Vectors Based on Modified Vaccinia Ankara Expressing Influenza H5N1 Hemagglutinin Induce Substantial Cross-Clade Protective Immunity

New highly pathogenic H5N1 influenza viruses are continuing to evolve with a potential threat for an influenza pandemic. So far, the H5N1 influenza viruses have not widely circulated in humans and therefore constitute a high risk for the non immune population. The aim of this study was to evaluate the cross-protective potential of the hemagglutinins of five H5N1 strains of divergent clades using a live attenuated modified vaccinia Ankara (MVA) vector vaccine.The replication-deficient MVA virus was used to express influenza hemagglutinin (HA) proteins. Specifically, recombinant MVA viruses expressing the HA genes of the clade 1 virus A/Vietnam/1203/2004 (VN/1203), the clade 2.1.3 virus A/Indonesia/5/2005 (IN5/05), the clade 2.2 viruses A/turkey/Turkey/1/2005 (TT01/05) and A/chicken/Egypt/3/2006 (CE/06), and the clade 2.3.4 virus A/Anhui/1/2005 (AH1/05) were constructed. These experimental live vaccines were assessed in a lethal mouse model. Mice vaccinated with the VN/1203 hemagglutinin-expressing MVA induced excellent protection against all the above mentioned clades. Also mice vaccinated with the IN5/05 HA expressing MVA induced substantial protection against homologous and heterologous AH1/05 challenge. After vaccination with the CE/06 HA expressing MVA, mice were fully protected against clade 2.2 challenge and partially protected against challenge of other clades. Mice vaccinated with AH1/05 HA expressing MVA vectors were only partially protected against homologous and heterologous challenge. The live vaccines induced substantial amounts of neutralizing antibodies, mainly directed against the homologous challenge virus, and high levels of HA-specific IFN-γ secreting CD4 and CD8 T-cells against epitopes conserved among the H5 clades and subclades.The highest level of cross-protection was induced by the HA derived from the VN/1203 strain, suggesting that pandemic H5 vaccines utilizing MVA vector technology, should be based on the VN/1203 hemagglutinin. Furthermore, the recombinant MVA-HA-VN, as characterized in the present study, would be a promising candidate for such a vaccine

A Pandemic Influenza H1N1 Live Vaccine Based on Modified Vaccinia Ankara Is Highly Immunogenic and Protects Mice in Active and Passive Immunizations

The development of novel influenza vaccines inducing a broad immune response is an important objective. The aim of this study was to evaluate live vaccines which induce both strong humoral and cell-mediated immune responses against the novel human pandemic H1N1 influenza virus, and to show protection in a lethal animal challenge model.For this purpose, the hemagglutinin (HA) and neuraminidase (NA) genes of the influenza A/California/07/2009 (H1N1) strain (CA/07) were inserted into the replication-deficient modified vaccinia Ankara (MVA) virus - a safe poxviral live vector – resulting in MVA-H1-Ca and MVA-N1-Ca vectors. These live vaccines, together with an inactivated whole virus vaccine, were assessed in a lung infection model using immune competent Balb/c mice, and in a lethal challenge model using severe combined immunodeficient (SCID) mice after passive serum transfer from immunized mice. Balb/c mice vaccinated with the MVA-H1-Ca virus or the inactivated vaccine were fully protected from lung infection after challenge with the influenza H1N1 wild-type strain, while the neuraminidase virus MVA-N1-Ca induced only partial protection. The live vaccines were already protective after a single dose and induced substantial amounts of neutralizing antibodies and of interferon-γ-secreting (IFN-γ) CD4- and CD8 T-cells in lungs and spleens. In the lungs, a rapid increase of HA-specific CD4- and CD8 T cells was observed in vaccinated mice shortly after challenge with influenza swine flu virus, which probably contributes to the strong inhibition of pulmonary viral replication observed. In addition, passive transfer of antisera raised in MVA-H1-Ca vaccinated immune-competent mice protected SCID mice from lethal challenge with the CA/07 wild-type virus.The non-replicating MVA-based H1N1 live vaccines induce a broad protective immune response and are promising vaccine candidates for pandemic influenza

