Protective immunity against influenza in HLA-A2 transgenic mice by modified vaccinia virus Ankara vectored vaccines containing internal influenza proteins.

BACKGROUND: The emergence of novel strains of influenza A viruses with hemagglutinins (HAs) that are antigenically distinct from those circulating in humans, and thus have pandemic potential, pose concerns and call for the development of more broadly protective influenza vaccines. In the present study, modified vaccinia virus Ankara (MVA) encoding internal influenza antigens were evaluated for their immunogenicity and ability to protect HLA-A2.1 transgenic (AAD) mice from infection with influenza viruses. METHODS: MVAs expressing NP (MVA-NP), M1 (MVA-M1) or polymerase PB1 (MVA-PB1) of A/California/4/09 (CA/09) virus were generated and used to immunize AAD mice. Antibodies and CD8+T cell responses were assessed by ELISA and ELISPOT, respectively, and challenge experiments were performed by infecting vaccinated mice with CA/09 virus. RESULTS: CD8+T cells specific to immunodominant and subdominant epitopes on the internal influenza proteins were elicited by MVA-based vectors in AAD mice, whereas influenza-specific antibodies were detected only in MVA-NP-immunized mice. Both M1- and NP-based MVA vaccines, regardless of whether they were applied individually or in combination, conferred protection against lethal influenza virus challenge. CONCLUSION: Our data further emphasize the promising potential of MVA vector expressing internal antigens toward the development of a universal influenza vaccine.

Immunogenicity of modified vaccinia virus Ankara expressing the hemagglutinin stalk domain of pandemic (H1N1) 2009 influenza virus.

BACKGROUND: Vaccination offers protection against influenza, although current vaccines need to be reformulated each year. The development of a broadly protective influenza vaccine would guarantee the induction of heterosubtypic immunity also against emerging influenza viruses of a novel subtype. Vaccine candidates based on the stalk region of the hemagglutinin (HA) have the potential to induce broad and persistent protection against diverse influenza A viruses. METHODS: Modified vaccinia virus Ankara (MVA) expressing a headless HA (hlHA) of A/California/4/09 (CA/09) virus was used as a vaccine to immunize C57BL/6 mice. Specific antibody and cell-mediated immune responses were determined, and challenge experiments were performed by infecting vaccinated mice with CA/09 virus. RESULTS: Immunization of mice with CA/09-derived hlHA, vectored by MVA, was able to elicit influenza-specific broad cross-reactive antibodies and cell-mediated immune responses, but failed to induce neutralizing antibodies and did not protect mice against virus challenge. CONCLUSION: Although highly immunogenic, our vaccine was unable to induce a protective immunity against influenza. A misfolded and unstable conformation of the hlHA molecule may have affected its capacity of inducing neutralizing antiviral, conformational antibodies. Design of stable hlHA-based immunogens and their delivery by recombinant MVA-based vectors has the potential of improving this promising approach for a universal influenza vaccine.

A heat-inactivated H7N3 vaccine induces cross-reactive cellular immunity in HLA-A2.1 transgenic mice.

BACKGROUND: Cross-reactive immunity against heterologous strains of influenza virus has the potential to provide partial protection in individuals that lack the proper neutralizing antibodies. In particular, the boosting of memory CD8+ T cell responses to conserved viral proteins can attenuate disease severity caused by influenza virus antigenic variants or pandemic strains. However, little is yet known about which of these conserved internal antigens would better induce and/or recall memory CD8+ T cells after in vivo administration of an inactivated whole virus vaccine. METHODS: We explored the CD8 + T cell responses to selected epitopes of the internal proteins of an H7N3 influenza virus that were cross-reactive with A/PR/8/34 virus in HLA-A2.1 transgenic (AAD) mice. RESULTS: CD8+ T cells against dominant and subdominant epitopes were detected upon infection of mice with live H7N3 virus, whereas immunization with non-replicating virus elicited CD8+ T cell responses against mostly immunodominant epitopes, which were rapidly recalled following infection with A/PR/8/34 virus. These vaccine-induced T cell responses were able to reduce the lung viral load in mice challenged intranasally with the heterologous influenza virus. CONCLUSIONS: A single immunization with non-replicating influenza virus vaccines may be able to elicit or recall cross-reactive CD8+ T cell responses to conserved immunodominant epitopes and, to some extent, counteract an infection by heterologous virus.

