transgenes in gliomas.

Malignant gliomas are the most common primary brain tumors in adults and, with few exceptions, have a dismal prognosis despite the therapeutic use of surgery, radiation therapy, and chemotherapy. Because CNS gliomas rarely metastasize, they represent an attractive target for gene therapy through local gene delivery. Here we report on the use of two different fusogenic membrane glycoproteins (FMGs), the measles virus proteins F and H (MV-F and MV-H) and a mutated form of the retroviral envelope protein of the gibbon ape leukemia virus (GALV.fus), as a novel class of therapeutic transgenes in gliomas. Transfection of U87 and U118 cells with MV-F and MV-H cDNA or GALV.fus cDNA led in 48 hr to massive syncytial formation followed by cell death. FMG-mediated cytotoxicity in the U87 and U118 cell lines was superior to the cytotoxicity caused by transfection with HSV-tk cDNA followed by ganciclovir (GCV) treatment at all time points. At high-density cell seeding, addition of tumor cells transfected with MV-F and H killed at least 1 log more cells than by HSV-tk + GCV treatment, indicating higher bystander effect. Similar results were obtained with GALV.fus. The mechanism of syncytial death in cultured glioma cell lines was predominantly apoptotic. Transfection of U87 cells with F + H or GALV.fus expression constructs completely suppressed their tumorigenicity. Treatment of established U87 xenografts in nude mice with a combination of F and H adenoviruses at 1:1 ratio led to complete tumor regression, significantly higher antitumor effect, and prolongation of survival as compared with control animals treated with a GFP adenovirus. In summary, the viral fusogenic membrane glycoproteins (GALV and the MV-F + MV-H combination) are potent therapeutic transgenes with potential utility in the gene therapy of gliomas.

Successful DNA immunization against measles: neutralizing antibody against either the hemagglutinin or fusion glycoprotein protects rhesus macaques without evidence of atypical measles.

Measles remains a principal cause of worldwide mortality, in part because young infants cannot be immunized effectively. Development of new vaccines has been hindered by previous experience with a formalin-inactivated vaccine that predisposed to a severe form of disease (atypical measles). Here we have developed and tested potential DNA vaccines for immunogenicity, efficacy and safety in a rhesus macaque model of measles. DNA protected from challenge with wild-type measles virus. Protection correlated with levels of neutralizing antibody and not with cytotoxic T lymphocyte activity. There was no evidence in any group, including those receiving hemagglutinin-encoding DNA alone, of 'priming' for atypical measles.

Recombinant secreted haemagglutinin protects mice against a lethal challenge of influenza virus.

Balb/c mice were immunized with 2 x 2 micrograms of purified recombinant secreted haemagglutinin, derived from the A/Victoria/3/75 (H3N2) virus. In the first immunization, Ribi adjuvant was used, while for the booster injection a monophosphoryl lipid A/muramyl dipeptide combination was chosen. Mice immunized in this way were 90-100% protected against a challenge with 20 LD50 of mouse-adapted, homologous virus (strain X47). Bromelain-solubilized haemagglutinin gave only 70% protection under comparable conditions.

Antigenic properties and protective capacity of a cyclic peptide corresponding to site A of influenza virus haemagglutinin.

Two cyclic peptide analogues corresponding to residues 139-146 (site A) of influenza A virus haemagglutinin (strain X31) were synthesized. The ability of these peptides to react with anti-influenza virus antibodies was found to depend on the conformation of the loop and on the orientation in which the peptide was presented to antibodies. Antibodies raised to the peptides were able to bind in ELISA with influenza virus antigen that had been allowed to dry on the microtitre plate. When OF1 mice were immunized with cyclic peptides, approximately 80% of the animals were protected against an intranasal challenge with influenza virus.

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.

Protection of mice from fatal measles encephalitis by vaccination with vaccinia virus recombinants encoding either the hemagglutinin or the fusion protein.

Vaccinia virus recombinants encoding the hemagglutinin or fusion protein of measles virus have been constructed. Infection of cell cultures with the recombinants led to the synthesis of authentic measles proteins as judged by their electrophoretic mobility, recognition by antibodies, glycosylation, proteolytic cleavage, and presentation on the cell surface. Mice vaccinated with a single dose of the recombinant encoding the hemagglutinin protein developed antibodies capable of both inhibiting hemagglutination activity and neutralizing measles virus, whereas animals vaccinated with the recombinant encoding the fusion protein developed measles neutralizing antibodies. Mice vaccinated with either of the recombinants resisted a normally lethal intracerebral inoculation of a cell-associated measles virus subacute sclerosing panencephalitis strain.