Recombinant poxviruses expressing avian influenza H5N1 antigens : a tool to study the role of individual H5N1 antigens in protection

Hintergrund: Die hoch pathogenen H5N1 Influenza Viren verändern sich und infizieren Menschen fortwährend mit der potentiellen Gefahr einer Influenza Pandemie. Das Ziel dieser Studie war es die Rolle einzelner H5N1 Antigene im Schutz und das Potential des HA Proteins im Kreuzschutz zu erforschen. Darüber hinaus wurde der Einfluss einer existierenden Immunität gegen den Vektor untersucht. Außerdem wurde das schützende Potential von Lebendvakzinen gegen das humane H1N1 Influenza Pandemie Virus erforscht. Ergebnisse: Es wurden rekombinante Vaccinia Viren basierend auf dem Stamm Lister konstruiert, die die Influenza Gene Hemagglutinin (HA), Neuraminidase (NA), Nukleoprotein (NP), Matrix Protein 1 (M1), und Matrix Protein 2 (M2) des Stammes A/Vietnam/1203/2004 (H5N1) exprimieren. Diese Lebendvakzine wurden zusammen mit Kontrollen in einem letalen Mausmodel auf Ausbildung von Schutz und einer T-Zell Antwort geprüft. Mäuse, die mit dem HA Konstrukt immunisiert wurden, waren voll geschützt und entwickelten eine stabile T-Zell Antwort. Das NA Konstrukt induzierte, abhängig von der Immunisierungsdosis, sowohl vollen homologen als auch heterologen Schutz gegen ein H5N1 Challenge. Es konnten aber nur geringe Level einer spezifischen T-Zell Antwort gefunden werden. Die Immunisierung mit dem NP Konstrukt führt zu partialem Schutz und einer exzellenten T-Zell Antwort. Die Matrix Proteine M1 und M2 induzierten einen schwachen Schutz und nur eine schwache T-Zell Antwort konnte gefunden werden. Das schützende Potential des HA Proteins wurde im nächsten Schritt weiter im Hinblick auf Kreuzschutz untersucht. Dafür wurden rekombinante MVAs, die die HA Gene verschiedener Influenza Clades exprimieren, auf homologes und heterologes Challenge getestet. Die Mäuse, die mit dem MVA immunisiert wurden, welches das Clade 1 HA von A/Vietnam/1203/2004 exprimiert (MVA-HA-VN), waren vollständig gegen das homologe und heterologe Challenge mit dem Clade 2.1.3 Virus A/Indonesia/5/2005 (IN5/05), dem Clade 2.2 Virus A/turkey/Turkey/1/2005 (TT01/05) und dem Clade 2.3.4 Virus A/Anhui/1/2005 (AH1/05) geschützt. Auch die Mäuse, die mit den anderen MVAs, welche das HA von IN5/05, TT01/05, AH/1/05, oder A/chicken/Egypt/3/2006 (CE/06) exprimieren, entwickelten, wenn auch geringer, homologen und heterologen Schutz. Eine bereits vorhandene Immunität gegen den Vektor MVA hatte dabei keinen Einfluss auf das Schutzpotential von MVA-HA-VN, unter der Voraussetzung, dass das Zeitintervall zwischen der Vorimmunisierung und Immunisierung lang genug war. Das rekombinante MVA, welches das HA des H1N1 Stammes A/California/04/09 (MVA-H1-Ca) exprimiert, rief vollen Schutz gegen den H1N1 Wildtyp-Stamm hervor. Wohingegen das rekombinante MVA, dass das NA exprimiert, nur partiellen Schutz hervorrief. Beide Konstrukte induzierten hohe Mengen spezifischer CD4 und CD8 T-Zellen. Ein starker Anstieg HA-spezifische CD4 und CD8 T-Zellen wurde in der Lunge von MVA-H1-Ca immunisierten Mäusen beobachtet. Auch Seren von Mäusen die mit MVA-H1-Ca immunisiert wurden, führten nach dem Transfer in SCID Mäuse zu einem passiven Schutz gegen ein letales Challenge mit dem H1N1 Wildtyp-Stamm. Schlussfolgerung: Ein Lebendvakzin, beruhend auf einem replikations-inkompetenten MVA, welches das Influenza HA Gen exprimiert induziert eine breite homologe und