Exploring mucosal immunization with a recombinant influenza virus carrying an HIV-polyepitope in mice with pre-existing immunity to influenza.

HIV-1 vaccines based on recombinant vectors have been developed to elicit immune responses; however, the failure of the STEP HIV-1 vaccine trial has caused concern regarding the impact on vaccine efficacy of pre-existing vector seropositivity in humans. By using a mouse model of infection, we evaluated the immune responses elicited by intranasal and vaginal immunization with the recombinant influenza virus WSN/CKG carrying the PCLUS3-P18 peptide and a Gag epitope in its hemagglutinin, and the impact of pre-existing vector immunity on protection against recombinant vaccinia virus challenge. We found that despite the protective immunity induced in naïve mice by the WSN/CKG virus via either route, the vaginal immunization of mice with pre-existing influenza immunity restricted vPE16 replication more significantly in the ovaries than intranasal immunization. Thus, successful vaccination strategies under limiting conditions, such as pre-existing vector immunity, require the local induction of mucosal immunity at the site of virus infection.

Modified vaccinia virus Ankara expressing the hemagglutinin of pandemic (H1N1) 2009 virus induces cross-protective immunity against Eurasian 'avian-like' H1N1 swine viruses in mice.

OBJECTIVES: To examine cross-reactivity between hemagglutinin (HA) derived from A/California/7/09 (CA/09) virus and that derived from representative Eurasian "avian-like" (EA) H1N1 swine viruses isolated in Italy between 1999 and 2008 during virological surveillance in pigs. DESIGN: Modified vaccinia virus Ankara (MVA) expressing the HA gene of CA/09 virus (MVA-HA-CA/09) was used as a vaccine to investigate cross-protective immunity against H1N1 swine viruses in mice. SAMPLE: Two classical swine H1N1 (CS) viruses and four representative EA-like H1N1 swine viruses previously isolated during outbreaks of respiratory disease in pigs on farms in Northern Italy were used in this study. SETTING: Female C57BL/6 mice were vaccinated with MVA/HA/CA/09 and then challenged intranasally with H1N1 swine viruses. MAIN OUTCOME MEASURES: Cross-reactive antibody responses were determined by hemagglutination- inhibition (HI) and virus microneutralizing (MN) assays of sera from MVA-vaccinated mice. The extent of protective immunity against infection with H1N1 swine viruses was determined by measuring lung viral load on days 2 and 4 post-challenge. RESULTS AND CONCLUSIONS: Systemic immunization of mice with CA/09-derived HA, vectored by MVA, elicited cross-protective immunity against recent EA-like swine viruses. This immune protection was related to the levels of cross-reactive HI antibodies in the sera of the immunized mice and was dependent on the similarity of the antigenic site Sa of H1 HAs. Our findings suggest that the herd immunity elicited in humans by the pandemic (H1N1) 2009 virus could limit the transmission of recent EA-like swine HA genes into the influenza A virus gene pool in humans.

Enhancement of T cell-mediated immune responses to whole inactivated influenza virus by chloroquine treatment in vivo.

Current influenza vaccines induce poor cross-reactive CD8+ T cell responses. Cellular immunity is generally specific for epitopes that are remarkably conserved among different subtypes, suggesting that strategies to improve the cross-presentation of viral antigens by dendritic cells (DC) could elicit a broadly protective immune response. Previous studies have shown that limited proteolysis within the endocytic pathway can favorably influence antigen processing and thus immune responses. Herein, we demonstrate that chloroquine improves the cross-presentation of non-replicating influenza virus in vitro and T cell responses in mice following a single administration of inactivated HI-X31 virus. CD8+ T cells were also recruited to lymph nodes draining the site of infection and able to reduce viral load following pulmonary challenge with the heterologous PR8 virus. These findings may have implications for vaccination strategies aimed at improving the cross-presentation capacity of DCs and thus the size of effector and memory CD8+ T cells against influenza vaccines.

Immunogenicity of a recombinant influenza virus bearing both the CD4+ and CD8+ T cell epitopes of ovalbumin.