heterologe, subtyp-spezifische Immunantwort und ist ein vielversprechender Kandidat für einen pandemischen Impfstoff. Konstrukte die das Influenza NA und NP Gen enthalten sind vielversprechende Kandidaten für die Induktion hetero-subtypischer Immunität.Background: The highly pathogenic H5N1 influenza viruses continue to evolve and infect humans with a potential threat for an influenza pandemic. The aim of this study was to analyze the role of the individual H5N1 antigens in protection and to evaluate the cross-protective potential of different hemagglutinins. Furthermore, the impact of pre-existing vector immunity was evaluated. Additionally, the protective potential of live vaccines against the human pandemic H1N1 influenza virus was evaluated. Results: Recombinant vaccinia viruses based on the Lister vaccine strain expressing the hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), and matrix protein 2 (M2) of the influenza strain A/Vietnam/1203/2004 (H5N1) were constructed. These live vaccines, together with controls, were tested in a lethal challenge model for induction of protection and specific T cell responses. Mice immunized with the HA construct were fully protected and developed a robust T cell response. The NA construct induced full homologous and heterologous protection dependent of the immunization dose but only low levels of specific influenza T cell responses were detected. Immunizations with the NP construct lead to partial protection and an excellent T cell response. The matrix proteins M1 and M2 induced poor protection and only poor T cell responses were detected. The protective potential of the HA protein was further investigated in cross-protection. Therefore recombinant MVAs expressing the HA genes of different clades were tested in homologous virus and heterologous challenge experiments. Mice vaccinated with the MVA expressing the clade 1 HA of A/Vietnam/1203/2004 (MVA-HA-VN) were fully protected against the homologous and heterologous clade 2.1.3 virus A/Indonesia/5/2005 (IN5/05), the clade 2.2 viruses A/turkey/Turkey/1/2005 (TT01/05) and the clade 2.3.4 virus A/Anhui/1/2005 (AH1/05). Also mice immunized with the other MVAs expressing the HA of IN5/05, TT01/05, AH/1/05, or A/chicken/Egypt/3/2006 (CE/06) developed homologous and heterologous protection to a lesser extent. The pre-existing immunity to the MVA vector had no impact on the protective potential of MVA-HA-VN, if the time interval between pre-vaccination and immunization was long enough. The recombinant MVA expressing the HA of A/California/04/09 (MVA-H1-Ca) lead to full protection against the H1N1 wild-type strain whereas the MVA expressing NA lead only to partial protection. Both constructs induced high amounts of specific CD4 and CD8 T cells. In the lungs, a rapid increase of HA-specific CD4 and CD8 T cells was observed in mice immunized with the MVA-H1-Ca. Also passive transfer of antisera raised in MVA-H1-Ca immunized mice protected SCID mice from lethal challenge with the wild-type H1N1 strain. Conclusions: A non-replicating MVA-based live vaccine expressing the influenza HA gene induces a broad homologous and heterologous, subtype-specific immune response and is a promising candidate for a pandemic vaccine. The NA and NP containing constructs are promising candidates for inducing heterosubtypic immunity.submitted by Annett HesselAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2011OeBB(VLID)188345

MVA vectors expressing conserved influenza proteins protect mice against lethal challenge with H5N1, H9N2 and H7N1 viruses.

BACKGROUND: The availability of a universal influenza vaccine able to induce broad cross-reactive immune responses against diverse influenza viruses would provide an alternative to currently available strain-specific vaccines. We evaluated the ability of vectors based on modified vaccinia virus Ankara (MVA) expressing conserved influenza proteins to protect mice against lethal challenge with multiple influenza subtypes. METHODS: Mice were immunized with MVA vectors expressing H5N1-derived nucleoprotein (NP), the stem region of hemagglutinin (HA), matrix proteins 1 and 2 (M1 and M2), the viral polymerase basic protein 1 (PB1), or the HA stem fused to a quadrivalent matrix protein 2 extracellular domain (M2e). Immunized mice were challenged with lethal doses of H5N1, H7N1 or H9N2 virus and monitored for disease symptoms and weight loss. To investigate the influence of previous exposure to influenza virus on protective immune responses induced by conserved influenza proteins, mice were infected with pandemic H1N1 virus (H1N1pdm09) prior to immunization and subsequently challenged with H5N1 virus. Antibody and T cell responses were assessed by ELISA and flow cytometry, respectively. RESULTS: MVA vectors expressing NP alone, or co-expressed with other conserved influenza proteins, protected mice against lethal challenge with H5N1, H7N1 or H9N2 virus. Pre-exposure to H1N1pdm09 increased protective efficacy against lethal H5N1 challenge. None of the other conserved influenza proteins provided significant levels of protection against lethal challenge. NP-expressing vectors induced high numbers of influenza-specific CD4(+) and CD8(+) T cells and high titer influenza-specific antibody responses. Higher influenza-specific CD4(+) T cell responses and NP-specific CD8(+) T cell responses were associated with increased protective efficacy. CONCLUSIONS: MVA vectors expressing influenza NP protect mice against lethal challenge with H5N1, H7N1 and H9N2 viruses by a mechanism involving influenza-specific CD4(+) and CD8(+) T cell responses

Weight loss after H5N1, H9N2 and H7N1 challenge.

<p>Mice were immunized twice, three weeks apart, with MVA vectors and challenged three weeks later with (<b>A and B</b>) 42 LD₅₀ of H5N1 VN1203, (<b>C</b>) 32 LD₅₀ of mouse-adapted H9N2 HK/G9 or (<b>D</b>) 16 LD₅₀ of H7N1 RO/34. (<b>B</b>) Primed mice were infected intranasally with 100 TCID₅₀ H1N1pdm09 virus, six weeks before immunization. Animals were monitored for 14 days after challenge. Shown are the daily variations in weight, as percentages compared to before virus challenge.</p

Protective efficacy of MVA vectors in Balb/c mice.

<p>Data are n/N (%) protected mice, 14 days after challenge with with 42 LD₅₀ of H5N1 VN1203, 32 LD₅₀ of mouse-adapted H9N2 HK/G9 or 16 LD₅₀ of H7N1 RO/34.</p>a<p>mice were immunologically primed by infecting with H1N1 six weeks prior to immunization.</p

Symptom scores after H5N1, H9N2 and H7N1 challenge.

<p>Mice were immunized twice, three weeks apart, with MVA vectors and challenged three weeks later with (<b>A and B</b>) 42 LD₅₀ of H5N1 VN1203, (<b>C</b>) 32 LD₅₀ of mouse-adapted H9N2 HK/G9 or (<b>D</b>) 16 LD₅₀ of H7N1 RO/34. (<b>B</b>) Primed mice were infected intranasally with 100 TCID₅₀ H1N1pdm09 virus, six weeks before immunization. Animals were monitored for 14 days after challenge. Shown are the cumulative mean symptom scores whereby ruffled fur, curved posture, apathy and death were scored as 1, 2, 3 and 4, respectively.</p

Conserved M2e sequences used to generate quadrivalent M2e coding sequence.

a<p>Example of a specific virus strain coding the respective M2e sequence.</p>b<p>Amino acid sequence of the respective M2e. Differences to the H5N1 M2e sequence are highlighted in bold.</p>C<p>Amino acid sequence of H1N1 is identical to that of H2N2 A/Korea/426/68 and H3N2 A/NewYork/392/2004.</p