Recombinant influenza viruses that bear the single immunodominant CD8+ T cell epitope OVA(257-264) or the CD4+ T cell epitope OVA323₋339 of the model antigen ovalbumin (OVA) have been useful tools in immunology. Here, we generated a recombinant influenza virus, WSN-OVA(I/II), that bears both OVA-specific CD8+ and CD4+ epitopes on its hemagglutinin molecule. Live and heat-inactivated WSN-OVA(I/II) viruses were efficiently presented by dendritic cells in vitro to OT-I TCR transgenic CD8+ T cells and OT-II TCR transgenic CD4+ T cells. In vivo, WSN-OVA(I/II) virus was attenuated in virulence, highly immunogenic, and protected mice from B16-OVA tumor challenge in a prophylactic model of vaccination. Thus, WSN-OVA(I/II) virus represents an additional tool, along with OVA TCR transgenic mice, for further studies on T cell responses and may be of value in vaccine design.

Protective immunity to influenza: lessons from the virus for successful vaccine design.

The development of an effective influenza vaccine would require the ability to protect against infection with multiple influenza viral strains. In particular, mucosal and T-cell-mediated immunity may offer a more cross-reactive vaccine approach for the prevention of epidemic or potentially pandemic influenza. Thus, it is imperative to more fully understand the molecular events that occur in the host upon infection with a live virus and, in particular, to better evaluate the role of the distinct signaling pathways involved in developing protective immune responses. The paper under evaluation here introduces the notion that activation of caspase-1 inflammasomes in the hematopoietic cells in vivo are required for the establishment of Th1, cytotoxic T-lymphocyte and IgA responses to influenza virus infection.

Primary CD8+ T-cell response to soluble ovalbumin is improved by chloroquine treatment in vivo.

The efficiency of cross-presentation of exogenous antigens by dendritic cells (DCs) would seem to be related to the level of antigen escape from massive degradation mediated by lysosomal proteases in an acidic environment. Here, we demonstrate that a short course of treatment with chloroquine in mice during primary immunization with soluble antigens improved the cross-priming of naïve CD8(+) T lymphocytes in vivo. More specifically, priming of chloroquine-treated mice with soluble ovalbumin (OVA), OVA associated with alum, or OVA pulsed on DCs was more effective in inducing OVA-specific CD8(+) T lymphocytes than was priming of untreated mice. We conclude that chloroquine treatment improves the cross-presentation capacity of DCs and thus the size of effector and memory CD8(+) T cells during vaccination.

Efficient vagina-to-lower respiratory tract immune trafficking in a murine model of influenza A virus infection.

Effective vaccination strategies for infectious diseases take into account the induction, long-term maintenance and recall of memory T-cell populations. To understand the immunological cross-talk within the mucosal compartments, we compared intranasal to vaginal immunization and demonstrated that vaginal infection of BALB/c mice with influenza A virus provides protective mucosal immunity against both homosubtypic and heterosubtypic virus challenge in the respiratory tract. We found that, prior to the viral challenge, in vaginally primed mice, antigen-specific CD8+ T cells were not detected in the lung airways and levels of serum antibodies were lower than those observed in intranasally immunized mice. However, following pulmonary challenge, NP147-specific CD8+ T cells were recruited and amplified in vaginally primed mice to the same extent as those in intranasally primed mice. Thus, the long-term memory immune response elicited by vaginal immunization with influenza virus is efficiently recalled and offers reasonable protection against infection in the respiratory tract.

Mucosal and systemic immune responses to a human immunodeficiency virus type 1 epitope induced upon vaginal infection with a recombinant influenza A virus.

The humoral and cellular immune responses in the genital mucosa likely play an important role in the prevention of sexually transmitted infections, including infection with human immunodeficiency virus type 1 (HIV-1). Here we show that vaginal infection of progesterone-treated BALB/c mice with a recombinant influenza virus bearing the immunodominant P18IIIB cytotoxic T-lymphocyte (CTL) epitope of the gp160 envelope protein from an HIV-1 IIIB isolate (P18IIIB; RIQRGPGRAFVTIGK) can induce a specific immune response in regional mucosal lymph nodes, as well as in a systemic site (the spleen). A single inoculation of mice with the recombinant influenza virus induced long-lasting (at least 5 months) antigen-specific CTL memory detectable as a rapid recall of effector CTLs upon vaginal infection with recombinant vaccinia virus expressing HIV-1 IIIB envelope gene products. Long-term antigen-specific CTL memory was also induced and maintained in distant mucosal tissues when mice were intranasally immunized with the recombinant influenza virus. These results indicate that mucosal immunization and, in particular, local vaginal immunization with recombinant influenza virus can provide strong, durable immune responses in the female genital tract of mice.